MMP-13 and TIMP-1 determinations in progressive chronic periodontitis

Matrix metalloproteinase (MMP)-13 is a collagenase involved in extracellular matrix degradation either by its direct degradative effects or by processing bioactive substrates. The aim of this study was to determine the levels of MMP-13 and tissue inhibitor of metalloproteinase (TIMP)-1 in gingival crevicular fluid (GCF) and gingival biopsies obtained from active and inactive sites during chronic periodontitis progression. MATERIALS AND METHODS: This was a longitudinal study in which chronic periodontitis patients with moderate to severe disease were included and followed until they developed progression determined by the tolerance method. GCF samples were obtained from periodontitis, active, inactive and healthy sites and additional gingival biopsies were taken from active and inactive sites. MMP-13 and TIMP-1 determinations were carried out by immunodot blots and immunowestern blots. RESULTS: In progressive periodontitis, MMP-13 and TIMP-1 remained unchanged between active and inactive sites, but as the TIMP-1 relative levels increased together with MMP-13 elevation in inactive samples, an inverse correlation was observed in active sites. Besides, MMP-13 was undetectable in healthy controls. CONCLUSION: Chronic periodontitis is characterized by increased MMP-13 expression. During disease progression, active sites tended to decrease TIMP-1 levels in association with MMP-13 elevation.     

Levels of cytokine receptor activator of nuclear factor kappaB ligand in gingival crevicular fluid in untreated chronic periodontitis patients

BACKGROUND: Receptor activator of nuclear factor kappaB ligand (RANK-L) is a cytokine involved in the regulation of osteoclastogenesis in bone remodeling and inflammatory osteolysis. One of the major causes of tooth loss in humans is bone destruction. The aim of our study was to determine the presence of RANK-L in gingival crevicular fluid (GCF) samples from adult patients with untreated chronic periodontitis and in healthy controls. We also identified the RANK-L present in lesions undergoing episodic attachment loss from GCF. METHODS: GCF samples were collected from two periodontally affected sites (probing depth > or = 5 mm, attachment loss > or = 3 mm) in 20 patients (N = 40). After monitoring for 4 months, seven patients showed active periodontal disease, and GCF samples were collected from one active and one inactive site (N = 14 samples). The comparison with healthy controls was carried out by collecting GCF samples from 12 healthy volunteers (N = 24 samples). GCF was collected using a paper strip, and enzyme-linked immunosorbent assay (ELISA) was performed to determine the total amount of RANK-L. RESULTS: RANK-L was found in a higher proportion (85%) of samples from patients than from controls (46%). The total amount of RANK-L was significantly higher in patients (115.53 +/- 78.18 picograms [pg]) than in healthy subjects (63.08 +/- 55.08 pg) (P = 0.003). Active sites, presumably associated with tissue destruction, had significantly higher levels of RANK-L than their inactive counterparts (125.95 pg versus 91.80 pg, P = 0.007). CONCLUSION: GCF total amount of RANK-L is significantly increased in periodontal disease, supporting its role in the alveolar bone loss developed in this disease.     

Levels of interleukin-1 beta, -8, and -10 and RANTES in gingival crevicular fluid and cell populations in adult periodontitis patients and the effect of periodontal treatment

BACKGROUND: Various cytokines have been identified at sites of chronic inflammation such as periodontitis. Cytokines are synthesized in response to bacteria and their products, inducing and maintaining an inflammatory response in the periodontium. The purpose of the present study was to investigate the involvement of interleukin-1 beta (IL-1 beta), IL-8, and IL-10 and RANTES (regulated on activation, normally T cell expressed and secreted) and the cell populations associated with the immune response in destructive periodontitis, as well as the effect of periodontal therapy on cytokine levels in gingival crevicular fluid (GCF). METHODS: Data were obtained from 12 patients with moderate to advanced periodontitis and 6 healthy controls. Patients presenting at least 2 sites with > or =2 mm clinical attachment loss were included in the destructive periodontitis group. After monitoring for 4 months, only 6 patients showed destructive periodontitis and GCF samples and soft tissues biopsies were collected from these patients. GCF samples and biopsies were collected both from active (12 CGF samples and 6 biopsies) and inactive (12 CGF samples and 6 biopsies) sites. The comparison with healthy controls was carried out by collecting GCF samples from 6 healthy volunteers (12 samples) and biopsies during the surgical removal of wisdom teeth. In periodontal patients, clinical data and GCF samples were obtained prior to periodontal treatment (72 samples) and 2 months after periodontal therapy (72 samples). GCF was collected using a paper strip; eluted and enzyme-linked immunoabsorbent assays (ELISA) were performed to determine cytokine levels. The inflammatory infiltrate was analyzed by immunohistochemistry of gingival biopsy samples with monoclonal antibodies against CD3, CD8, CD4, CD11c, and CD19 antigens. RESULTS: Cellular components of the inflammatory infiltrate include B and T lymphocytes and monocyte/macrophages. Active sites contained a higher number of B lymphocytes and macrophages. IL-8 and IL-1 beta and RANTES in GCF were detected in the majority of sites from periodontal patients (100%, 94% and 87%, respectively); IL-10 was found in only 43%. IL-8 was the only cytokine detected in the GCF (75%) of the control group. Moreover, IL-1 beta levels were significantly higher in active sites versus inactive sites (P <0.05). IL-8 and IL-10 and RANTES were increased in active sites; however, differences were not significant (P>0.05). A positive correlation between the IL-8 and RANTES (r = 0.677, P<0.05) was observed in periodontitis patients. Periodontal therapy reduced the total amount of IL-1 beta, IL-8, and IL-10 and RANTES. Data showed a weak correlation between the clinical parameters and the total amount of cytokines in periodontitis. CONCLUSIONS: These data suggest that the amount of crevicular IL-1 beta, IL-8, and IL-10 and RANTES is associated with periodontal status. Removal of the bacterial plaque reduces the antigenic stimuli and consequently could modulate the chemokines present in GCF. We propose that the dynamic interactions between cytokines, their production rates, and their quantity could represent factors controlling the induction, perpetuation, and collapse of the cytokine network present in the periodontal disease.     

The subgingival microflora and gingival crevicular fluid cytokines in refractory periodontitis

Abstract Refractory periodontitis manifests as a rapid, unrelenting, progressive loss of attachment despite the type and frequency of therapy. This study examined possible relationships between cytokine levels in gingival crevicular fluid (GCF), occurrence of specific periodontopathic microflora. and disease activity in patients with refractory periodontitis. Refractory periodontitis patients (7 male and 3 female) were selected on the basis of history and longitudinal clinical observations. In each patient. 2 teeth with pocket depths greater than 6 mm were selected and individual acrylic stents were fabricated with reference grooves for each site. The sites were examined at both baseline and 3 months later. The pattern and amount of alveolar bone resorption were assayed by quantitative digital subtraction radiography. Pocket depth and attachment loss were measured with a Florida Probe. The gingival index was measured at 4 sites around each sample tooth. Sites were divided into active sites (2.1 mm loss of attachment in 3 months) or inactive sites (2.0 mm loss of attachment in 3 months). The distribution and prevalence of the predominant microflora in active and inactive sites were compared using anaerobic culture and indirect immunofluorescence. Interleukin-1β, 2, 4, 6 and tumor necrosis factor-α (TNF-α) levels in gingival crevicular fluid (GCF) were quantified by ELISA. Prevotella intermedia and Eikenella corrodens significantly decreased in inactive sites but remained the same in active sites after 3 months. The active sites revealed significantly higher GCF levels of IL-2 and IL-6 than inactive sites at both baseline and at 3 months. IL-1β was also significantly greater in active sites than in inactive sites at 3 months. Alveolar bone loss in active sites correlated with increased GCF levels of IL-1β and 1L-β. These results suggest that GCF levels of IL-1β, IL-2 and IL-6 and P. intermedia and E. corrodens in subgingival plaque may serve as possible indicators of disease activity in refractory periodontitis.     

Chemokine RANTES in gingival crevicular fluid of adult patients with periodontitis

Background, aims: This study presents the first evidence on the presence of the chemokine RANTES in the gingival fluid crevicular (GCF) of patients with periodontitis. RANTES is a chemokine that selectively attracts and activates macrophages and lymphocytes. Leucocytes play a critical rôle in the host response to the subgingival microflora. Method: In this study, the presence de RANTES in GCF was determined in samples obtained from adult patients with periodontitis and from control subjects with clinically healthy gingiva. GCF was collected from different probing depths (<3 mm, 4–6 mm, >6 mm) (n=72); and active (n=12) and inactive sites (n=12). An active site was defined as attachment loss >2 mm, as determined by sequential probing and the tolerance method. GFC was collected for 30 s using Periopaper^® strips, and RANTES was quantified by ELISA. Results: The presence of RANTES was detected exclusively in the group of patients with periodontitis, presenting a total amount of 40.43±16 pg and a concentration 67.80±41 pg/μl. RANTES concentration was significantly higher in probing depth <3 mm than in probing depth >6 mm (87.24 versus 51.87, p=0.014). Total amount and concentration in the GCF samples from active sites were higher that in inactive sites (p>0.05). Conclusions: The finding that RANTES is found only in patients with periodontitis, may represent a general feature of chronic inflammatory in periodontal diseases. Finally, RANTES may be implicated in the biological mechanisms underlying the pathogenesis and progression of periodontal disease.     

Monitoring periodontal disease status in smokers and nonsmokers using a gingival crevicular fluid matrix metalloproteinase-8-specific chair-side test

BACKGROUND AND OBJECTIVE: With current periodontal diagnostic tools it is difficult to identify susceptible individuals or sites at risk. The aim of this study was to evaluate the efficacy of the matrix metalloproteinase (MMP)-8-specific chair-side dip-stick test in longitudinally monitoring the periodontal status of smoking (S) and nonsmoking (NS) patients with chronic periodontitis, using their gingival crevicular fluid (GCF) MMP-8 concentrations. MATERIAL AND METHODS: Clinical parameters, MMP-8 test results and concentrations were monitored in 16 patients after initial treatment and in 15 patients after scaling and root planing (SRP), every other month, over a 12-mo time period. Progressing and stable sites, and sites with exceptionally high MMP-8 concentrations, were analysed in smokers and nonsmokers. RESULTS: SRP reduced the mean GCF MMP-8 levels, test scores, probing depth (PD), attachment loss (AL) and bleeding on probing (BOP). In sites of periodontal disease progression, the distribution of MMP-8 concentrations was broader than in stable sites, indicating a tendency for elevated concentrations in patients with periodontal disease. The mean MMP-8 concentrations in smokers were lower than in nonsmokers, but in smokers' and nonsmokers' sites with progressive disease, MMP-8 concentrations were similar. Sites with exceptionally elevated MMP-8 concentrations were clustered in smokers who also showed a poor response to SRP. In these sites, the MMP-8 concentration did not decrease with SRP and these sites were easily identified by the MMP-8 test. CONCLUSION: Persistently elevated GCF MMP-8 concentrations may indicate sites at risk, as well as patients with poor response to conventional periodontal treatment (e.g. SRP). MMP-8 testing may be useful as an adjunct to traditional periodontal diagnostic methods during the maintenance phase.     

Longitudinal evaluation of GCF MMP-3 and TIMP-1 levels as prognostic factors for progression of periodontitis

BACKGROUND: To determine whether matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in gingival crevicular fluid (GCF) could serve as prognostic factors for the progression of periodontitis, we monitored GCF MMP-3 and TIMP-1 and periodontal status of selected sites in 40 medically healthy subjects over a 6-month period. METHOD: Clinical measurements including gingival index (GI), plaque index, bleeding on probing, suppuration, probing depth (PD), attachment loss (AL), and GCF samples were taken from 2 healthy sites (including sites with gingival recession, GI=0 PD < or =3 mm; AL < or =2 mm) and 2 periodontitis sites (GI > or =1; PD > or =5 mm; AL > or =3 mm) of each patient at baseline, 3-month and 6-month visits by means of sterile paper strips. GCF levels of MMP-3 and TIMP-1 were determined by sandwich ELISA assays. RESULTS: The mean amounts of MMP-3 and TIMP-1 in diseased sites were significantly higher than in healthy sites (p<0.0001). Significantly higher GCF levels of MMP-3 and TIMP-1 were found at progressing sites than in nonprogressing periodontitis sites (0.001 or =2 mm loss of attachment during 6- month study period. GCF levels of MMP-3 were highly correlated with clinical measurements taken at baseline, 3-month and 6-month visits (p<0.001). TIMP-1 levels were only moderately correlated with probing depth and attachment level (p<0.01). Step-wise multiple regression analysis was performed to construct models for the prediction of probing depth and attachment loss increases. The most parsimonious regression models which had the best R2 values included the following variables and accounted for the indicated % of variability. The regression model for the prediction of probing depth increase included MMP-3, smoking pack-years, TIMP-1 and accounted for 53% of the variability. The best model for the prediction of attachment loss increase included MMP-3, smoking pack-years, age, TIMP-1 and explained 59% of the variability. CONCLUSION: These data indicate that sites with high GCF levels of MMP-3 and TIMP-1 are at significantly greater risk for progression of periodontitis.     

A longitudinal study of aspartate aminotransferase in human gingival crevicular fluid

Previous studies have shown that aspartate aminotransferase (AST), an established serum marker for cardiac and liver damage in humans, appears in elevated concentrations in samples of gingival crevicular fluid (GCF) from ligated vs. non-ligated teeth in beagle dogs and in elevated quantities in cross-sectional GCF sampling, adjusted for collection time, from human sites with clinical signs of past or present periodontal disease as compared to healthy sites. This paper describes a longitudinal study in which AST was monitored quarterly over a 2-year period at 2 sites/tooth in 31 patients with mild to moderate adult periodontitis. In this study sample, 40 (2.6%) of 1536 sites exhibited confirmed loss of at least 2 mm of attachment during the 2-yr observation period. In comparison with healthy sites within the same patients, AST standardized to a 30-second collection interval (AST30) was elevated at these sites with new confirmed attachment loss, and at sites with past attachment loss or gingivitis in the absence of periodontitis. When both within- and between-patient variation were taken into account, observed odds-ratios associating enzyme with disease were higher for sites with new attachment loss (9-16 depending on test cut-point) than for sites with pre-study attachment loss (3-12), or gingivitis in the absence of periodontitis (5-8). AST in GCF is strongly related to human periodontal disease. The data are consistent with the hypothesis that the relationship is strongest during episodes of cumulative tissue breakdown, but the small numbers of sites with confirmed attachment loss during the study period, or with gingivitis in the absence of periodontitis, means that further clinical studies are necessary to clarify this issue.     

Gingival crevicular fluid gelatinase and its relationship to periodontal disease in human subjects

Collagenolytic enzymes released by neutrophils are associated with the destruction of periodontium in periodontal diseases. Measurement of these enzymes in gingival crevicular fluid (GCF) could be used to test for periodontal diseases and thereby simplify diagnosis. To test this hypothesis, gelatinase (MMP-9) was analyzed in GCF samples with a simple assay system. GCF was collected by a mouthrinse method from 10 patients with gingivitis (G); 10 well-treated and maintained periodontitis patients (TP) without detectable loss of attachment; and 9 patients with recurrent loss of periodontal attachment (greater than 2 mm) and/or abscess formation (RP). Clinical measurements including tooth mobility (MOB) and gingival attachment level (GAL) were made monthly for a maximum of 10 months. Active and latent forms of gelatinase were measured by a functional assay using gelatin substrate-gel enzymography and the activities were quantified by laser densitometry. Reproducibility analysis demonstrated that the assay (inter-gel, inter-assay, inter-scan) and diurnal variations were small compared to biological variation. The presence of active gelatinase was detected in 97.8% of TP samples, 86.4% of RP samples, but in only 11.4% of G samples. In addition, the mean active gelatinase activity was found to be significantly higher (p less than 0.001) in the RP (71,006 U) than the TP (43,814 U) groups, both of which were higher (p less than 0.001) than the G group (2824 U). During periods of attachment loss, samples from the RP group exhibited a 2-fold increase of mean active gelatinase activity (129,414 U).(ABSTRACT TRUNCATED AT 250 WORDS)     

Collagenolytic fragments and active gelatinase complexes in periodontitis

Background: Periodontal tissues remodel rapidly, which enables quick adaptation to mechanical changes. Matrix metalloproteinases (MMPs) are involved in these remodeling processes under control of tissue inhibitor of metalloproteinases (TIMPs). In periodontitis, overactivity of MMPs results in pathologic tissue degradation. The aim of this study was to analyze MMPs and TIMPs in healthy and diseased gingiva, periodontal ligament (PDL), and gingival crevicular fluid (GCF). Methods: Samples of gingiva, PDL, and GCF were obtained from healthy controls (gingiva: n = 18; PDL: n = 15; GCF: n = 8) and subjects with periodontitis (gingiva: n = 11; PDL: n = 18; GCF: n = 12). MMPs and TIMPs were analyzed by gelatin-, collagen-, and reverse zymography and by Western blotting. Total MMP activity was analyzed using a fluorogenic substrate. Results: TIMP-1 and -2, active and pro-MMP-2 and -9, and active MMP-1 and -8 were present in all samples. Large amounts of active MMP-2 complexes and collagenolytic fragments were also found. Their levels were higher in PDL and GCF from subjects with periodontitis. In general, TIMP levels were lower in diseased periodontal tissues. Especially diseased GCF contained more MMPs. Surprisingly, some MMPs were more abundant in healthy gingiva and PDL than in diseased tissue. Conclusions: Unexpected variations in MMP and TIMP levels in gingiva, PDL, and GCF may result from differences in subject characteristics and disease activity. The levels of active MMP-2 complexes and collagenolytic fragments are higher in the periodontium of subjects with periodontitis and might contribute significantly to periodontal destruction.     

Gingival crevicular fluid collagenase-2 (MMP-8) test stick for chair-side monitoring of periodontitis

BACKGROUND: A rapid chair-side test based on the immunological detection of elevated levels of collagenase-2 (matrix metalloproteinase-8, MMP-8) in gingival crevicular fluid (GCF) was developed to identify and monitor the course and treatment of adult periodontitis. METHODS: MMP-8 was determined in GCF from periodontitis (11 patients, 90 sites), gingivitis (10 patients, 58 sites) and healthy control (8 patients, 59 sites) sites (i) by a test stick incorporating monoclonal antibodies to two epitopes on MMP-8 and (ii) by measuring MMP-8 concentration by a quantitative immunofluorometric assay. Patients with adult periodontitis were treated by scaling and root planing (SRP) and received oral hygiene instructions. GCF MMP-8 testing and clinical measurements were done before and after SRP. RESULTS: MMP-8 GCF levels and chair-side test differentiated periodontitis from gingivitis and healthy control sites. MMP-8 GCF levels > 1 mg/l and positive chair-side test identified especially severe periodontitis sites. A positive and negative test stick result, the outcome of which was rapidly detectable in 5 mins, in GCF correlated well with MMP-8 immunofluorometric assay analysis from the collected GCF samples and the severity of periodontitis. Scaling and root planing reduced the MMP-8 levels in severe periodontitis sites with positive MMP-8 test and gingival probing pocket depth (PD) > 5 mm before treatment. The test stick result and the quantitative assay were discrepant in only 18 of the 207 sites tested, thus agreement was very good (kappa = 0.81). With a threshold of 1 mg/l MMP-8 activity the chair-side test provided a sensitivity of 0.83 and specificity of 0.96 (n = 207). CONCLUSION: The MMP-8 test can be used to differentiate periodontitis from gingivitis and healthy sites as well as to monitor treatment of periodontitis. A reduction in GCF MMP-8 levels and a change in test stick result provide a means to optimize patient control during maintenance of periodontal treatment.     

IgG subclasses in gingival crevicular fluid from active versus stable periodontal sites

Since IgG subclasses are common immunoglobulins associated with the periodontium and have different biological characteristics, these subclasses were measured in gingival crevicular fluid (GCF) from periodontally active (greater than or equal to 2 mm clinical attachment loss within three months of sample) versus clinically similar but stable or healthy sites. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was performed to quantitate IgG subclass and albumin concentrations in serum and interproximal GCF samples from at least one each of the three disease categories from each of 20 periodontal maintenance patients. Although much variability existed among sites, mean IgG1 (p less than 0.05) and IgG4 (p less than 0.01) concentrations were higher in GCF from active periodontitis areas than stable sites, even though both had similar clinical characteristics. When IgG subclass concentrations were adjusted per mg albumin, both IgG1 and IgG4 levels in GCF from active sites were still significantly elevated over stable areas (p less than 0.05). Mean adjusted concentrations in GCF were generally greater than in serum, especially for IgG4 (active site GCF:serum = 24.2:1). GCF IgG4 concentrations may be useful as an indicator of the immunopathological changes which occur in active periodontitis.     

Collagenase activity in recurrent periodontitis: relationship to disease progression and doxycycline therapy

Previous reports have suggested that active progression of periodontitis may be correlated with increased collagenolytic activity, and that improved clinical conditions after tetracycline treatment may be explained by inhibition of host collagenase. Eighty-two patients with a recent history of periodontal abscesses and/or loss of gingival attachment level (GAL) despite active periodontal therapy were enrolled in a double-blind, randomized, placebo-controlled trial. Clinical measurements, sampling of gingival crevicular fluid (GCF) and subgingival scaling were performed every 2 months. If any site exhibited greater than 2 mm loss of GAL or a periodontal abscess, patients were administered either 100 mg doxycycline per day for 3 weeks or placebo. During 12 months of monitoring, 55 patients exhibited recurrent active disease and were then randomly assigned to either the doxycycline (n = 30) or placebo (n = 25) groups. Analysis of active collagenase and latent collagenase in GCF samples were determined by functional assays and quantitated after SDS-PAGE and fluorography. Collagenase activities were assayed at sites exhibiting active destruction (study site), at sites with pocket depth comparable to the study site but without active destruction, and at healthy sites. Clinical measurements of GAL and collagenase activity were made at intervals between 1 wk and 7 months after completion of the drug regime. Within 7 months, 15 out of 19 patients on placebo exhibited recurrent disease compared to 13 out of 29 patients on doxycycline. Collagenase activity exhibited large variations among patients and was analyzed as presence or absence of active collagenase with a logistic model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between levels of aspartate aminotransferase in gingival crevicular fluid and conventional measures of periodontal status assessed using PocketWatch: a cross-sectional study

The aim of this cross-sectional study was to determine, using PocketWatch, the relationship between the level of aspartate aminotransferase (AST) in gingival crevicular fluid (GCF) and conventional measures of periodontal status, such as probing depth, attachment level, bleeding on probing and gingival index, in patients with untreated chronic periodontitis. A total of 15 patients with chronic periodontitis were enrolled. Their periodontal status and AST levels in their GCF were measured (n = 93) and statistically analyzed. There was a statistically significant difference in AST levels between diseased periodontal sites and healthy sites (p < 0.0001). The coefficients of correlation between AST levels and probing depth, attachment level and gingival index at all sites were 0.436, 0.266 and 0.468 (Spearman rank correlation). The correlation coefficients were too small to show a definite relationship between AST levels and individual measures of clinical periodontal status. However, AST levels may help to confirm clinical observations in patients with chronic periodontitis before therapy, since AST levels differentiate active and inactive periodontal diseased sites.     

Collagenase-2 (MMP-8) and collagenase-3 (MMP-13) in adult periodontitis: molecular forms and levels in gingival crevicular fluid and immunolocalisation in gingival tissue

AIM: To determine the cellular and molecular forms of MMP-8 (collagenase-2) and MMP-13 (collagenase-3) associated with chronic adult periodontitis by examining the species present in gingival crevicular fluid (GCF) and enzyme distribution in gingival tissue. METHODS: 30-s GCF samples were collected directly from the periodontal pockets of 12 untreated patients using filter paper strips. After elution into buffer, the samples were examined by Western immunoblotting with polyclonal antibodies for MMP-8 and MMP-13 and quantification by scanning image analysis. Individual band intensities were expressed as a percentage of total sample absorbance and mean patient values were calculated. Gingival tissue from 6 patients was fixed in formalin and embedded in paraffin wax. MMP-8 and MMP-13 were localised using the same antibodies and an avidin-biotin-peroxidase detecting system. Double staining was performed with a contrasting substrate reaction. RESULTS: The majority of MMP-8 staining in pre-treatment GCF was present in 80, 75 and 60 kD bands corresponding to prepro-, pro- and active forms of PMN-type enzyme. 43 and 38 kD bands evidently represented active, fibroblast-type MMP-8. Immunoreactivities at >100 kD and < or =30 kD were probably enzyme-inhibitor complex and degraded fragments, respectively. MMP-13 was seen mainly as 60 kD proenzyme with some 40 kD active enzyme and a small proportion of >100 kD complex. The percentages of MMP-8 PMN-type enzyme and MMP-13 proenzyme bands correlated significantly with gingival and bleeding indices (p<0.05). Immunohistochemistry demonstrated MMP-8 in PMNs, sulcular epithelial and also plasma cells in inflamed gingival connective tissue. MMP-13 immunoreactivity was detected in the sulcular epithelium and in macrophage-like cells. CONCLUSION: Multiple species and elevated levels of both MMP-8 and MMP-13 from many rather than single cellular sources in the diseased periodontium are identified in untreated periodontitis GCF and active forms contribute to GCF collagenase activity.     

Gingival crevicular fluid matrix metalloproteinase-25 and -26 levels in periodontal disease

BACKGROUND: Tissue destruction associated with the progression of periodontal disease is caused by a cascade of host and microbial proteolytic enzymes. Host-derived matrix metalloproteinases (MMPs) play an important role in the degradation of the extracellular matrix. Leukolysin/membrane-type 6 (MT-6)/MMP-25, the latest member of the MT-MMP subgroup of the MMP family, is primarily expressed by neutrophils and involved in extracellular matrix turnover. Matrilysin-2/MMP-26 (endometase), a novel member of the matrilysin subgroup of the MMP family, can degrade the extracellular matrix, alpha1-antitrypsin, and activate pro-MMP-9. Our study aimed to examine the levels, molecular forms, and degrees of activation of MMP-25 and MMP-26 in gingival crevicular fluid (GCF) from patients with different periodontal diseases. METHODS: A total of 105 subjects, 35 with generalized aggressive periodontitis (GAgP), 29 with chronic periodontitis (CP), 20 with gingivitis, and 21 periodontally healthy subjects, were included in this study. Periodontal status was evaluated by measuring probing depth, clinical attachment loss, presence of bleeding on probing, and plaque. GCF MMP-25 and MMP-26 levels were analyzed by computer-quantitated Western immunoblotting using specific antibodies. RESULTS: The 57-kDa soluble pro-MMP-25 and 45- to 47-kDa active form of MMP-25 were detected by Western immunoblots in CP and GAgP GCF, and lesser levels of these soluble MMP-25 immunoreactive bands were detected in gingivitis GCF. An enhanced and similar degree of MMP-25 activation was found in GAgP, CP, and gingivitis groups. There were no detectable MMP-25 immunoreactivities in the healthy subjects' GCF. GAgP and CP groups had elevated GCF MMP-26 levels and degrees of activation compared to the gingivitis and healthy groups (P <0.008). The gingivitis group had higher GCF MMP-26 levels and degree of activation compared to the healthy group (P <0.008). CONCLUSIONS: The present study demonstrated the presence of soluble or shed forms of MMP-25 and MMP-26 in GCF of patients with different periodontal diseases. Increased levels and activation of MMP-25 and MMP-26 in GCF are associated with an enhanced severity of periodontal inflammation, suggesting that these novel MMPs can participate in the progression of periodontal diseases. They may prove to be diagnostically useful and could be targets of medication in the future.     

Prediction and diagnosis of attachment loss by enhanced chemiluminescent assay of crevicular fluid alkaline phosphatase levels

The current study aimed to apply a novel enhanced chemiluminescence assay in the analysis of gingival crevicular fluid (GCF) alkaline phosphatase (ALP) levels from patients with untreated adult periodontitis. 3666 sites in 25 patients were monitored prior to and after attachment loss was detected with a Florida disc probe. Parameters assessed were, relative attachment level, probing pocket depth, occurrence of bleeding on probing (single episode), GCF volume (microliter), total ALP levels (microIU/30 s sample time) and ALP concentration (IU/l). After recruiting patients to the study, all measures were taken at baseline and 3 months later, prior to the institution of non-surgical periodontal therapy at active sites. Thresholds for determining attachment loss were calculated using a modification of the tolerance method. The mesio-buccal sites of all teeth had GCF samples collected. The size of individual patient thresholds used to define whether attachment loss had occurred, was dependent upon the discomfort felt by that patient during electronic probing, with a positive correlation existing between discomfort on probing (10 cm visual analogue scale) and threshold size (R = 0.52, p < 0.049). A total of 274 sites (7.5%) experienced attachment loss of which 39 sites had GCF samples available for analysis. Total ALP levels were significantly higher at baseline for sites that progressed to attachment loss than paired controls (p < 0.003), but all other parameters showed no differences (p > 0.1). There were significant increases in total ALP levels and GCF volumes for active sites between baseline and 3 month measures (p < 0.01), but not for control sites or test site ALP concentration (p > 0.8). The diagnostic accuracy for GCF ALP as a predictor of future attachment loss (threshold 900 microIU/30 s) was 64%, with +ve and -ve predictive values of 62% and 68%. When a threshold of 1300 microIU/30 s was selected for ALP as a marker of recent or currently active disease, diagnostic accuracy and +ve/-ve predictive values were 77% and 77%/76%, respectively. These results indicate that total GCF ALP levels may serve as a predictor of future or current disease activity.     

Longitudinal evaluation of prostaglandin E2 (PGE2) and periodontal status in HIV+ patients

The study aim was to determine whether prostaglandin E(2) (PGE(2)) in gingival crevicular fluid (GCF) could serve as a risk factor for periodontitis in human immunodeficiency virus-positive (HIV(+)) patients. Clinical measurements, including gingival index (GI), plaque index, bleeding index, probing depth (PD), attachment loss (AL) and GCF samples were taken from two healthy sites (including sites with gingival recession, GI=0; PD< or =3 mm; AL< or =2 mm), three gingivitis sites (GI>0; PD< or =3 mm; AL=0) and three periodontitis sites (GI>0; PD> or =5 mm; AL> or =3 mm) of each of the 30 patients at baseline and 6-month visits. GCF samples were also taken by means of paper strips. GCF PGE(2) levels were determined by a sandwich ELISA. The progressing site was defined as a site which had 2 mm or more attachment loss during the 6-month study period. The mean amounts of PGE(2) were significantly higher in gingivitis and periodontitis sites than in healthy sites (p<0.0001). GCF levels of PGE(2) were significantly correlated with probing depth, attachment loss, CD4(+) cells, viral load, age and smoking pack-years at baseline and 6-month visits (0.0001相似文献   

Longitudinal evaluation of GCF IFN-gamma levels and periodontal status in HIV+ patients

BACKGROUND/AIM: Loss of periodontal support and related tooth loss is a common finding among HIV+ patients. The etiology of this destruction may be an increase in the levels of pro-inflammatory cytokines and subsequent increase in periodontal disease activity. The purpose of this study was to investigate the associations between gingival crevicular fluid interferon gamma (GCF IFN-gamma) and clinical measures of periodontal disease in HIV+ individuals. We monitored GCF IFN-gamma and periodontal status of selected sites in 33 HIV+ subjects over a 6-month period. METHOD: Clinical measurements including gingival index, plaque index, bleeding on probing, probing depth, attachment loss (AL), and GCF samples were taken from four lower incisors and the upper right posterior sextant of each patient at baseline and 6-month visits by means of sterile paper strips. GCF levels of IFN-gamma were determined by sandwich ELISA assays. A progressing site was defined as a site that had 2 mm or more AL during the 6-month study period. RESULTS: Twenty-five of the 264 examination sites showed 2 mm or more clinical AL during the 6-month study period. Significantly higher GCF levels of IFN-gamma were found at progressing sites than in nonprogressing sites (p < 0.001). GCF levels of IFN-gamma were highly correlated with clinical measurements taken at baseline and 6-month visits (0.001相似文献