Cathepsin B, α2-macroglobulin and cystatin levels in gingival crevicular fluid from chronic periodontitis patients

Abstract. Gingival crevicular fluid (GCF) was collected from 16 molar and premolar sites in each of 20 chronic periodontitis patients before and after periodontal therapy using filter paper strips. These were eluted individually into buffer for determination of cathepsin B and its endogenous inhibitors, α₂-macroglobulin and cystatin. Cathepsin B activity was assayed with a fluorogenic peptide substrate, α₂-macroglobulin by enzyme-linked immunosorbent assay and cystatin activity by inhibition of papain. Total amounts of enzyme and inhibitor per GCF sample decreased after treatment and correlated positively with pocket depth and gingival, bleeding and plaque indices. These comparisons were nearly always statistically significant for pooled site data and sometimes so for mean patient values. The amounts of α₂-macroglobulin and cystatin were greater than those of cathepsin B and, surprisingly, enzyme and inhibitor levels correlated positively with each other. Experiments with purified reagents, however, demonstrated that the cathepsin B: α₂-macroglobulin complex was still active against the low molecular weight substrate and that cystatin levels in GCF are probably insufficient to inhibit the enzyme substantially. These factors may explain why GCF cathepsin B activity reflects the clinical status of periodontal lesions and has been identified in another study as a promising indicator of disease progression.     

Phylloquinone in gingival crevicular fluid in adult periodontitis

Abstract. Phylloquinone is a lipid soluble vitamin which is an absolute growth requirement for black-pigmented anaerobes, many of which are implicated in the aetiology of periodontal diseases. This cross-sectional study aimed to detect the levels of phylloquinone in GCF from healthy and diseased sites in subjects with adult periodontitis, in order to investigate further its potential role in the disease process. The sample consisted of eighteen patients with adult periodontitis. Periodontal probing depths, attachment levels and gingival indices were recorded from one healthy and one diseased site in each subject. GCF was sampled and the amount of phylloquinone in each sample was determined using reverse-phase high performance liquid chromatography coupled to electrochemical detection. The mean amount of phylloquinone in accumulated GCF from diseased sites was 406 pg/site and 80 pg/site from healthy sites ( p =0.013). When the amounts of phylloquinone in GCF were expressed as concentrations the values were 228 ng/ml and 3350 ng/ml for diseased and healthy sites respectively ( p =0.084). These findings suggest the levels of phylloquinone in GCF differs in periodontal health and disease in subjects with adult periodontitis. The total phylloquinone at diseased sites may provide the nutritional requirements favouring the growth of black-pigmented anaerobes.     

Cathepsin G in gingival tissue and crevicular fluid in adult periodontitis

Abstract The presence, localization and activities of cathepsin G in gingival tissue specimens and crevicular fluid (GCF) from 9 adult periodontitis patients and 6 controls with clinically healthy periodontium were studied by use of avidin-biotin-peroxidase complex method. Western and dot blotting, and spectrophotometric activity assay. In contrast to healthy gingival tissue specimens, gingival tissue specimens collected from adult periodontitis patients contained inflammatory cells in lamina propria, beneath the oral sulcular epithelium, 10–50% of which were cathepsin G positive polymorphonuclear neutrophilic leukocytes (PMNs) and monocyte/macrophage-like cells. Cathepsin G activities were increased in adult periodontitis GCF when compared to periodontally healthy controls' GCF (p>0.05). In adult periodontitis GCF, Western blotting disclosed free cathepsin G but also clear complexes of cathepsin G with its predominant endogenous inhibitor α₁antichymotrypsin (α₁-ACT). The present results demonstrate that part of the cathepsin G, despite the presence of increased concentrations of α₁-ACT, was in an uncomplexed, free and functionally active form. Our results suggest that GCF cathepsin G reflects the disease process in adjacent inflamed gingiva and also increased host response to microbiota and/or dental plaque in the periodontitis lesions. Cathepsin G may contribute to periodontal tissue destruction directly and indirectly, via proteolytic activation of latent neutrophil procollagenase (promatrix metalloproteinase-8 [proMMP-8]).     

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.     

Gingival crevicular fluid glycosaminoglycan levels in patients with chronic adult periodontitis

Abstract This study investigated levels of hyaluronan and chondroitin-4-sulphate in the crevicular fluid of patients with chronic adult periodontitis at diseased and healthy sites before and after treatment. The relationship between clinical diagnostic parameters and levels of glycosaminoglycans in gingival crevicular fluid were also analysed. Within each patient. 4 sites either mesial or distal and on single rooted teeth were classified as diseased or healthy using a modified gingival index, pocket depth and attachment loss. Crevicular fluid was collected from each site using glass micropipettes and analysed for glycosaminoglycan content by cellulose acetate electrophoresis. Significantly higher levels of chondroitin-4-sulphate were detected at diseased sites prior to treatment correlating with increased pocket depth or attachment levels. Following a period of treatment consisting of oral hygiene instruction and root planing, the patients were reassessed for their response to treatment by measuring the modified gingival index, pocket depth, attachment loss and levels of glycosaminoglycans. Analysis of glycosaminoglycan levels at diseased sites that demonstrated a poor response to treatment also demonstrated significantly higher levels of chondroitin-4-sulphate than those sites that responded well to treatment. Hyaluronan levels were less significantly associated with clinically succesful treatment. This study confirmed the use of the sulphated glycosaminoglycan chondroitin-4–sulphate as a potential diagnostic aid of periodontal tissue destruction; however, further longitudinal studies are required to assess their performance.     

Subantimicrobial-dose doxycycline and cytokine-chemokine levels in gingival crevicular fluid

Background: The present randomized, double‐masked, placebo‐controlled, parallel‐arm study examines the impact of adjunctive subantimicrobial‐dose doxycycline (SDD) on the local inflammatory response through cytokine and chemokine levels in gingival crevicular fluid (GCF) samples from patients with chronic periodontitis. Methods: Forty‐six patients with chronic periodontitis received scaling and root planing with or without adjunctive SDD. GCF samples were collected and clinical parameters including probing depth, clinical attachment level, gingival index, and plaque index were recorded every 3 months for 12 months. GCF tumor necrosis factor‐α, interleukin (IL)‐6, IL‐4, IL‐10, IL‐13, IL‐17, macrophage inhibitory protein 1α, macrophage inhibitory protein 1β, monocyte chemoattractant protein 1, and regulated on activated normal T‐cell expressed and secreted protein levels were determined by xMAP multiplex immunoassay. Results: Significant improvements were observed in all clinical parameters in both groups over 12 months (P <0.0125), whereas the SDD group showed significantly better reduction in gingival index, probing depth, and gain in clinical attachment compared to the placebo group (P <0.05). Decrease in IL‐6 in the SDD group was significantly higher compared to the placebo group at 6 and 9 months in deep pockets (P <0.05), whereas tumor necrosis factor‐α was significantly reduced in moderately deep pockets (P <0.05). SDD resulted in a stable IL‐4 and IL‐10 response while reducing the monocyte chemoattractant protein 1 levels at 3 months (P <0.05). Conclusions: These results show that SDD, as an adjunct to non‐surgical periodontal therapy, stabilizes the inflammatory response by promoting the suppression of proinflammatory cytokines and increasing the anti‐inflammatory cytokines. The chemokine activity would account for the regulation of the inflammatory response to SDD therapy.     

Efficacy of systemically administered acetylsalicylic acid plus scaling on periodontal health and elastase-α1-proteinase inhibitor in gingival crevicular fluid

Abstract The purpose of this proof of principle trial was to assess whether conventional periodontal therapy and systemically administrated acetylsalicylic acid (ASA) are functionally synergistic when combined in the treatment of periodontitis, A total of 30 patients with untreated moderate to severe adult periodontitis were enrolled into the study and were given placebo q.i.d. between the baseline and 6-week examination, and acetylsalicylic acid (ASA) 500 mg q.i.d. between the 6-week and 12-week examinations. In addition, they received supra-and subgingival scaling in 1 quadrant after baseline examination and in 2 further randomly selected quadrants after the 6-week examination. The study design resulted in the following 4 therapies: (1) scaling plus ASA 500 mg q.i.d.: (2) scaling plus placebo q.i.d: (3)ASA 500 mg q.i.d. alone: (4) placebo q.i.d. alone. Two-way analysis of variance showed functional synergism of ASA and scaling, resulting in a therapeutic efficacy approximately equivalent to the sum of each individual therapeutic efficacy (i.e., ASA alone and scaling alone) in reducing gingival inflammation and pocket probing depth over the 6-week observation period (interaction: p >0.05). Only the effect of ASA was significant m reducing the concentration of elastase-α₁-proteinase inhibitor in gingival crevicular fluid (GCF E-α₁-PI) (p >0.001), reduction in GCF E-α₁-PI concentrations by ASA may indicate a decreased risk in periodontal disease progression. The results suggest that the combination of therapies and their different mechanisms of action, i.e., reduction of bacterial plaque and inhibition of destructive components of the immune responses, may result in functionally synergistic therapeutic efficacies in patients with untreated adult periodontitis.     

Altered relation between granulocyte elastase and α-2-macroglobulin in gingival crevicular fluid from sites with periodontal destruction

Abstract Granulocyte elastase activity and α-2-macroglobulin (α-2-MG) were studied in the gingival crevicular fluid (GCF) from 3 categories of sites in 6 patients with gingivitis and 6 patients with periodontitis. 6 inflamed sites in each gingivitis patient were sampled on paper strips and 12 sites, 6 with and 6 without attachment loss and periodontal pockets, were selected in each periodontitis patient. To avoid the influence of increase GCF volume from deep pockets, the elastase activity and the α-2-MG were calculated per μl of GCF. The proteolytic activity of elastase was measured with a low molecular weight substrate and the antiprotease, α-2-MG, with ELISA. The measured activity could be ascribed to elastase that had been released into the gingival tissues and into the GCF prior to sampling. In the periodontitis patients, the sites with tissue destruction had a significantly higher elastase activity per site and per μl GCF and a significantly lower α-2-MG per μl than the 2 other categories of sites without tissue destruction. The destructive inflammation seems to be associated with increased release of elastase, either from more numerous or from more active granulocytes and with an increased proteolytic consumption of the inhibitor accompanied by the fast elimination of the protease-inhibitor-complex. In conclusion, the study shows a strong relationship between elastase activity and tissue destruction, a finding that supports the pathogenic theory of an involvement of granulocytes and their proteolytic enzymes in the mechanism of periodontal destruction.     

Gingival crevicular fluid osteocalcin in adult periodontitis

This study aimed to detect the levels of osteocalcin in gingival crevicular fluid (GCF) from healthy (< or =3 mm sulcus depth and non-bleeding) and diseased sites (> or =6 mm probing depth and bleeding) in subjects with adult periodontitis, in order to further investigate its potential as a possible marker of the disease process. Periodontal probing depths, attachment levels and gingival indices were recorded from one healthy and one diseased site in each of 20 subjects with adult periodontitis. Both GCF accumulated in the periodontal pocket or sulci and GCF flowing into the periodontal pocket or sulci over a three-minute interval were sampled. The amounts of osteocalcin in each GCF sample was determined using immunoassays. A mean of 2.34 ng/site (2.7 microg/ml) osteocalcin was found at diseased sites and a mean of 2.47 ng/site (5.47 microg/ml) was found at healthy sites for the accumulated GCF collection method. A mean of 0.17 ng/ site (2.17 microg/ml) osteocalcin was found at diseased sites and a mean of 0.14 ng/ site (1.85 microg/ml) at healthy sites for the flow method of GCF collection. There were no statistically significant differences between osteocalcin levels in diseased and healthy sites in subjects with adult periodontitis.     

The effect of topical doxycycline usage on gingival crevicular fluid MMP-8 levels of chronic and aggressive periodontitis patients: a pilot study

Abstract:  The aim of this study was to evaluate the efficacy of topical subgingival application of doxycycline hyclate (DH) gel adjunctive to non-surgical periodontal therapy on gingival crevicular fluid (GCF) matrix metalloproteinase (MMP)-8 levels in chronic and aggressive periodontitis patients. Forty teeth of 10 chronic periodontitis patients and 32 teeth of eight aggressive periodontitis patients were screened for 6 months. Scaling and root planing (SRP) was applied to the control sites and DH gel adjunctive to SRP was applied to the test sites of each patient simultaneously. GCF MMP-8 levels were analysed at baseline, 7 days; and at 1, 3 and 6 months by Sandwich Elisa Method. At 1, 3 and 6 months, probing depth ( P  < 0.0051) and plaque scores and bleeding on probing values ( P  = 0.000) significantly decreased in each group when compared with the baseline, but there was no statistically significant difference between the test and control sites. GCF MMP-8 levels reduced presenting statistically significant differences on 7 days, 1, 3 and 6 months in four of the groups ( P  < 0.05); however, intergroup differences were not statistically significant. Developing functional and immunological-based chair-side MMP tests might serve as useful adjunctive diagnostic tools when monitoring the effects of DH gel application.     

牙周炎患者龈沟液中乳酸脱氢酶水平分析

目的：探讨龈沟液中乳酸脱氢酶（ＬＤＨ）水平对于慢性成人牙周炎患者诊断及预后观察的意义。方法：酶动力学方法。结果：患病部位和健康部位龈沟液中ＬＤＨ水平有非常显著差异（Ｐ〈０．００１）。探诊深度和龈沟液中ＬＤＨ水平呈正相关（Ｐ〈０．０５）。附着丧失水平和龈沟液中ＬＤＨ水平呈正相关（Ｐ〈０．０５）。结论：龈沟液中ＬＤＨ水平对于慢性成人牙周炎的诊断和疗效监测具有一定的临床意义。     

Granulocyte elastase activity and PGE2 levels in gingival crevicular fluid in relation to the presence of subgingival periodontopathogens in subjects with untreated adult periodontitis

This study aimed to determine the association between the levels of granulocyte elastase and prostaglandin E2 (PGE2) in GCE and the concomitant presence of periodontopathogens in untreated adult periodontitis (AP). GCF and subgingival plaque were sampled by paper strips and paper points respectively, from various periodontal sites in 16 AP subjects. Granulocyte elastase activity in GCF was analyzed with a low molecular weight substrate specific for granulocyte elastase, pGluProVal-pNA, and the maximal rate of elastase activity (MR-EA, mAbs/min/site) was calculated. PGE2 levels in GCF were determined by radioimmunoassay. 5 species-specific DNA probes were used to detect the presence of A. actinomyceterncomitans (A.a., ATCC 43718), B. forsythus (B.f, ATCC 43037), P. gingivalis (P.g., ATCC 33277), P. intermedia (P.i., ATCC 33563), and T. denticola (T.d., ATCC 35405), with a sensitivity of 10(3) cells/paper point. No A.a. was detectable from all sites sampled. The predominant combination of species detected was B.f., P.g., P.i. & T.d. and it was significantly higher at periodontitis sites (68%) than at healthy (7%) or gingivitis sites (29%) (p<0.05). Overall, MR-EA values were strongly correlated with PGE2 levels (r=0.655, p<0.001), especially at these periodontitis sites co-infected by B.f., P.g., P.i. & T.d. (r=0.722, p<0.001). The periodontitis sites co-infected by the 4 species were observable from 15 subjects. These sites were sub-grouped into 8 subjects with a high MR-EA and 7 subjects with a low MR-EA. The PGE2 levels in the high MR-EA group were significantly higher than in the low MR-EA group (p<0.05). No significant differences in clinical or bacterial data were found between the two groups. While within the high MR-EA group, similar results were found between the paired periodontitis sites in each subject with highest and lowest MR-EA values. This study shows that the local host response to bacterial challenge in untreated periodontal pockets is diverse in terms of the intensity of inflammatory response measured by granulocyte elastase and PGE2 levels in GCE A more thorough evaluation of the risk for active periodontal disease may involve the combined approaches to the test of the dynamic bacteria-host relations.     

Intracytoplasmic enzymes in gingival crevicular fluid of patients with aggressive periodontitis

Castro CE, Koss MA, López ME. Intracytoplasmic enzymes in gingival crevicular fluid of patients with aggressive periodontitis. J Periodont Res 2011; 46: 522–527. © 2011 John Wiley & Sons A/S Background and Objective: Biochemical parameters of crevicular fluid could provide evidence of periodontal tissue disease. The aim of this study was to analyze enzymes in crevicular fluid in aggressive localized and generalized periodontitis. Material and Methods: One hundred and twenty‐four subjects were classified as having localized (n = 36) or generalized aggressive periodontitis (n = 38) and subclassified into moderate and severe groups. Controls were 50 periodontitis‐free subjects. Activities of the enzymes lactate dehydrogenase, neutrophil elastase, alkaline phosphatase and aspartate aminotransferase were determined. Data were analyzed using one‐way ANOVA and Tukey’s test. Results: Among the subjects with localized aggressive periodontitis, values of lactate dehydrogenase and alkaline phosphatase increased notably in moderate and severe periodontitis compared with control subjects. Values for aspartate aminotransferase increased with the severity of the disease, and neutrophil elastase was increased in the moderate and severe states. In generalized aggressive periodontitis, lactate dehydrogenase showed higher values than in control subjects in both periodontal subgroups. Alkaline phosphatase and neutrophil elastase showed higher significant differences between moderate and severe periodontitis compared with the control group. Aspartate aminotransferase showed differences between the severe and moderate periodontitis groups compared with the control group. Of all the enzymes analyzed, only lactate dehydrogenase showed higher values in localized than in generalized aggressive periodontitis. Conclusion: Lactate dehydrogenase may distinguish localized and generalized aggressive periodontitis. Alkaline phosphatase increases from moderate to severe states in both types of periodontitis. Aspartate aminotransferase and neutrophil elastase only increase with strong evidence of periodontal destruction.     

Local and systemic biomarkers in gingival crevicular fluid increase odds of periodontitis

Aim: To determine the independent and combined associations of interleukin-1 β (IL-1 β ) and C-reactive protein (CRP) in gingival crevicular fluid (GCF) on periodontitis case status in the Australian population.
Materials and Methods: GCF was collected from 939 subjects selected from the 2004–2006 Australian National Survey of Adult Oral Health: 430 cases had examiner-diagnosed periodontitis, and 509 controls did not. IL-1 β and CRP in GCF were detected by enzyme-linked immunosorbent assays. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated in bivariate and stratified analysis and fully adjusted ORs were estimated using multivariate logistic regression.
Results: Greater odds of having periodontitis was associated with higher amounts of IL-1 β (OR=2.4, 95% CI=1.7–3.4 for highest tertile of IL-1 β relative to lowest tertile) and CRP (OR=1.9, 95% CI=1.5–2.5 for detectable CRP relative to undetectable CRP). In stratified analysis, there was no significant interaction between biomarkers ( p =0.68). In the multivariate analyses that controlled for conventional periodontal risk factors, these relationships remained (IL-1 β OR=1.8, 95% CI=1.1–2.6; CRP OR=1.7, 95% CI=1.3–2.3).
Conclusions:  Elevated odds of clinical periodontitis was associated independently with each biomarker. This suggests that people with elevated biomarkers indicative of either local (IL-1 β ) or systemic (CRP) inflammation are more likely to suffer from periodontal disease.     

Post-treatment effects of subantimicrobial dose doxycycline on clinical parameters and gingival crevicular fluid transforming growth factor-beta1 in severe, generalized chronic periodontitis

Abstract: Objective: Present study aimed to evaluate the effect of 3‐month adjunctive subantimicrobial dose doxycycline (SDD) on clinical parameters and gingival crevicular fluid (GCF) transforming growth factor‐beta1 (TGF‐β₁) levels in chronic periodontitis patients over 12 months. Methods: Thirty‐five patients with severe, generalized periodontitis participated in the present randomized, placebo‐controlled study. Patients received scaling and root planing (SRP) plus 3 months adjunctive SDD or placebo. Clinical measurements and GCF sampling were performed at baseline, 3, 6, 9 and 12 months. Eleven periodontally healthy subjects served as controls for GCF TGF‐β₁ analysis. Results: Clinical parameters of both SDD and placebo groups significantly improved during the study (P < 0.0125). SDD group exhibited significantly higher PD reduction at deep sites (baseline PD ≥7 mm) compared with placebo group at 6 months (P < 0.05). In SDD group significantly higher percentage of deep pockets resolved (PD reduction ≥3 mm from baseline) when compared with placebo group at 6 and 9 months (73.4% versus 49.7%; 79.9% versus 50.6%, respectively, P < 0.05). PD reduction ≥4 mm for deep pockets from baseline was also greater in SDD group than placebo at 6 months (53.4% versus 36.3%, P < 0.05). GCF TGF‐β₁ levels of SDD group was significantly higher than baseline (P < 0.0125) and placebo group (P < 0.017) at 3 months. Conclusions: These results ensure further data for beneficial effects of adjunctive SDD therapy in the management of severe chronic periodontitis.     

Changes of alpha1-protease inhibitor and secretory leukocyte protease inhibitor levels in gingival crevicular fluid before and after non-surgical periodontal treatment

OBJECTIVES: The possible contribution of alpha1-protease inhibitor (alpha1-PI) and secretory leukocyte protease inhibitor (SLPI) in gingival crevicular fluid (GCF) to predict the periodontal disease activity was evaluated. DESIGN: GCF samples were collected at each site before scaling and root planning (SRP), 2 and 4 weeks after SRP. SUBJECTS AND METHODS: Forty-one sites that initially bled on probing in 21 patients with moderate to severe periodontitis were studied. Sites were classified according to the presence or absence of bleeding on probing (BOP) at 4 weeks. In GCF alpha1-PI and SLPI were determined by enzyme-linked immunosorbent assays. RESULTS: A significant decrease was observed in alpha1-PI at 2 and 4 weeks in BOP(-) sites and at 4 weeks in BOP(+) sites. SLPI significantly increased at 2 weeks in BOP(+) site, while SLPI did not significantly differ at both time points in BOP(-) sites and at 4 weeks in BOP(+) sites. GCF alpha1-PI was significantly less at 2 weeks in BOP(-) than in BOP(+) sites. CONCLUSION: At 2 weeks GCF alpha1-PI may reflect the healing response of the periodontal tissues following nonsurgical periodontal treatment. GCF SLPI levels may be influenced by healing.     

Elevated conversion of alpha-2-macroglobulin to the complexed form in gingival crevicular fluid from adult periodontitis patients

The broad spectrum protease inhibitor, α₂-macgrolobulin (α2M), is one of the host's principal regulators of both endogenous and exogenous proteases and is likely to have an important role in the regulation of proteolytic activity at inflammatory sites. We have determined the amount of complexed (comα₂M) and total α₂M (totα₂M) in gingival crevicular fluid (GCF) harvested from shallow and deep sites in adult periodontitis (AP) patients (n=21). An ELISA technique was developed to measure both forms of α₂M in the same sample utilizing a monoclonal antibody (MAb) specific for the complexed form. In addition, protease activity towards human serum albumin (Protl), transferrin (Prot2) and Nα-benzoyl-L-arginine 7-amido-4-methylcoumarin-hydrochloride (BAAMc; Prot3) were determined in a second GCF sample from the same site. Plasma α₂M concentrations were only positively correlated (p=0.0163) with GCF totα₂M from highly inflamed sites. We observed a significant positive correlation between totα₂M and proteolytic activity in GCF from deep sites but not from shallow sites (Protl: p=0.002; Prot2: p=0.005). A similar correlation between totα₂M and proteolytic activity was found at highly inflamed sites (Protl: p=0.014; Prot2: p=0.002). A very high proportion of the totα₂M in GCF was in the complexed form at both shallow (71.14%±29.13) and deep sites (68.17%±28.5) Comα₂M was positively correlated with proteolytic activity only in deep sites (Protl: p=0.015; Prot2: p=0.031). Our results suggest that the concentration of totα₂M in the gingival crevice is positively associated with the amount of proteolytic activity at the site and that protease activities in GCF may only partly explain the high percentage conversion α₂M to the complexed form. The high level of α₂M inactivation in GCF from AP patients reported here may have significance not only in view of its role as a broad spectrum protease inhibitor but also through the differential effects of native vs complexed α₂M on the regulation of immune responses.     

Characterization of bone resorbing activity in gingival crevicular fluid from patients with periodontitis

BACKGROUND: In attempts to elucidate factors stimulating bone resorption in patients with different inflammatory diseases in the vicinity of the skeleton, e.g., peridontal disease and rheumatoid arthritis, we are investigating the presence of bone-resorbing activity in a variety of inflammatory exudates. The aim of the present study was to characterize the bone-resorbing activity present in patients with periodontitis. METHODS: Bone-resorbing activity was assessed in gingival crevicular fluids (GCFs) collected from patients with periodontitis and from patients with no signs of gingivitis. Bone-resorbing activity was evaluated by analyzing the capacity of GCFs to stimulate the release of minerals and the breakdown of bone matrix proteins in cultured neonatal mouse calvariae. The concentrations of IL-1alpha, IL-1beta and PGE2 were determined with ELISA and RIA techniques, respectively. RESULTS: GCF eluates from 24 different healthy sites caused a 1.23+/-0.05 fold stimulation of 45Ca release, whereas GCF eluates from 45 different diseased (periodontitis) sites caused a 2.46+/-0.10 fold stimulation. The effect on 45Ca release was time- and concentration-dependent, inhibited by 3 different osteoclast inhibitors and associated with enhanced release of 3H from [3H]-proline-labelled bones. The activity in GCF causing enhanced 45Ca release was unaffected, or in some samples partially reduced, by ultrafiltration using a filter with a molecular weight cut-off of 3000 Daltons. The bone-resorbing activity was temperature sensitive (+90degrees C, 10 min). The concentrations of prostaglandin E2 (PGE2) in the diluted GCF eluates, used in the bone resorption bioassay, were too low to be responsible for the release of 45Ca. Antisera specifically neutralizing human IL-1a inhibited the stimulatory effect of GCF pooled from several diseased sites. The specific, recombinant human IL-1 receptor antagonist completely inhibited the effect of pooled GCFs. GCF eluates from diseased sites contained human IL-1alpha and IL-1beta at concentrations of 1838+/-294 pg/ml and 512+/-91 pg/ml, respectively. CONCLUSIONS: These data show that GCF contains activity(ies) stimulating osteoclastic bone resorption in vitro. The factor primarily responsible for this activity seems to be IL-1alpha, but IL-1alpha is not the sole activator of bone resorption in GCF.     

慢性牙周炎患者龈沟液中白细胞介素-4的检测和意义

目的检测慢性牙周炎患者牙周基础治疗前后龈沟液中白细胞介素-4(IL-4)的质量浓度,探讨IL-4与牙周炎的关系及其在牙周炎发病机制、病情进展等方面所起的作用。方法用滤纸条浸润法采集成年健康者和牙周炎患者治疗前后的龈沟液样本,用酶联免疫吸附测定检测样本中IL-4的质量浓度。结果慢性牙周炎患者龈沟液中IL-4的质量浓度低于健康对照组(P<0.05)。经牙周基础治疗1个月后,IL-4的质量浓度无明显变化,治疗前后的差异无统计意义(P>0.05);IL-4的质量浓度与探诊深度呈显著负相关,与牙龈指数和附着丧失无明显相关性。结论IL-4缺乏可能会导致牙周病的发生,IL-4可作为早期诊断牙周病和检测易患人群的敏感性指标。