Differential expression of mitogen activating protein kinases in periodontitis.

AIM: Following toll-like receptor (TLR) engagement, lipopolysaccharide (LPS) can stimulate the expression of pro-inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase-induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen-activated protein kinases (MAPK) described in mammals, p38, c-Jun N-terminal activated kinases (JNK1-3) and extracellular activated kinases (ERK1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre-clinical animal models. MATERIALS & METHODS: In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho-specific antibodies against p38, JNK and ERK kinases. RESULTS: Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive (p-value < 0.05) for total p38 (correlations ranging 0.49-0.68), phospho-p38 (range 0.44-0.56), and total ERK (range 0.52-0.59) levels, and correlations with JNK levels also supported association (range 0.42-0.59). Phospho-JNK and phospho-ERK showed no significant positive correlation with clinical parameters of disease. CONCLUSION: These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.

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.

Presence of aspartate aminotransferase in peri-implant crevicular fluid with and without mucositis.

The aim of this study was to assess the presence of aspartate aminotransferase (AST) in peri-implant crevicular fluid, with or without clinical signs of mucositis, to determine its predictive diagnostic value, sensitivity, and specificity. The AST levels were determined (at a threshold of 1200 µIU/mL) for 60 clinically successful implants in 25 patients with or without peri-implant mucositis. Samples were taken prior (AST1) to peri-implant probing with a manual constant-pressure probe (0.2 N) and 15 minutes after probing (AST2). Clinical assessments included radiographic determination of preexisting bone loss, probing, and the evaluation of mucositis, plaque, and bleeding upon probing. Analysis was performed at both the level of the implant and the patient as a unit. We detected a significant difference between AST1 and AST2 at both levels. A significant difference was observed at AST1 between implants that bled upon probing and those that did not. However, when we considered the patient as a unit, there were no significant differences. The plaque index was not significant at either level. AST1 had high specificity and positive predictive diagnostic value (80%) for bleeding upon probing. Probing induces a greater release of AST from inflamed tissues compared with healthy tissues in situ but not at the systemic level. At the implant level, the implant position could be responsible for this difference. Aspartate aminotransferase was a reliable predictor of patients with mucositis.

Analysis of cathepsin-K activity at tooth and dental implant sites and the potential of this enzyme in reflecting alveolar bone loss.

BACKGROUND: Cathepsin-K is an enzyme involved in bone metabolism which may make this feature important for both natural teeth and dental implants. The aims of the present study are to comparatively analyze the gingival crevicular fluid (GCF)/peri-implant sulcus fluid (PISF) cathepsin-K levels of natural teeth and dental implants, and to assess the potential relationship between this biochemical parameter and alveolar bone loss around natural teeth and dental implants. METHODS: Probing depth, bleeding on probing, gingival index, and plaque index clinical parameters were assessed, and GCF/PISF samples were obtained from natural teeth/dental implants presenting with either clinical health, gingivitis/peri-implant mucositis, or chronic periodontitis/peri-implantitis. Cathepsin-K activity levels of 42 GCF samples and 54 PISF samples were determined, and marginal bone loss (MBL) measures were calculated from digitalized standardized intraoral periapical radiographs obtained from natural teeth and dental implants by using cemento-enamel junction and the actual distance between two consecutive threads of the dental implant as reference points for natural teeth and dental implants, respectively. RESULTS: Comparing the natural teeth group with dental implant group with regard to MBL measure, cathepsin-K activity, and GCF/PISF volume revealed no significant differences. In both natural teeth and dental implant groups, despite higher MBL measures, cathepsin-K activity, and GCF/PISF volumes with the presence of inflammation, it was the presence of alveolar bone loss that lead to significantly higher values for these parameters. CONCLUSION: We suggest cathepsin-K as a biochemical parameter for monitoring periodontal/peri-implant alveolar bone loss.

Protease inhibitor levels in periodontal health and disease.

BACKGROUND AND OBJECTIVE: Our previous study showed that protease inhibitors were attenuated by the periodontal pathogen Porphyromonas gingivalis in cultured gingival epithelial cells. We hypothesize that fewer protease inhibitors would be present in more advanced periodontal disease sites, where the level of P. gingivalis may be high. The goal of this study was to investigate the relationship between the protease inhibitor [secretory leukocyte protease inhibitor (SLPI), elastase-specific inhibitor (ELAFIN) and squamous cell carcinoma antigen (SCCA)] levels in gingival crevicular fluid and the number of P. gingivalis micro-organisms in subgingival plaque. MATERIAL AND METHODS: Plaque samples from subjects without (n = 18) and with moderate to advanced periodontitis (n = 41) were used to quantify P. gingivalis using real-time PCR. Protease inhibitor levels in the gingival crevicular fluid of all the subjects were determined by ELISA. RESULTS: P. gingivalis was detected in 68.3% of patients with periodontitis, while 16.7% of subjects without periodontitis had a detectable level of P. gingivalis. Patients with periodontitis and P. gingivalis in their plaque exhibited lower SLPI and ELAFIN levels (p < 0.001) compared with control subjects without periodontitis. Secretory leukocyte protease inhibitor was also reduced (p < 0.05) in gingival crevicular fluid of periodontitis patients without a detectable level of P. gingivalis. Periodontitis patients with high vs. low levels of P. gingivalis exhibited reciprocal mean levels of SLPI and ELAFIN concentrations. CONCLUSION: The reduced concentrations of SLPI and ELAFIN may contribute to the loss of host protective capacity and increase susceptibility to breakdown from chronic infection. The work of this investigation may aid in finding diagnostic and prognostic markers in periodontal health and disease and may also help in finding pharmacological targets directed against periodontal inflammation.

Analysis of cathepsin-K levels in biologic fluids from healthy or diseased natural teeth and dental implants.

PURPOSE: Cathepsin-K is an enzyme involved in bone metabolism. This feature may make it important both for natural teeth and dental implants. The aims of the present study were to comparatively analyze cathepsin-K levels in gingival crevicular fluid (GCF) and peri-implant sulcus fluid (PISF) and to determine whether GCF and PISF cathepsin-K profiles reflect the clinical periodontal/peri-implant status. MATERIALS AND METHODS: Clinical parameters (probing depth, Gingival Index, Plaque Index, and bleeding on probing) were recorded, and GCF/PISF samples were obtained from natural teeth (group T) and dental implants (group I), which were divided into groups based on health (clinically healthy, gingivitis/peri-implant mucositis, and periodontitis/peri-implantitis). Cathepsin-K activity was determined with a commercially available cathepsin-K activity assay kit (BioVision). RESULTS: Sixty natural teeth and 68 dental implants were examined. Teeth with periodontitis (group T-3) showed significantly higher total cathepsin-K activity (10.39 units) than teeth with gingivitis (group T-2, 1.71 units) and healthy teeth (group T-1, 1.90 units). The difference in cathepsin-K activity between groups T-2 and T-1 was not significant. Implants with peri-implantitis (group I-3) had higher total enzyme activity (10.26 units) than healthy implants (group I-1) (3.44 units). Although the difference between clinical parameters was not significant, group I-3 had higher cathepsin-K levels than group I-2 (4.74 units). When natural teeth (T-1, T-2, T-3) were compared to implants (I-1, I-2, I-3), no significant differences were observed for cathepsin-K levels. CONCLUSION: More cathepsin-K activity was clearly observed with inflammatory periodontal and peri-implant destruction. The highest cathepsin-K levels detected in GCF and PISF samples, obtained from sites with periodontitis and peri-implantitis, suggests the potential involvement of cathespin-K in increased bone metabolism around natural teeth and dental implants.

The impact of smoking status on antioxidant enzyme activity and malondialdehyde levels in chronic periodontitis.

BACKGROUND: The aim of this study is to investigate the impact of smoking status on the systemic and local superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities and malondialdehyde (MDA) levels in subjects with chronic periodontitis (CP). METHODS: Sixty-five CP patients (23 smokers [CP-S], 23 former smokers [CP-FS], and 19 non-smokers [CP-NS]) and 20 periodontally healthy non-smoker controls (PH-NS) were included in the study. After the clinical measurements, serum and gingival tissue samples were collected. SOD, GSH-Px, and CAT activities and MDA levels in hemolysates and gingival tissue samples were spectrophotometrically assayed. RESULTS: Blood MDA levels in all the periodontitis groups were higher than in the PH-NS group but only the difference between CP-FS and PH-NS groups was significant (P <0.01). Gingival tissue MDA levels in the periodontitis groups were significantly higher than that in the control group (P <0.01). However, the control group had the highest gingival SOD, GSH-Px, and CAT activities compared with all the periodontitis groups (P <0.01). The CP-S group had the highest gingival MDA levels and SOD, GSH-Px, and CAT activities among the periodontitis groups, whereas the lowest values were observed in the CP-NS group (P <0.01). The blood and gingival MDA levels in the CP-FS group were similar in the CP-NS group, whereas they were lower than in the CP-S group. CONCLUSIONS: Systemic and local MDA levels are increased by smoking in addition to the impact of periodontitis. The decreased local SOD, GSH-Px, and CAT activities observed in periodontitis patients may increase with smoking.

Crevicular fluid matrix metalloproteinase-8, -13, and TIMP-1 levels in type 2 diabetics.

OBJECTIVES: To evaluate whether type 2 diabetes mellitus (DM) enlarged and if so the quantum of such increase in the gingival crevicular fluid (GCF) levels of matrix metalloproteinase-8 (MMP-8), MMP-13 and tissue inhibitor of metalloproteinases-1 (TIMP-1). METHODS: Subjects (n = 73) were divided into five groups as follows: 12 DM patients with gingivitis (DM-G), 12 DM patients with periodontitis (DM-P), 12 systemically healthy patients with gingivitis (H-G), 13 systemically healthy patients with periodontitis (H-P) and 24 periodontally, systemically healthy volunteer subjects (H-C). Full-mouth clinical periodontal measurements were performed at six sites per tooth. Gingival crevicular fluid samples were obtained from two sites representing the clinical periodontal diagnosis in single-rooted teeth. Gingival crevicular fluid levels of MMP-8, MMP-13 and TIMP-1 were analysed by immunofluorometric MMP assay (IFMA), enzyme-linked immunosorbent assay (ELISA). Data were tested statistically by parametric tests. RESULTS: All clinical periodontal measurements were similar in both diabetic and systemically healthy patients with periodontal disease (all P > 0.05). Total amounts of MMP-8 in GCF samples were significantly lower in H-C group than DM-G, DM-P, H-P groups (all P < 0.05). Matrix metalloproteinase-13, TIMP-1 total amounts were similar in study groups (P > 0.05). Diabetes mellitus patients exhibited similar levels of MMP-8, MMP-13, TIMP-1 with systemically healthy gingivitis/periodontitis patients (P > 0.05). CONCLUSIONS: Within the limits of this study, DM does not seem to significantly affect GCF levels of MMP-8, MMP-13, TIMP-1 or clinical periodontal status.

Alanine aminopeptidase and dipeptidyl peptidase IV in saliva of chronic periodontitis patients.

BACKGROUND: Alanine aminopeptidase (ALAP) and dipeptidyl peptidase IV (DPPIV) are ectopeptidases that play a role in collagen degradation and are thought to be involved in the destruction of periodontal tissue. This study compared the activities of salivary ALAP and DPPIV in patients with periodontitis and periodontally healthy subjects. The correlations of enzyme activities with clinical variables and the presence of Porphyromonas gingivalis were also evaluated. METHODS: Whole saliva was collected from 30 periodontally healthy subjects, 30 localized chronic periodontitis (LCP) patients, and 30 generalized chronic periodontitis (GCP) patients to determine the activities of ALAP and DPPIV. The presence of P. gingivalis in subgingival plaque was detected by polymerase chain reaction. Periodontal clinical assessments included probing depth, clinical attachment level, and bleeding on probing. RESULTS: The activities of DPPIV in the LCP and GCP groups were not significantly different from one another, but both groups had significantly higher enzyme activities than the periodontally healthy group (P = 0.001). DPPIV activity was positively correlated with all clinical parameters and the prevalence of P. gingivalis. The ALAP activities were not significantly different among the three study groups. There was no significant correlation of ALAP activity with any of the clinical and bacterial parameters. CONCLUSION: DPPIV, but not ALAP, activity is associated with periodontitis and the presence of P. gingivalis.

Increased expression of extracellular matrix metalloproteinase inducer is associated with matrix metalloproteinase-1 and -2 in gingival tissues from patients with periodontitis.

BACKGROUND AND OBJECTIVE: Extracellular matrix metalloproteinase inducer (EMMPRIN), an immunoglobulin-like cell surface glycoprotein, could promote collagenolytic balance in favor of the expression and activation of matrix metalloproteinases (MMPs). This study was to investigate the expression of EMMPRIN in gingival tissues from different periodontal conditions and to correlate it with the production of MMP-1 and MMP-2. MATERIAL AND METHODS: Gingival biopsies were collected from 15 patients with untreated advanced chronic periodontitis and 15 patients with aggressive periodontitis (AgP). The control group consisted of 12 subjects diagnosed either as periodontally healthy individuals or as individuals with a gingival index of one (H/G1). The peptides and mRNA of EMMPRIN, MMP-1 and MMP-2 were detected by immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction, respectively. RESULTS: The expression of EMMPRIN, MMP-1 and MMP-2 peptides in periodontally healthy tissues was mainly confined to the gingival epithelium. The EMMPRIN was strongly expressed in the cell membrane of the basal layer. Immunoreactivity for EMMPRIN was more intensive and more widespread in periodontitis, extended from the epithelial layers to the underlying connective tissues, and was essential in both inflammatory and fibroblast-like cells. In addition, MMP-1 and MMP-2 showed the same localized expression. The chronic periodontitis group had a significantly higher mRNA expression of EMMPRIN and MMP-2 compared with the H/G1 subjects (p < 0.05). Production of MMP-1 and MMP-2 by gingival tissues was correlated with the mRNA level of EMMPRIN (r = 0.463, p = 0.013 for MMP-1 and r = 0.404, p = 0.033 for MMP-2). CONCLUSION: The expression of EMMPRIN in human normal and diseased gingiva might contribute to periodontal physiological and pathological processes; moreover, its increased production might be associated with MMP-1 and MMP-2 expression.

Tumor necrosis factor-alpha-converting enzyme (TACE) levels in periodontal diseases.

Tumor necrosis factor-alpha-converting enzyme (TACE) is a metalloprotease which can shed several cytokines from the cell membrane, including receptor activator of NF-kappaB ligand (RANKL). This study aimed to investigate the hypothesis that TACE would be elevated in the gingival crevicular fluid (GCF) of persons with periodontitis. Total TACE amounts in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis or in healthy persons. TACE concentrations in GCF were higher in persons with chronic and aggressive periodontitis than in those with gingivitis, although not significantly higher than in healthy persons. Persons with chronic periodontitis receiving immunosuppressive treatment exhibited over 10-fold lower TACE levels than the other periodontitis groups. TACE was positively correlated with probing pocket depth, clinical attachment levels, and RANKL concentrations in GCF. In conclusion, the increased GCF TACE levels in persons with periodontitis and their positive correlation with RANKL may indicate an association of this enzyme with alveolar bone loss, and may warrant special attention in future therapeutic approaches.

Expression of cathepsin-K in gingival crevicular fluid of patients with periodontitis.

Cathepsin-K is a highly expressed cysteine protease, and it plays a key role in bone remodeling and cartilage breakdown in bone. Cathepsin-K is used as a well-known marker of osteoclast activity, because this enzyme is mainly derived from osteoclasts. The receptor activator for NF-kappaB ligand (RANKL) plays an important role in osteoclast formation. Although a recent study suggests the involvement of RANKL in the pathogenesis of periodontal disease, no one has previously examined the level of cathepsin-K in the body fluid of human subjects. If the presence of cathepsin-K, as well as RANKL, can be detected in body fluids, it would be indirect proof of the differentiation and/or activation of osteoclasts in the tissues bathed by these fluids. This communication reports on the in vivo concentrations of cathepsin-K and RANKL in the gingival crevicular fluid (GCF) of normal subjects and those patients with severe, moderate, and mild forms of the disease. Increased concentrations of cathepsin-K and RANKL were detected in the GCF from patients with periodontitis (P<0.005 versus control subjects). Also, there was a positive correlation between cathepsin-K and RANKL levels (r=0.726), suggesting that both of them contribute to osteoclastic bone destruction in periodontal disease.

Gingival crevicular fluid alkaline phosphatase levels in postmenopausal women: effects of phase I periodontal treatment.

BACKGROUND: The aim of this study was to determine how estrogen status may possibly influence gingival crevicular fluid (GCF) alkaline phosphatase (ALP) levels in estrogen-deficient (ED) and -sufficient (ES) postmenopausal women at baseline (BL) and 1 year after periodontal phase I treatment (AT). METHODS: Thirty-six postmenopausal women on estrogen supplements (mean serum estradiol levels >30 pg/ml; estrogen sufficient) and 37 postmenopausal women not on estrogen supplements (mean serum estradiol levels <30 pg/ml; ED) were divided into two subgroups as chronic periodontitis and clinically healthy controls after clinical and radiographic examination. The ES group consisted of 19 control (ES/C) and 17 chronic periodontitis (ES/P) patients, and the ED group consisted of 20 control (ED/C) and 17 chronic periodontitis (ED/P) patients. Plaque (PI) and gingival (GI) indices, bleeding on probing (BOP), probing depths (PD), clinical attachment loss (CAL) scores, and GCF samples were recorded at BL and AT. The levels of ALP in the GCF were measured photometrically. The paired samples Student t and Wilcoxon tests were used to compare the ALP levels and clinical parameters between BL and AT. The correlation among the ALP and clinical parameters was analyzed using the Pearson correlation. RESULTS: The mean of all clinical parameters (PI, GI, BOP, PD, and CAL) was significantly (P <0.05) higher in periodontitis groups (ES/P and ED/P) than controls (ES/C and ED/C). Periodontitis groups (ES/P and ED/P) demonstrated significantly increased GCF volumes and GCF ALP levels (P <0.05) compared to controls (ES/C and ED/C). There were no significant differences in the concentrations of ALP between periodontitis and control groups (P >0.05). The BL GCF ALP total levels of the ED/P group were significantly higher than the ES/P group (P <0.05). The BL and AT serum ALP levels of the ED/P group were not significantly but were numerically higher than the ES/P group. One year after periodontal treatment, the GCF volume, GCF ALP total, and concentrations decreased significantly in both periodontitis groups (P <0.05). However, the GCF ALP levels were still numerically higher in the ED/P group. A positive statistical correlation was found between total ALP levels and PD (r = 0.621; P <0.05). CONCLUSION: These data suggest that the presence of ALP in GCF is not simply a reflection of the local inflammation state and that a patient's estrogen status may possibly influence local ALP levels in GCF.

Cathepsin K levels in the crevicular fluid of dental implants: a pilot study.

OBJECTIVE: To determine the concentration of cathepsin K secreted into the crevicular fluid around dental implants and its correlation with clinical parameters of healthy implants and implants showing clinical signs of peri-implantitis. MATERIAL AND METHODS: Nineteen patients with 40 implants with and without peri-implantitis were enrolled in the study. Peri-implantitis was diagnosed by the pocket probing depth (PD), the modified bleeding index (MBI), the modified plaque index (MPI) and by radiographic signs of bone loss. Gingival crevicular fluid collected from the buccal and lingual sites was adsorbed to filter strips. Cathepsin K levels and total protein within the crevicular fluid were determined by immunoassay and the bicinchoninic method, respectively. RESULTS: Cathepsin K per filter strip normalized to the time of collection was 10.1 (0-33.5) pmol/sample around control implants and 22.4 (3.7-56.3) pmol/sample in the peri-implantitis group. The difference between the medians was significant (p < 0.01). Absolute cathepsin K levels in the crevicular fluid of all implants investigated showed a positive correlation with PD (R = 0.25; p = 0.03), MPI (R = 0.28; p = 0.01) and MBI (R = 0.32; p < 0.01). Absolute cathepsin K levels in the crevicular fluid also correlated with the adsorbed volume of gingival crevicular fluid (R = 0.51; p < 0.01). When normalized to the adsorbed volume of gingival crevicular fluid, the concentration of cathepsin K was 2.2 (0.01-6.4) nM around control implants and 1.7 (0.4-4.6) nM in the peri-implantitis group (p = 0.33). Patients' age correlated with sample volume and with cathepsin K normalized to the adsorbed volume of gingival crevicular fluid (R = 0.39; p < 0.01). Moreover, significant differences between male and female (p < 0.01, p < 0.01), and between mandible and maxilla (p < 0.05, p < 0.01), but not between buccal and lingual sites (p = 0.99, p= 0.93), were observed when analysed for the parameters adsorbed volume and absolute cathepsin K levels. CONCLUSION: Clinical parameters of peri-implantitis are associated with a higher amount of cathepsin K and a higher volume adsorbed to filters strips. To establish cathepsin K as a biochemical parameter to monitor peri-implant tissue health, age, sex and collection site should be considered to avoid interfering influences because of sample inhomogenity. Also a prospective study over time including more patients would be necessary.

Analysis of superoxide dismutase activity levels in gingiva and gingival crevicular fluid in patients with chronic periodontitis and periodontally healthy controls.

OBJECTIVES: Superoxide dismutase (SOD) is an antioxidant enzyme that acts against superoxide, an oxygen radical, released in inflammatory pathways and causes connective tissue breakdown. In this study, SOD activities in gingiva and gingival crevicular fluid (GCF) from patients with chronic periodontitis (CP) and periodontally healthy controls were compared. MATERIAL AND METHODS: Twenty-six CP patients and 18 controls were studied. In patients, teeth with moderate-to-severe periodontal breakdown and > or =5 mm pockets that required full-thickness flap surgery in the right or left maxillary quadrant, and in controls, teeth scheduled for extraction for orthodontic reasons were studied. After the clinical measurements (probing depth, clinical attachment level, gingival index, gingival bleeding index, plaque index), GCF samples were collected. Tissue samples were harvested from the same teeth, during flap operation in patients and immediately after tooth extraction in controls. SOD activities were spectrophotometrically assayed. The results were statistically analysed. RESULTS: Gingival SOD activity was significantly higher in the CP group than in controls (p<0.05). No significant difference was found in GCF SOD activity between the groups (p>0.05). Correlations between gingival and GCF SOD activities were not statistically significant in CP and control groups (p>0.05). CONCLUSION: In CP, SOD activity seems to increase in gingiva, probably as a result of a higher need for SOD activity and protection in gingiva in CP than in periodontal health, while not significantly changing in GCF, suggesting a weak SOD activity in GCF in periodontal disease state. The weak correlation between gingival and GCF SOD activities suggests distinct actions of these SODs.

Crevicular fluid elastase levels in relation to periodontitis and metabolic control of diabetes.

The purpose of this study was to investigate the associations between gingival crevicular fluid (GCF) elastase levels, clinical measures of periodontal status, and metabolic control of diabetes in insulin dependent (type 1) diabetes (IDDM) and non-insulin dependent (type 2) diabetes (NIDDM) patients. Sixty patients were recruited from the Diabetes Center at the University of California in San Francisco. Thirty subjects were type 1 diabetics and 30 subjects were type 2 diabetics. Metabolic control was evaluated by glycosylatted hemoglobin (HbA1c) levels. Demographic information was obtained using a structured interview with the subjects. Clinical measurements and GCF samples were taken from the mesio-buccal surfaces of 2 premolars and 2 molars from the most diseased sextant. GCF elastase was determined by measurement of p-Nitroanalide resulting from hydrolysis of elastase specific peptide. Crevicular fluid elastase levels were significantly correlated with gingival index, bleeding index, probing depth and attachment level in both type 1 and type 2 diabetes groups (0.01

Expression of cytokines and inducible nitric oxide synthase in inflamed gingival tissue.

BACKGROUND: Periodontopathic bacteria induce inflammation of periodontal tissues. The cytokines and nitric oxide released in periodontal lesions have been reported to play a protective role in bacterial infection and to relate to the process of inflammation. To clarify the relationship between colonization of periodontopathic bacteria and cytokines, we evaluated profiles of inflammatory cytokines, chemokine, anti-inflammatory cytokines, and inducible nitric oxide synthase (iNOS) and colonization by Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, which are major pathogens of periodontitis. METHODS: mRNA expression of cytokines and iNOS in inflamed and healthy gingival tissue was determined using reverse transcription-polymerase chain reaction (RT-PCR), and the relationship between their profiles and the detection of specific bacteria was analyzed. RESULTS: The relative expression of interleukin (IL)-6 and iNOS mRNAs in periodontal lesions was significantly higher than those in healthy individuals. IL-6 mRNA expression was also significantly higher at bleeding on probing (BOP)-positive sites than at BOP-negative sites. The expressions of IL-1alpha and IL-8 increased, but IL-10 expression decreased at sites where A. actinomycetemcomitans was detected. We found no correlation between the expression of cytokine and iNOS mRNA and infection by P. gingivalis. CONCLUSIONS: The expression of IL-6 may reflect inflammation in gingival tissue, and iNOS may be involved in the inflammatory process in periodontitis. The presence of A. actinomycetemcomitans or P. gingivalis might relate to the different cytokine profiles of IL-1alpha, IL-8, and IL-10.

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.

Neutrophil elastase in crevicular fluid: comparison of a middle-aged general population with healthy and periodontitis groups.

Neutrophil elastase (NE) was measured in crevicular fluid (GCF) collected from 3 subject groups. GCF was harvested at a single visit of subjects with periodontal health (n = 21) and with periodontitis (n = 28). Samples were obtained from 132 middle-aged, middle-class health conscious patients of a health maintenance organization (HMO) at baseline and 1 year later. GCF NE was higher in periodontitis than in health. Mean GCF NE of HMO subjects was much closer to health than to periodontitis. Few members of the HMO population had enzyme levels typical of periodontitis. Subjects and sites of the HMO population were segregated into 3 categories based on enzyme levels of the healthy and periodontitis subjects. Most HMO subjects and sites were in the activity category corresponding to healthy subjects. Only a small portion were in the activity category common in periodontitis. Enzyme levels in the highest activity category at both samplings were infrequent. High enzyme levels in the HMO population were not associated with attachment loss. Thus, assay of GCF NE provided little evidence of disease in a middle-aged, middle-class health conscious general population. This finding confirms an analysis of epidemiological surveys which concluded that a population such as studied here would not benefit from periodontal diagnostic testing.

Cathepsin B- and L-like activities at local gingival sites of chronic periodontitis patients.

The cysteine proteinases cathepsins B and L have the potential to degrade connective tissue in chronic periodontitis and this may progress episodically at individual tooth sites. The activities of cathepsin B- and L-like proteinases in homogenised gingival tissue from control and periodontitis patients were measured biochemically using the selective peptide substrate Z-Phe-Arg-AFC and the selective cathepsin L inhibitor Z-Phe-Phe-CHN2. Each tooth site was divided, where appropriate, into gingival tissue and granulomata. These were assayed separately and the measurements related to the DNA and protein contents of the tissues. Enzyme activity in healthy control tissue was significantly lower than in diseased tissue. Enzyme activity in gingival tissue and total tissue from periodontitis patients decreased with increasing pocket depth, clinical attachment level, gingival index and bleeding index whilst cathepsin B activity in granulomata increased with increasing pocket depth and clinical attachment level but not with increasing gingival index or gingival bleeding index. Mean enzyme activity in gingival tissue was 1.6-2.8 times greater than in granulomata. Mean patient enzyme activity in diseased patients did not correlate positively with their mean pocket depth, clinical attachment level, gingival index or gingival bleeding index. These results are best explained by the probable cellular origins of the enzymes and the likely influence of their serum and tissue inhibitors during the disease process.