Secretory leukocyte protease inhibitor and its potential interactions with elastase and cathepsin B in gingival crevicular fluid and saliva from patients with chronic periodontitis.

BACKGROUND AND OBJECTIVE: Elastase is carried into the oral cavity by gingival crevicular fluid (GCF) from periodontal lesions. Our study investigated the regulation of elastase activity by secretory leukocyte protease inhibitor (SLPI) and the possible action of another GCF protease on this protective salivary component. MATERIAL AND METHODS: Whole-mouth saliva (WMS), parotid saliva (PS) and GCF were obtained from 19 patients with periodontitis. The concentrations of active elastase and cathepsin B were determined using peptide substrates. SLPI and alpha1-proteinase inhibitor (alpha1PI) concentrations were determined using enzyme-linked immunosorbent assays (ELISAs). The molecular forms of SLPI were examined by immunoblotting. RESULTS: The molar concentrations of elastase, cathepsin B and alpha1PI were higher in GCF than in WMS and especially PS (p < 0.0002). The GCF SLPI concentrations were also higher than the WMS SLPI concentrations (p < 0.05). All WMS components increased with GCF content, significantly for elastase and SLPI (p < 0.002). In GCF, the concentration of alpha1PI was higher than the concentration of SLPI (p < 0.0002), while there was no significant difference for WMS. SLPI and elastase levels in GCF and WMS were inversely related (p < 0.005). In SLPI immunoblots, PS contained only the intact 14-kDa molecule of SLPI, while WMS also contained an 8-kDa fragment. For WMS there was a positive correlation between SLPI degradation and cathepsin B (p < 0.002). Incubation of WMS alone or of PS with GCF in the presence of cysteine proteinase activators caused SLPI immunoreactivity to shift to 8 kDa. CONCLUSION: For GCF, serum-derived alpha1PI is the major elastase inhibitor, but in WMS SLPI probably reduces activity. The inflamed gingivae can be an additional source of SLPI in the oral cavity, but here the molecule is apparently cleaved by GCF cysteine proteinases, such as cathepsin B.

Collagen degradation by interleukin-1beta-stimulated gingival fibroblasts is accompanied by release and activation of multiple matrix metalloproteinases and cysteine proteinases.

OBJECTIVE: Several collagenolytic matrix metalloproteinases (MMPs) have recently been identified in gingival fibroblasts, while secreted cysteine proteinases could also participate in connective tissue destruction in periodontitis. To clarify their involvement, we examined enzyme release during collagen breakdown by cultured cytokine-stimulated fibroblasts. MATERIALS AND METHODS: Gingival fibroblasts were derived from four chronic periodontitis patients and cultured on collagen gels in serum-free medium for 1-4 days. Collagenolysis was measured by hydroxyproline release into the medium. Proteinases were assessed by electrophoresis and immunoblotting. RESULTS: Adding interleukin-1beta resulted in progressive gel breakdown. This was associated particularly with a shift in MMP-1 band position from proenzyme to active enzyme and the appearance of active as well as proenzyme forms of cathepsin B. There was also partial processing of pro-MMP-13 and increased immunoreactivity for active cathepsin L. In addition, both pro-forms and active forms of MMP-8, membrane-type-1-MMP and MMP-2 were present in control and treated cultures. CONCLUSIONS: Fibroblast MMP-1 was most likely responsible for collagen dissolution in the culture model, while cathepsin B may have been part of an activation pathway. All studied proteinases contribute to extracellular matrix destruction in inflamed gingival tissue, where they probably activate each other in proteolytic cascades.

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.

Matrix metalloproteinase-8 levels and elastase activities in gingival crevicular fluid from chronic adult periodontitis patients.

AIM: To make an initial assessment of the periodontal diagnostic potential of immunoreactive matrix metalloproteinase-8 (MMP-8) in gingival crevicular fluid (GCF) by comparison with elastase activity which has previously been associated with disease severity and progression. METHODS: GCF was collected from molar and premolar sites of 16 chronic adult periodontitis patients before treatment and 13 of this group 2 weeks after scaling and root planing. Samples were analysed for MMP-8 by immunofluorometric assay and for elastase activity with a fluorogenic substrate. RESULTS: Mean patient clinical parameters and GCF enzyme totals both decreased significantly after treatment. Total MMP-8 levels and elastase activities generally correlated significantly with gingival and bleeding indices. For GCF concentrations, only MMP-8 showed a significant fall after treatment, and some significant correlations with clinical parameters. Amounts of the 2 enzymes correlated significantly with each other. CONCLUSIONS: Similarities between MMP-8 and elastase probably reflect the fact that both enzymes are associated mainly with neutrophils: MMP-8 levels may have fallen more after treatment because the assay, unlike that for elastase, would most likely not have detected much enzyme bound to alpha-macroglobulin. The immunoassay for MMP-8 is more specific and convenient than functional collagenase assays, and might be suitable for monitoring the periodontal condition.

Antibacterial agents in the control of supragingival plaque--a review.

This review considers the main agents which have been used as antibacterial agents in mouthwashes and other vehicles to inhibit the growth of supragingival plaque. The agents discussed are bisguanide antiseptics, quaternary ammonium compounds, phenolic antiseptics, hexetidine, povidone iodine, triclosan, delmopinol, salifluor, metal ions, sanguinarine, propolis and oxygenating agents. The plaque inhibitory, anti-plaque and anti-gingivitis properties of these agents are considered along with their substantivity, safety and possible clinical usefulness. Clinical trials of these agents that have been published are also reported. The possible clinical uses of antiseptic mouthwashes are finally considered along with some advice about assessing manufacturers claims. Throughout this review the terms plaque inhibitory, anti-plaque and anti-gingivitis have been used according to the clarification of terminology suggested by the European Federation of Periodontology at its second workshop. This defines a plaque inhibitory effect as one reducing plaque to levels insufficient to prevent the development of gingivitis; an anti-plaque effect as one which produces a prolonged and profound reduction in plaque sufficient to prevent the development of gingivitis; and anti-gingivitis as an anti-inflammatory effect on the gingival health not necessarily mediated through an effect on plaque.

Advances in periodontal diagnosis. 9. Potential markers of cell death and tissue degradation.

This paper describes the potential markers of cell death and connective tissue degradation which might serve as markers of periodontal disease activity. The first section deals with enzymes released by dead and degenerating cells. Firstly, it describes how these pass from the periodontal tissues into gingival crevicular fluid (GCF) and explains that these enzymes have been used as markers of cell death in medicine for several decades. It then discusses the main enzymes in this group, aspartate amino transferase (AST) and lactate dehydrogenase (LDH) and reviews those studies which have attempted to relate these enzymes to periodontal disease severity and activity. Secondly, it describes the potential markers of connective tissue degradation, fibronectin, hydroxyproline-containing peptides and glycosaminoglycans (GAGs) and explains how these are produced. Finally, it describes the only commercial test kit for markers in this group (GCF-AST).

Advances in periodontal diagnosis. 10. Potential markers of bone resorption.

This paper describes the markers of bone resorption which might serve as potential markers for periodontal disease activity. It firstly describes the bone specific proteins which are involved in bone mineralisation and the role they play in this process. It then explains how they may pass into GCF and reviews those studies which have attempted to relate these factors to periodontal disease severity and activity. It next discusses the difficulties in isolating and detecting these factors in GCF and their possible use as markers for periodontal disease activity. As the final part in the series it lastly discusses the possible uses of predictive diagnostic tests of periodontal disease activity in dental practice.

Advances in periodontal diagnosis. 7. Proteolytic and hydrolytic enzymes link with periodontitis.

Biomarkers of periodontal disease activity may be obtained from potential proteolytic and hydrolytic enzymes of inflammatory cell origin. Studies that have sought to correlate these enzymes with periodontal disease activity are reviewed with special consideration given to collagenases, cysteine, aspartate and serine proteinases, beta-glucuronidase, arylsulphate, alkaline and acid phosphatases, myeloperoxidase, lysozyme and lactoferrin.

Advances in periodontal diagnosis. 5. Potential inflammatory and immune markers.

The potential inflammatory and immune markers that might detect periodontal disease severity or activity are examined. The role of inflammatory and immune factors passing from the tissues into the gingival crevicular fluid (GCF) are considered. GCF sampling is a necessary part of all diagnostic tests based on gingival and periodontal tissue factors. Inflammatory and immune factors found in GCF are reviewed with special reference to the humoral immune response, complement, cytokines and prostaglandins. The possible development of diagnostic tests based on these factors are discussed.

Advances in periodontal diagnosis. 6. Proteolytic and hydrolytic enzymes of inflammatory cell origin.

Potential proteolytic and hydrolytic enzymes of inflammatory cell origin might serve as biomarkers of periodontal disease activity. The role of these enzymes in periodontal pathology, particularly in respect of collagen and proteoglycan degradation, is discussed. The cellular location of these enzymes and their normal control mechanisms by endogenous inhibitors is described.

Advances in periodontal diagnosis. 8. Commercial diagnostic kits based on GCF proteolytic and hydrolytic enzyme levels.

Biomarkers of periodontal disease activity may be obtained from potential proteolytic and hydrolytic enzymes of inflammatory cell origin. Commercial diagnostic tests and those under development are discussed along with their advantages and disadvantages.

Advances in periodontal diagnosis. 4. Potential microbiological markers.

The potential of bacterial markers for the detection of periodontal disease activity is discussed. Chronic periodontitis has been associated with subgingival bacteria. Techniques are available to detect and distinguish different bacteria. In subgingival plaque, bacterial proteases can be detected. Commercial diagnostic tests based on bacterial factors are described.

Advances in periodontal diagnosis. 3. Assessing potential biomarkers of periodontal disease activity.

The methods for assessing potential biomarkers of periodontal disease activity are considered. This is necessary because a detailed examination of all the relevant research evidence is an essential process in assessing the possible clinical usefulness of a periodontal diagnostic test system based on any one of these markers.

Advances in periodontal diagnosis. 2. New clinical methods of diagnosis.

This paper describes the new methods of periodontal diagnosis. An outline of the problems of periodontal probing techniques is followed by a discussion of the advances made by the use of constant pressure electronic probes. We finally highlight the problems of periodontal radiographical techniques, leading on to a description of computer-aided radiographic methods--in particular, digital subtraction radiology and computer-assisted linear radiology.

Advances in periodontal diagnosis. 1. Traditional clinical methods of diagnosis.

This series will consider the limitations of traditional periodontal diagnostic techniques and the recent advances which have sought to overcome them. It will cover improvements in probing and radiographic techniques and also discusses the attempts to find bacterial or tissue-derived markers which will diagnose or predict periodontal disease activity. The first paper describes the traditional methods of periodontal diagnosis.

Cathepsin B, alpha2-macroglobulin and cystatin levels in gingival crevicular fluid from chronic periodontitis patients.

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, alpha2-macroglobulin and cystatin. Cathepsin B activity was assayed with a fluorogenic peptide substrate, alpha2-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 alpha2-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: alpha2-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.

Characterization of protease activities in Capnocytophaga spp., Porphyromonas gingivalis, Prevotella spp., Treponema denticola and Actinobacillus actinomycetemcomitans.

Protease activities in cell sonicates of defined bacterial strains were examined using peptide substrates and class-specific inhibitors. Capnocytophaga spp. all produced serine dipeptidyl peptidase activity and arginine/lysine, elastase- and chymotrypsin-like enzymes with some metalloprotease characteristics. The elastase-like activity was strongest in Capnocytophaga sputigena, but the others were greatest in Capnocytophaga gingivalis. The latter also had a separate arginine-specific enzyme which appeared not to be present in the other two species. Porphyromonas gingivalis showed serine dipeptidyl peptidase activity and very strong arginine and lysine cysteine protease activities. Prevotella spp. had inhibitor-resistant dipeptidyl peptidase activity and arginine cysteine protease activity that was much weaker but biochemically similar to P. gingivalis. Treponema denticola possessed a strong trypsin-like serine protease activity as well as very weak dipeptidyl peptidase and chymotrypsin-like activities that were sensitive to some cysteine protease reagents. Actinobacillus actinomycetemcomitans showed a novel alanine- and lysine-specific activity, but its nature was unclear.

The future of dental amalgam: a review of the literature. Part 7: Possible alternative materials to amalgam for the restoration of posterior teeth.

This is the last in a series of articles on the future of dental amalgam. It considers possible alternative materials to amalgam for the restoration of posterior teeth. The materials discussed are gold inlays, gold foil, gallium alloys, and tooth coloured non-metal alternatives including glass-ionomer cements, composite resins, glass-ionomer-resin hybrids, compomers and ceramics. The clinical indications for these restorations are first described along with their potential clinical problems and their mean survival rates in comparison with dental amalgam. Secondly, the safety of composite resins is considered and potential toxic and hypersensitive effects of these materials are discussed. Finally, it is concluded that the present evidence does not appear to demonstrate that dental amalgam is hazardous to the health of the general population. It does, however, recommend that in continuing to use amalgam dentists must use strict mercury hygiene procedures to avoid risk to their staff and contamination of the environment. It seems that mercury contamination of the environment is likely to be the main reason for any future government action against the continued clinical use of dental amalgam.

Investigations into the cellular contribution to host tissue proteases and inhibitors in gingival crevicular fluid.

Gingival crevicular fluid (GCF) was collected from chronic periodontitis patients using plastic micropipettes and coverslip smears stained with antibodies for leukocyte markers and Toluidine Blue for mast cells. The smears consisted of 70-80% granulocytes, 10-20% monocytes/macrophages, 5% mast cells and 5% T lymphocytes; no B lymphocytes were found. Proteases and inhibitors in GCF cells were investigated by enzyme cytochemistry using 2-methoxy-4-naphthylamine-linked peptide substrates and simultaneous coupling to Fast Blue B and immunocytochemistry using biotinylated secondary antibodies and an alkaline phosphatase/new fuchsin detecting system. Elastase was detected in granulocytes, cathepsin B in macrophages, dipeptidyl peptidases II and IV in a small proportion of macrophages, dipeptidyl peptidase IV in a few T lymphocytes, tryptase in mast cells and alpha-1-proteinase inhibitor and alpha-2-macroglobulin in some macrophages. GCF was also collected on filter paper strips and eluted into buffer for biochemical enzyme assays. Lysis of cells by addition of detergent to the elution buffer increased activities to 140-240% of control values. Removal of cells by centrifugation reduced measured activities to 1-30% of original figures; this effect was less if samples were pre-treated with detergent. Proteases from inflammatory cells therefore appear to make up most of the measured enzyme activity in GCF, and this association may explain recent correlations with periodontal disease progression.

The future of dental amalgam: a review of the literature. Part 6: Possible harmful effects of mercury from dental amalgam.

This is the sixth article in a series of seven on the future of dental amalgam. It considers the possible toxic and allergic effects which could occur as a result of exposure to mercury from dental amalgam. The main toxic effects covered are neurotoxicity, kidney dysfunction, reduced immunocompetence, effects on the oral and intestinal bacterial flora, fetal and birth effects and effects on general health. The relevant studies in all these areas are described and extensively discussed. In addition, the possible development of hypersensitivity to mercury from amalgam is described and the production of delayed hypersensitivity contact reactions on the skin and mucous membrane, including lichenoid lesions, are considered.