Methodological evaluation of gingival crevicular fluid volume and neutrophil elastase levels: sequential sampling, length of sampling time and two different sampling methods.

Objectives: The mechanisms underlying the formation and composition of gingival crevicular fluid (GCF) and its flow into and from periodontal pockets are not understood very well. The aim of this study was to evaluate the length of sampling time and sequential sampling of GCF neutrophil elastase (NE) enzyme levels by using intracrevicular and orifice methods.Material and methods: Twenty adults (mean age of 41.8 years, ranged 31-60 years, 18 males and 2 females) with chronic periodontitis were enrolled and all completed the 3-d study. GCF was collected by both intracrevicular and intrasulcular methods, 720 samples of GCF were collected. In first, second and third day, the length of sampling time in seconds (s) and order were '5- 10-30-s'; '10- 30- 5-s' and '30- 5- 10-s,' respectively. GCF elastase levels were determined by hydrolysis of neutrophil specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide.Results: NE activity (µU) and NE activity/volume (µU/µl) were significantly different for order of sampling (p < .05), but not for the length of sampling time (p>.05).Conclusions: Within the limits of this study, the choice of sampling technique in GCF-profile studies seems to be a critical decision as it has the potential to affect the GCF volume and NE activity.

Structural insights unravel the zymogenic mechanism of the virulence factor gingipain K from Porphyromonas gingivalis, a causative agent of gum disease from the human oral microbiome.

Skewing of the human oral microbiome causes dysbiosis and preponderance of bacteria such as Porphyromonas gingivalis, the main etiological agent of periodontitis. P. gingivalis secretes proteolytic gingipains (Kgp and RgpA/B) as zymogens inhibited by a pro-domain that is removed during extracellular activation. Unraveling the molecular mechanism of Kgp zymogenicity is essential to design inhibitors blocking its activity. Here, we found that the isolated 209-residue Kgp pro-domain is a boomerang-shaped all-ß protein similar to the RgpB pro-domain. Using composite structural information of Kgp and RgpB, we derived a plausible homology model and mechanism of Kgp-regulating zymogenicity. Accordingly, the pro-domain would laterally attach to the catalytic moiety in Kgp and block the active site through an exposed inhibitory loop. This loop features a lysine (Lys129) likely occupying the S1 specificity pocket and exerting latency. Lys129 mutation to glutamate or arginine led to misfolded protein that was degraded in vivo Mutation to alanine gave milder effects but still strongly diminished proteolytic activity, without affecting the subcellular location of the enzyme. Accordingly, the interactions of Lys129 within the S1 pocket are also essential for correct folding. Uniquely for gingipains, the isolated Kgp pro-domain dimerized through an interface, which partially overlapped with that between the catalytic moiety and the pro-domain within the zymogen, i.e. both complexes are mutually exclusive. Thus, pro-domain dimerization, together with partial rearrangement of the active site upon activation, explains the lack of inhibition of the pro-domain in trans. Our results reveal that the specific latency mechanism of Kgp differs from those of Rgps.

Role of indoleamine 2,3-dioxygenase in an inflammatory model of murine gingiva.

BACKGROUND AND OBJECTIVE: Indoleamine 2,3-dioxygenase (IDO) is one of the major pathways for metabolism of tryptophan in a variety of cells, including immune cells. Increasing evidence indicates that IDO is a critical player in establishing the balance between immunity and tolerance and ultimately in the maintenance of homeostasis. By inducing inflammation in gingival tissue, we tested the hypothesis that IDO is a pivotal player in regulating the immune and inflammatory responses of gingiva. MATERIAL AND METHODS: We utilized the IDO knockout mouse model in conjunction with lipopolysaccharide (LPS)-induced inflammation. Accordingly, wild-type and IDO knockout mice were injected with LPS or vehicle in the anterior mandibular gingiva, twice over a 2-wk period, which was followed by procurement of gingival tissue for histopathology and preparation of tissue for flow cytometry-based studies. RESULTS: Clinical and histological examinations revealed a marked adverse impact of IDO deficiency on gingival inflammation. These observations were consistent with a more marked increase in the number of cells positive for the proinflammatory cytokine interleukin (IL)-17, but no significant change in the number of cells positive for the anti-inflammatory cytokine IL-10, in LPS-treated IDO knockout mice. Consistent with the more marked proinflammatory impact of IDO deficiency, the percentage of regulatory T cells was much reduced in gingival tissue of LPS-treated IDO knockout mice than in gingival tissue of wild-type mice. These proinflammatory changes were accompanied with a prominent increase in apoptotic and necrotic cell death in gingival tissue of IDO knockout mice compared with wild-type mice. CONCLUSION: Collectively, our findings support a major role for IDO in the development of gingival inflammation, as an example of an inflammatory condition, and lay the foundation for subsequent studies to explore it as a novel immunotherapy target.

Evaluation of a novel point-of-care test for active matrix metalloproteinase-8: agreement between qualitative and quantitative measurements and relation to periodontal inflammation.

BACKGROUND AND OBJECTIVES: Severe periodontitis affects about 10% of the world population. In addition, associations between periodontitis and systemic diseases exist. Therefore, the diagnosis should be made quickly and at an early stage. Matrix metalloproteinase-8 (MMP-8) is the most prominent collagenase found in inflamed periodontal tissues. Its active form (aMMP-8) is increasingly used as a diagnostic biomarker. Aim of the present study is to evaluate the diagnostic accuracy of a novel aMMP-8 point-of-care (POC) test in comparison to the standard laboratory test to diagnose the disease rapidly and reliably. MATERIAL AND METHODS: In a prospective, mono-center, double-blinded, case-control study, participants with healthy gums (n = 35), gingivitis (n = 60) and periodontitis (n = 35) were investigated before and after therapy. Beside clinical variables for plaque and inflammation, aMMP-8 concentrations were determined in oral rinsing specimens by the enzyme-linked immunosorbent assay (ELISA) and by POC. Positive and negative percent agreements with their exact one-sided lower 95% confidence limits were calculated. RESULTS: Of 130 participants, 111 finished the study. Overall, positive percent agreements were 75.8% (57.7-88.9) before treatment and 73.7% (56.9-86.6) after treatment. Negative percent agreements were 92.8% (85.7-97.0) before and 93.3% (85.1-97.8) after treatment. Positive test results (POC and ELISA) ranged from 5.7% to 8.6% in healthy patients, 25.0-29.8% in patients with gingivitis and 40.0-48.1% in patients with periodontitis. Patients who had positive aMMP-8 test results (POC) showed higher scores for plaque and inflammation. CONCLUSIONS: The novel POC test to detect aMMP-8 has proved to agree strongly with the standard method, ELISA. The test can be recommended to screen patients at risk for periodontitis in dental offices, at the general practitioner and at specialists for associated diseases.

Association between serum and oral matrix metalloproteinase-8 levels and periodontal health status.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase-8 (MMP-8) is involved in a wide range of pathologies including periodontitis and cardiovascular diseases (CVD). The association between periodontitis and CVD has been repeatedly recognized. The aim of the study was to analyze to what extent circulating active MMP-8 (aMMP-8) is associated with periodontal disease status and oral fluid aMMP-8 levels in otherwise healthy subjects. MATERIAL AND METHODS: In a cross-sectional study, aMMP-8 was measured in serum of 59 volunteers, comprising 19 periodontally healthy subjects, 20 patients with gingivitis as well as 20 with periodontitis. All study subjects were characterized regarding aMMP-8 concentrations in different oral fluids as well as clinically and microbiologically with respect to periodontal disease. aMMP-8 levels in gingival crevicular fluid were measured using the enzyme-linked immunosorbent assay. Saliva enzyme levels as well as circulating aMMP-8 were determined by a time-resolved immunofluorometric assay. Both methods utilized the same monoclonal antibodies. Correlation and regression analyses were performed to study the potential association between serum aMMP-8 and oral parameters. RESULTS: Oral aMMP-8 levels were significantly higher in patients with periodontitis compared to periodontally healthy or gingivitis subjects. Highest serum aMMP-8 concentration was also found in the periodontitis group. The serum levels correlated significantly with oral aMMP-8 as well as with clinical parameters in a dose-dependent manner. These results were confirmed in a multivariate regression analysis. After adjusting for potential confounders, saliva aMMP-8 concentrations as well as periodontitis severity were significant predictors of serum aMMP-8. CONCLUSION: The associations between circulating aMMP-8 and oral aMMP-8 as well as periodontal findings in a dose-dependent manner may contribute to linking periodontal disease with increased CVDsusceptibility.

Expression of cathepsin K in periodontitis and in gingival fibroblasts.

OBJECTIVE: To study non-osteoclastic sources of cathepsin K in periodontitis. MATERIALS AND METHODS: Tissue samples were obtained from 10 otherwise healthy periodontitis pati-ents during routine periodontal flap operations and 10 systemically and periodontally healthy individuals who underwent extraction operations for retained third molars. Methods used were immunohistochemistry, image analysis, immunofluorescence double-staining, gingival fibroblast culture, tumour necrosis factor-α (TNF-α) stimulation and Western blotting. RESULTS: Macrophage-like cells, fibroblast-like cells, vascular endothelial cells and gingival epithelial cells were more intensively stained for cathepsin K and also more frequent in periodontitis than in controls (665 ± 104 vs 258 ± 40 cells mm(-2) , P < 0.01). Some cathepsin K(+) cells in periodontal tissues were CD68(+) , but some were CD68(-) and probably fibroblasts. Indeed, in gingival fibroblast culture, resting fibroblasts released cathepsin K, more 43 kD procathepsin K than 29 kD active cathepsin K. TNF-α increased the release of the activated cathepsin K 4- to 5-fold. CONCLUSIONS: Results suggest that GCF-cathepsin K is not only osteoclast-derived, but in periodontitis, also other cells contribute to it. GCF-cathepsin K, perhaps together with intracellular, lysosomal collagenolytically active cathepsin K in fibroblasts, macrophages and gingival epithelial cells, can contribute to the loss of attachment and destruction of the periodontal ligament.

Site-specific dental plaque pH in 13-year-old Thai schoolchildren.

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.

The relationship between matrix metalloproteinases (MMP-3, -8, -9) in serum and peripheral lymphocytes (CD8+ , CD56+ ) in Down syndrome children with gingivitis.

BACKGROUND AND OBJECTIVE: Altered immune response may be a major contributor to periodontal disease in Down syndrome. This study investigated the relationship between peripheral lymphocytes and matrix metalloproteinases (MMPs) in serum in Down syndrome children with gingivitis. MATERIAL AND METHODS: Children with Down syndrome (n = 10) and healthy controls (n = 10) were clinically and radiographically examined during dental treatment under general anaesthesia. Peripheral blood and gingival crevicular fluid were collected from each subject and concentrations were determined: serum MMP-2, -3, -8 and -9; serum tissue inhibitors of metalloproteinases (TIMP) -1, -2 and -3; and gingival crevicular fluid. Leukocytes were isolated from peripheral blood and the relative amounts (%) of the various cell phenotypes were analysed using flow cytometry. In addition, peripheral blood cells were treated with Porphyromonas gingivalis lipopolysaccharide and levels of MMPs and TIMPs measured. RESULTS: Concentrations of MMP-3, MMP-8 and TIMP-1 in serum were significantly higher (p < 0.05) in the Down syndrome group compared to the controls. When peripheral blood leukocytes were cultured in the presence or absence of P. gingivalis lipopolysaccharide, MMP-8 levels were significantly (p < 0.05) higher in the Down syndrome group compared to controls. Children with Down syndrome exhibited significant positive correlations between CD8(+) T cells and MMP-8 (r = 0.630; p = 0.050), between CD8(+) T cells and MMP-9 (r = 0.648; p = 0.043), and between CD56(+) NK cells and MMP-3 (r = 0.828; p = 0.003) compared to controls. CONCLUSIONS: The positive relationship of serum MMP-3, -8 and -9 with immune cells in children with Down syndrome may facilitate migration of CD8(+) T cells and CD56(+) NK cells into the periodontal tissue, which may contribute to the increased degradation of periodontal tissue in individuals with Down syndrome.

The effect of adjunctive chlorhexidine mouthrinse on GCF MMP-8 and TIMP-1 levels in gingivitis: a randomized placebo-controlled study.

BACKGROUND: The aim of the present study was to evaluate the effect of adjunctive chlorhexidine (CHX) mouthrinse on gingival crevicular fluid (GCF) MMP-8 and TIMP-1 levels in plaque-associated gingivitis. METHODS: A total of 50 gingivitis patients were included in the present study. In addition to daily plaque control, CHX group rinsed with CHX, while placebo group rinsed with placebo mouthrinse for 4 weeks. GCF samples were collected, and clinical parameters including plaque index, papillary bleeding index, calculus index and pocket depth were recorded at baseline and 4 weeks. GCF MMP-8 and TIMP-1 levels were determined by immunofluorometric assay (IFMA) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: In both groups, GCF MMP-8 levels of anterior and posterior sites at four weeks were not different from baseline (p > 0.05). There were no significant differences in GCF MMP-8 levels between the study groups at four weeks (p > 0.05). GCF TIMP-1 levels of anterior and posterior sites at four weeks were higher compared to baseline in both groups (p < 0.05). There was no significant difference in GCF TIMP level between the study groups at four weeks (p > 0.05). CONCLUSIONS: CHX usage had no significant effects on the GCF MMP-8 and TIMP-1 levels in plaque-associate gingivitis. However, daily plaque control resulted in the increase of GCF TIMP-1 levels regardless of CHX usage.

Periodontal disease and gene-expression levels of metalloendopeptidases in human buccal mucosal epithelium.

BACKGROUND AND OBJECTIVE: Endopeptidases, such as neutral endopeptidase (NEP), endothelin-converting enzyme-1 (ECE-1) and a disintegrin and metalloprotease 17 (ADAM17), are believed to have various important roles in oral mucosal and epidermal tissue for the regulation of defensive biological responses in the oral cavity, and their expression and activity are influenced by various factors, including oral diseases. However, knowledge concerning these endopeptidases in the oral cavity has been minimal until now. This study focused on three metalloendopeptidases - NEP, ECE-1 and ADAM17 - in the oral buccal mucosal epithelium of patients with periodontal diseases and investigated the relationship between their gene-expression levels and periodontal disease. MATERIAL AND METHODS: The levels of expression of NEP, ECE-1 and ADAM17 mRNAs in tissue samples collected from the oral buccal mucosal epithelium of 61 patients were investigated by relative quantification using real-time RT-PCR analysis. information on oral and systemic health was obtained from the clinical record of each patient. RESULTS: Among the three groups, classified based on the diagnosis of periodontal diseases (healthy/gingivitis, early periodontitis and moderate/advanced periodontitis), the relative expression level of NEP mRNA was significantly increased in the early periodontitis group and in the moderate/advanced periodontitis group compared with that in the healthy/gingivitis group. Moreover, the relative expression levels of ECE1 and ADAM17 mRNAs were significantly increased in the moderate/advanced periodontitis group compared with those in the healthy/gingivitis group. The correlation coefficients between the mean relative expression levels of NEP and ECE1 mRNAs, NEP and ADAM17 mRNAs, and ECE1 and ADAM17 mRNAs were r = 0.758, r = 0.707 and r = 0.934, respectively (p < 0.001). Furthermore, among the oral-related factors, there was a significant correlation between the number of sites with probing pocket depths of more than 4 mm and of more than 6 mm and the relative expression levels of NEP, ECE1 and ADAM17 mRNAs. In stepwise logistic regression models, high relative expression levels of ECE1 and ADAM17 mRNAs were significantly associated with moderate/advanced periodontitis. CONCLUSION: The present study suggests that the severity of periodontal disease may be associated with the expression of metalloendopeptidase genes, including NEP, ECE1 and ADAM17, in the buccal mucosal epithelium.

Altered relationship between MMP-8 and TIMP-2 in gingival crevicular fluid in adolescents with Down's syndrome.

BACKGROUND AND OBJECTIVE: Periodontitis is more frequently found in subjects with Down's syndrome. The aim was to investigate whether the relationship between MMPs and TIMPs) in the gingival crevicular fluid of subjects with Down's syndrome is altered compared with controls. MATERIAL AND METHODS: Twenty-one adolescents with Down's syndrome and gingivitis (DS-G), 12 subjects with Down's syndrome and periodontitis (DS-P), 26 controls with gingivitis (HC-G) and eight controls with periodontitis (HC-P) were clinically examined. All patients were between 11 and 20 years of age. Gingival crevicular fluid was collected from each subject and the concentrations of MMPs (2, 3, 8, 9 and 13) and TIMPs (1, 2 and 3) (expressed as pg/µL adjusted for volume of gingival crevicular fluid) were determined using multianalyte kits from R&D Systems. RESULTS: The concentrations of MMP-2, MMP-3, MMP-8, MMP-9 and TIMP-2 in gingival crevicular fluid were significantly higher (p < 0.005) in the DS-G group compared with the HC-G group. The correlation coefficient between MMP-8 and TIMP-2 differed significantly (p = 0.006) between the DS-G group and the HC-G group. On the contrary, the correlation coefficients between MMPs and TIMPs did not differ significantly between the DS-P group and the HC-P group. However, the DS-P group exhibited a significantly lower concentration of TIMP-2 in the gingival crevicular fluid compared with the HC-P group. CONCLUSION: Down's syndrome subjects with gingivitis exhibit higher concentrations of MMPs in gingival crevicular fluid with an altered relationship between MMP-8 and TIMP-2, which might impair the periodontal tissue turnover.

Over-expression and potential role of cyclophilin A in human periodontitis.

BACKGROUND AND OBJECTIVE: We previously demostrated that EMMPRIN participates in the periodontitis and its interaction with Cyclophilin A possibly exists in animal periodontitis models. This study is aimed to address the expression and potential role of cyclophilin A (CypA) in human periodontitis. MATERIAL AND METHODS: Gingival tissues and peripheral blood were collected from patients with moderate to severe periodontitis or from healthy donors. Western blotting and immunohistochemistry were performed to detect the expression and distribution of CypA in the gingival tissues. Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated from the peripheral blood by Ficoll-Paque density-gradient centrifugation. Chemotaxis assays were applied to evaluate the effects of different concentrations of CypA (100, 300 and 500 ng/mL) on the migration of PBMCs and neutrophils. Supernatants of human THP-1 cells were collected after treatment with 200 ng/mL of CypA for different periods of time (1, 3, 6, 12 and 24 h) to detect the levels of interleukin (IL)-1ß, IL-8 and tumor necrosis factor alpha (TNF-α) by ELISA. RESULTS: Western blot analyses revealed an increase of CypA expression in inflamed gingival tissues compared with healthy tissues. Immunohistochemistry identified that the over-expressed CypA was localized in the infiltrating cells and/or in the extracellular matrix in the inflamed gingival connective tissues. The positive infiltrating cells contained mononuclear cells and lobulated-nuclei neutrophils. Chemotactic assays showed that 300 ng/mL of CypA apparently facilitated the chemotaxis of PBMCs/neutrophils from healthy donors, compared with the no-treatment control (p < 0.01 for PBMCs, p < 0.05 for neutrophils), whereas 100 and 500 ng/mL of CypA only weakly enhanced the chemotaxis of PBMCs/neutrophils (p > 0.05 for PBMCs/neutrophils, not significant). The PBMCs/neutrophils from patients with periodontitis exhibited a stronger ability to migrate when stimulated with 300 ng/mL of CypA than did PBMCs/neutrophils from healthy donors (p < 0.05 for PBMCs, p < 0.01 for neutrophils). ELISA revealed that the level of TNF-α secreted by THP-1 cells was elevated after treatment with 200 ng/mL of CypA for 12 h compared with the no-treatment 0-h control (p < 0.05). The IL-8 level was sharply raised after 3 h of stimulation with 200 ng/mL of CypA (p < 0.01 compared with 0 h), but no significant change was observed at the other time points (p > 0.05). There was no statistical difference at any of the treatment time points for the secretion of IL-1ß (p > 0.05 for 1, 3, 6, 12 and 24 h compared with 0 h). CONCLUSIONS: CypA participates in the pathogenesis of human periodontitis. It may be involved in the inflammatory response of periodontal tissues through inducing the chemotaxis of PBMCs/neutrophils and the secretion of TNF-α/IL-8.

Clinical parameters and aMMP-8-concentrations in gingival crevicular fluid in pregnancy gingivitis.

BACKGROUND: During pregnancy hormonal changes may increase the risk for developing gingivitis. The aim of this study was to evaluate the signs of gingival inflammation and the enzyme activity of matrix metalloproteinase-8 (aMMP-8) in the gingival crevicular fluid of pregnant women. METHODS: After approval by the ethics commission, a total of 40 volunteers participated in the study; group 1 (n = 20, age: 32 +/- 4 years) with pregnant women, and group 2 (n = 20, age: 30 +/- 10 years) with age-matched non-pregnant women as controls. After obtaining anamnestic data, the dental examination included assessment of oral hygiene, gingival inflammation, probing pocket depth, and recession. Gingival crevicular fluid was collected from both groups. A quantitative determination of aMMP-8 concentrations in the gingival crevicular fluid samples was performed. RESULTS: The aMMP-8 values of group 1 were higher (median 6.25 ng/mL aMMP-8 eluate) compared with group 2 (median 3.88 ng/mL aMMP-8 eluate), but the difference was not statistically significant (p = 0.265). Group 1 showed significantly increased probing pocket depths (p = 0.001). Gingival inflammation was present in 80% of the pregnant women, but only in 40% of the control subjects. CONCLUSIONS: It was shown that during pregnancy changes related to periodontal health could be observed. Higher aMMP-8 values, elevated probing pocket depths, and an increase of gingival inflammation could be detected in comparison with non-pregnant women.

Treponema denticola associates with increased levels of MMP-8 and MMP-9 in gingival crevicular fluid.

OBJECTIVES: The aim was to assess the association between the presence of site-specific subgingival micro-organisms and the levels of matrix metalloproteinases-8 and matrix metalloproteinases-9 (MMP-8 and MMP-9) in gingival crevicular fluid (GCF). MATERIALS AND METHODS: The patient group consisted of 56 subjects with periodontitis and the control group of 43 subjects without periodontitis. GCF samples from four test sites for each subject were collected. Polymerase chain reaction was used to detect the presence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Treponema denticola. MMP-8 concentrations were analyzed by a time-resolved immunofluorometric assay, and MMP-9 levels were determined by enzyme-linked immunosorbent assay. Student's unpaired t-test, chi-square test, and Fisher's exact P-value were calculated. RESULTS: The presence of T. denticola in the test sites was significantly higher in the patient group than in the control group. The presence of T. forsythia and T. denticola was associated with increased levels of MMP-8 in the test sites. Respectively, site-specific presence of T. denticola was associated with an increase in MMP-9 levels in three of the four test sites. CONCLUSIONS: The presence of subgingival micro-organisms in GCF, particularly T. denticola, appeared to induce a host response with an increased release of MMP-8 and MMP-9 in the test sites.

Acute myocardial infarction elevates serine protease activity in saliva of patients with periodontitis.

BACKGROUND AND OBJECTIVE: There are indications that acute myocardial infarction (AMI) may have an effect on the oral environment, which is reflected in the expression of salivary and gingival proteinases. According to our knowledge, no studies have been carried out to investigate the effect of AMI on the activities of two major tissue-destructive serine protease and microbial effectors, elastase and cathepsin G, produced by oral fluid polymorphonuclear granulocytes (PMN). Therefore, we compared the activities of elastase and cathepsin G in saliva from patients with AMI and from systemically healthy subjects (non-AMI) with similar periodontal conditions. MATERIAL AND METHODS: A total of 92 patients (47 AMI and 28 non-AMI patients with gingivitis or periodontitis, and 17 systemically and periodontally healthy subjects as a control group) were recruited. Clinical periodontal measurements were recorded, and stimulated whole-saliva samples were collected. The patients with AMI were clinically examined within 3-4 d after admission to the coronary care unit. The activities of saliva neutrophil elastase and cathepsin G were measured after collection, at specific time-points during incubation (from baseline to 23 h) by specific synthetic peptide substrate assays. RESULTS: The saliva of patients with AMI and periodontitis had a significant trend for the highest elastase activities among the study groups. Elastase and cathepsin G activities correlated significantly with each other in the AMI periodontitis group (r = 0.8, p < 0.01). In a logistic regression analysis, the level of salivary elastase activity associated significantly with periodontitis. CONCLUSION: AMI may be reflected in PMN serine protease elastase activity in saliva, despite its strong association with periodontitis.

Matrix metalloproteinase-8 expression in periodontal tissues surgically removed from diabetic and non-diabetic patients with periodontal disease.

BACKGROUND: Although it is known that periodontal matrix metalloproteinase-8 (MMP-8) expression is associated with periodontal disease, the information concerning the periodontal MMP-8 expression in diabetic patients with periodontal disease is insufficient. MATERIALS AND METHODS: Periodontal tissue specimens were collected from seven patients without periodontal disease and diabetes (Group 1), 15 patients with periodontal disease alone (Group 2) and 10 patients with both periodontal disease and diabetes (Group 3). The frozen sections were prepared and MMP-8 protein expression was detected using immunohistochemistry and quantified. For in vitro study, human U937 mononuclear cells were pre-exposed to normal or high glucose and then treated with lipopolysaccharide (LPS). RESULTS: The nonparametric Kruskal-Wallis test showed that the difference in MMP-8 protein levels among the three groups were statistically significant (p = 0.003). Nonparametric analysis using Jonckheere-Terpstra test showed a tendency of increase in periodontal MMP-8 levels across Group 1 to Group 2 to Group 3 (p = 0.0002). In vitro studies showed that high glucose and LPS had a synergistic effect on MMP-8 expression. CONCLUSION: Our current study showed an increasing trend in MMP-8 protein expression levels across patients without both periodontal disease and diabetes, patients with periodontal disease alone and patients with both diseases.

[Granulocyte elastase levels in saliva and gingival crevicular fluid of subjects with various periodontal conditions].

OBJECTIVE: To compare the granulocyte elastase (EA) levels in saliva and/or gingival crevicular fluid (GCF) of subjects with various periodontal conditions and analyze the relation between EA levels in GCF and in saliva. METHODS: GCF and salivary samples were collected from 17 subjects with healthy periodontium, 14 with gingivitis, 24 with chronic periodontitis (CP) and 24 with aggressive periodontitis (AgP). The EA levels in GCF and saliva were analyzed. RESULTS: The GCF-EA level in AgP were significantly higher than that in CP (0.485 3 ± 0.225 0 vs. 0.288 4 ± 0.193 1, P<0.01); the levels of EA in saliva of periodontitis patients (AgP and CP) were higher than those of healthy and gingivitis subjects (0.844 5 ± 0.660 6, 0.637 3 ± 0.648 9 vs. 0.031 6 ± 0.020 6, 0.012 2 ± 0.005 8, P<0.001). A positive correlation was found between EA levels in saliva and those in GCF (r=0.660). CONCLUSION: GCF-EA level may serve as a marker for clinical assessment of periodontal conditions. The measurement of EA levels in saliva may facilitate to overall screen periodontitis patients in epidemiological study or to monitor periodontal conditions in clinical practice.

Localized reduction of gingival inflammation using site-specific therapy with a topical gingival patch.

METHODS: Sites of inflammation were identified on subjects with moderate-to-severe chronic periodontitis, and were allocated to either patch placement or untreated controls, both for 24 hours. Conventional treatment with scaling and root planing was postponed during the study period. Inflammation was evaluated measuring neutrophilic activity using gingival crevicular fluid (GCF) beta-glucuronidase (b-glu) levels, and clinical response was evaluated using the gingival index (GI). RESULTS: A total of 26 patients were recruited and 36 sites examined, with 22 sites on which the patch was placed and 14 controls. GCF b-glu levels at 24 hours were reduced following patch placement, significantly more so than with controls (17/22 vs. 3/14 sites, respectively; p = 0.002). The patch placement resulted in a significant reduction in mean b-glu levels (-2.52 +/- 1.62), with a reduction from baseline of 29.7%. This compared to untreated controls, for whom the mean b-glu levels and percent change from baseline increased (2.14 +/- 0.89 and 33%, respectively). At 24 hours, GI response rate for treated sites was better than for control sites (18/21 vs. 7/14; p = 0.053). No adverse events were reported in either group. CONCLUSION: This pilot study indicates that a topical gingival patch promotes reduction of gingival inflammation. Further clinical testing of this novel treatment of gingival inflammation is warranted.

The effect of Parodontax® on the MMP-8 concentration in gingivitis patients.

The aim of the study was to evaluate the efficacy of Parodontax® (GlaxoSmith-Kline, Bühl, Germany) on the signs gingival inflammation and the enzyme activity of matrix metalloproteinase-8 (aMMP-8) in the gingival crevicular fluid. After approval by the ethics commission, a total of 50 volunteers participated in the study; group 1 (n = 25, age: 43 ± 12 years) with moderate gingivitis (BOP +) and group 2 (n = 25, age: 29 ± 11 years) with clinically healthy gingival conditions (BOP -). After obtaining anamnestic data, the dental examination included assessment of oral hygiene (Quigley & Hein 1962), gingival inflammation (Saxer & Mühlemann 1975), probing pocket depth and clinical attachment level. Gingival crevicular fluid was collected from both groups. A quantitative assessment of aMMP-8 in the gingival crevicular fluid samples was performed (DentoAnalyzer, Dentognostics GmbH, Jena, Germany). Study participants were instructed to use only Parodontax®. After three weeks, all parameters were measured again. The aMMP-8 values of group 1 were significantly reduced after the use of Parodontax® toothpaste and mouthwash (p < 0.001; baseline median 41.25 ± 38.16 ng/ml, final post-treatment median 7.73 ± 7.58 ng/ml aMMP-8 eluate; group 2: baseline median 3.75 ± 3.16 ng/ml, final post-treatment median 3.73 ± 1.54 ng/ml aMMP-8 eluate). Gingival inflammation and plaque accumulation were reduced. It was shown that Parodontax® was effective in reducing the enzymatic activity of inflammation.

Management of gingival inflammation in orthodontic patients with ozonated water irrigation--a pilot study.

OBJECTIVE: Ozonated water irrigation has recently been tried for its antimicrobial and anti-inflammatory effects in treatment of periodontitis. During orthodontic treatment, gingival inflammation occurs along with increased lactate dehydrogenase (LDH) enzyme levels in gingival crevicular fluid (GCF). Thus, the aim of this pilot study was to evaluate the clinical effects of a single subgingival irrigation with ozonated water on gingival inflammation in orthodontic patients and also to correlate the clinical effects with LDH enzyme activity in GCF. METHODS: Fifteen systemically healthy orthodontic patients (seven men and eight women, mean age 17.3 years) with full-mouth brackets were included in this prospective, cross-sectional, clinical and laboratory investigation. Clinical parameters, LDH enzyme activity and GCF volume were measured at baseline (0 day) followed by subgingival irrigation with 0.01 mg l(-1) ozonated water. These parameters were again assessed on 14th and 28th day. RESULTS: There was significant (P < 0.05) reduction in values of clinical parameters, GCF LDH activity and GCF volume after subgingival irrigation with ozonated water. Also, a significant correlation (r = 0.50, P = 0.01) was observed only between the post-treatment changes of plaque index and LDH values, among the clinical parameters assessed. CONCLUSIONS: A single subgingival irrigation of 0.01 mg l(-1) ozonated water can effectively reduce the gingival inflammation in orthodontic patients, which is also reflected in the reduction of LDH enzyme levels. However, further randomized controlled trials are required to validate the use of ozone irrigation in orthodontic patients for plaque control measures.