cathepsin-g and Periodontitis

The objective of this research was to determine the effectiveness of a biochemical assay which measures proteolytic enzyme activity in gingival crevicular fluid (GCF) and to relate this enzyme activity to clinical parameters traditionally utilized for periodontitis detection. A clinical trial was conducted on 8 periodontitis subjects with > or =4 sites exhibiting a loss of attachment of > or =5 mm and probing depths of > or =5 mm with bleeding on probing. On each subject, a plaque index was performed, followed by GCF sampling at those sites which exhibited a loss of attachment and probing depths. GCF was analyzed for activity against benzoyl-L-arginine-p-nitroanilide in the presence (BAPNA w/gly-gly) and the absence (BAPNA w/o gly-gly) of glycyl-glycine and against MeOSuc-Ala-Ala-Pro-Val-pNA and Suc-Ala-Ala-Pro-Phe-pNA for neutrophil serine proteinases activity (elastase and cathepsin G, respectively). Subsequently, a gingival index was performed, attachment levels and probing depths were recorded using a constant force probe with bleeding on probing being noted. A split-mouth design was employed and half mouths were randomly assigned to the following treatment groups: group A, half of the mouth received scaling/root planing and polishing: group B, half of the mouth received no treatment (control). Subjects were treated, then instructed on toothbrushing and interdental cleaning. After 4 weeks, subjects returned to receive a plaque index; GCF sampling, gingival index, attachment levels, probing depths and bleeding on probing as described above. Using a paired Student t-test, the findings suggest that BAPNA w/gly-gly was significantly less in treatment sites than in non-treated control sites (p=0.05). No such correlation was found for other activities, including neutrophil serine proteinases which were shown to occur in GCF in free, proteolytically active forms. In addition, significant treatment effects were detected for probing depths (p= 0.03) which reduced by 1.3 mm and attachment levels (p=0.02) which gained 0.7 mm. The reduction of P. gingivalis from treated periodontitis sites as detected by a significant decrease in BAPNA w/ gly-gly may prove to be a valuable marker for periodontal disease activity.

Topics: Benzoylarginine Nitroanilide; Cathepsin G; Cathepsins; Chromogenic Compounds; Dental Plaque Index; Dental Prophylaxis; Dental Scaling; Double-Blind Method; Endopeptidases; Follow-Up Studies; Gingival Crevicular Fluid; Gingival Hemorrhage; Glycylglycine; Humans; Neutrophils; Oral Hygiene; Pancreatic Elastase; Periodontal Attachment Loss; Periodontal Index; Periodontal Pocket; Periodontitis; Prospective Studies; Root Planing; Serine Endopeptidases; Toothbrushing

Periodontitis is a chronic disease which affects at least 10% of the population. If untreated, periodontitis can lead to teeth loss. Unfortunately, current diagnostic tests are limited in their sensitivity and specificity. In this study, a novel multiplex hand-held colorimetric diagnostic biosensor, using two typical inflammatory salivary biomarkers, Human Neutrophil Elastase (HNE) and Cathepsin-G, was constructed as proof of concept to potentially detect periodontitis. The biosensing method was based on the measurement of proteolytic activity using specific proteases probes. These probes consist of specific proteases substrates covalently bound to a magnetic bead from one end and to the gold sensor surface by the other end. When intact, this renders the golden sensor black. Upon proteolysis, the cleaved magnetic beads will be attracted by an external magnet revealing the golden color of the sensor surface observable by the naked eye. The biosensor was capable of specific and quantitative detection of HNE and Cathepsin-G in solution and in spiked saliva samples with a lower detection limit of 1 pg/mL and 100 fg/mL for HNE and Cathepsin-G, respectively. Examination of periodontitis patients' sample and a healthy control showed the potential of the multiplex biosensor to detect the presence of HNE and Cathepsin-G activity in situ. This approach is anticipated to be a useful biochip array amenable to low-cost point-of-care devices.

Topics: Biomarkers; Biosensing Techniques; Cathepsin G; Colorimetry; Humans; Leukocyte Elastase; Magnetite Nanoparticles; Periodontitis

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

Topics: Adult; Aged; Cathepsin G; Chronic Periodontitis; Female; Follow-Up Studies; Gingival Hemorrhage; Gingivitis; Humans; Leukocyte Elastase; Male; Middle Aged; Myocardial Infarction; Periodontal Pocket; Periodontitis; Saliva; Salivary Proteins and Peptides; Tooth Loss

The presence of lysozyme in human gingiva has not previously been demonstrated. In this study, we looked for evidence for the potential role of lysozyme as a protector of gingival elastic fibres. The objective of this study was also to determine the ex vivo susceptibility to hydrolysis of gingival elastic fibres from patients with or without periodontal disease by human leukocyte elastase and by human cathepsin G.. Using gingival tissue sections from eight control, 10 gingivitis and 10 periodontitis patients, we evaluated the area fraction occupied by gingival elastic fibres (after selective staining) by the use of automated image analysis. In the ex vivo experiments, serial tissue sections from four control, four gingivitis, four young periodontitis and four aged periodontitis patients were submitted to the action of human leukocyte elastase and cathepsin G, after which enzymatic activities were determined by image analysis. Indirect immunodetection of lysozyme was also done on tissue sections for all patients included in this study.. Large variations of the area fraction occupied by elastic fibres were observed in human gingiva from young and aged patients with and without periodontal disease. In control and gingivitis patients, leukocyte elastase and cathepsin G had high comparable elastin solubilizing activities. With young and aged periodontitis patients, the two serine proteinases had weak elastin solubilizing activities. Lysozyme appeared to be present at the periphery of gingival elastic fibres in periodontitis patients.. Lysozyme can be considered an important natural protector of elastic fibres in pathological gingiva.

Topics: Adolescent; Adult; Age Factors; Aged; Cathepsin G; Cathepsins; Contractile Proteins; Elastic Tissue; Elastin; Enzyme Inhibitors; Extracellular Matrix Proteins; Female; Fluorescent Antibody Technique, Indirect; Gingiva; Gingival Hemorrhage; Gingivitis; Humans; Hydrolysis; Image Processing, Computer-Assisted; Leukocyte Elastase; Male; Middle Aged; Muramidase; Periodontal Attachment Loss; Periodontal Pocket; Periodontitis; Serine Endopeptidases; Young Adult

Thrombomodulin, a cell transmembrane glycoprotein, binds to thrombin and converts it from a procoagulant protease to an anticoagulant enzyme that activates protein C. Thrombomodulin is very important in regulating the function of thrombin. Elevated soluble thrombomodulin is present in the gingival crevicular fluid of subjects with periodontitis. The objective of the present study was to investigate the mechanisms about the elevated soluble thrombomodulin in gingival crevicular fluid.. Gingival sections from six patients with chronic periodontitis and from three periodontally healthy subjects were immunostained for thrombomodulin detection. Thrombomodulin levels were investigated in the gingival crevicular fluid of 11 subjects with chronic periodontitis. The effects of neutrophil enzymes on thrombomodulin release and on thrombomodulin in the gingival crevicular fluid were examined by an enzyme-linked immunosorbent assay or by Western blotting.. The expression of gingival epithelial thrombomodulin was lost or decrease near infiltrating neutrophils. Thrombomodulin was rapidly released from gingival epithelial cells by neutrophil enzymes, and gingival crevicular fluid with periodontitis included the proteolytic cleavage thrombomodulin using immunoblotting analysis. The thrombomodulin release was not caused by rapid cell damage, on lactate dehydrogenase assay. There were significant differences in thrombomodulin content between gingival crevicular fluid samples from healthy and diseased sites, regardless of the degree of probing depth.. Neutrophil enzymes induced rapid thrombomodulin release from the membrane surface of gingival epithelial cells. This might explain the thrombomodulin increase in gingival crevicular fluid with local diseased gingiva. Elevation of thrombomodulin in gingival crevicular fluid may be a potential marker of epithelial cell membrane injury.

Topics: alpha 1-Antitrypsin; Alveolar Bone Loss; Blotting, Western; Cathepsin G; Cathepsins; Chronic Disease; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Gingiva; Gingival Crevicular Fluid; Gingival Hemorrhage; Humans; Immunohistochemistry; L-Lactate Dehydrogenase; Leukocyte Elastase; Male; Middle Aged; Neutrophils; Periodontal Pocket; Periodontitis; Serine Endopeptidases; Serine Proteinase Inhibitors; Thrombomodulin

This investigation was designed to determine and compare the distribution pattern of anti-neutrophil cytoplasmic antibodies (ANCA) in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in the presence or absence of periodontal disease.. Sera of 30 patients with SLE and 30 with RA were tested for ANCA utilizing an indirect enzyme immunosorbent assay (ELISA) directed to a neutrophil granular extract and 6 neutrophil granule proteins. A control group of 20 healthy individuals showing neither evidence of periodontal disease nor systemic compromise was also included in this study.. For RA, the number of ANCA-positive sera was very low but was evenly distributed among patients with and without periodontitis. Conversely, a high number of ANCA-positive sera in SLE was found mostly in individuals presenting periodontal compromise. A statistically significant association between ANCA and periodontitis in SLE patients was found (P <0.005, chi square test).. A marked difference in the number and distribution of ANCA with respect to periodontitis between RA and SLE was found. Hyperresponsiveness of B cells and polyclonal B activation to periodontopathic bacteria in SLE might be accountable for the high numbers of ANCA and the close association observed between those autoantibodies and periodontitis in SLE.

Topics: Adult; Antibodies, Antineutrophil Cytoplasmic; Arthritis, Rheumatoid; Autoantigens; B-Lymphocytes; Bacteria; Cathepsin G; Cathepsins; Chi-Square Distribution; Enzyme-Linked Immunosorbent Assay; Female; Humans; Lactoferrin; Leukocyte Elastase; Lupus Erythematosus, Systemic; Lymphocyte Activation; Male; Middle Aged; Muramidase; Myeloblastin; Neutrophils; Periodontitis; Peroxidase; Serine Endopeptidases

Cathepsin G and medullasin are 2 major serine proteinases associated with the granular fraction of polymorphonuclear leukocytes (PMNs). To know their possible involvement in the pathophysiological gingival connective tissue turnover, we have determined the distribution and localization of these 2 enzymes in inflamed gingival tissues from periodontal patients by immunohistochemistry with discriminating antibodies specific for each enzyme. The gingival connective tissues were obtained from periodontitis patients with various inflammatory conditions and control healthy subjects without any clinical signs of periodontal inflammation. In all gingival specimens examined, cathepsin G and medullasin were found mainly in neutrophil-like cells and partly in macrophage-like cells. No positive staining for both enzymes was obtained in endothelial cells and fibroblasts in every part of the gingival tissues. Immunoreactivity for each enzyme in the gingival tissues from the periodontitis group was stronger and greater in the intensity and frequency than that from the control group and appeared to be increased with the severity of the disease In both groups, the number of immunoreactive cells for each enzyme was greater in the vicinity of pocket epithelium (zone I) than in the area of central connective tissue (zone II) or the area subjacent to the oral epithelium (zone III). While both enzymes in zones II and III were exclusively found in coarse granules, their stainings in zone I were not only coarse but also diffuse. These results strongly suggest that both enzymes may have some association with inflamed gingival tissue degradation.

Topics: Adult; Antibodies; Cathepsin G; Cathepsins; Connective Tissue; Cytoplasmic Granules; Endothelium, Vascular; Epithelial Attachment; Epithelium; Female; Fibroblasts; Gingiva; Gingivitis; Humans; Immunoenzyme Techniques; Immunohistochemistry; Macrophages; Male; Middle Aged; Neutrophils; Periodontal Pocket; Periodontitis; Serine Endopeptidases

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 avidinbiotin-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 alpha 1-antichymotrypsin (alpha 1-ACT). The present results demonstrate that part of the cathepsin G, despite the presence of increased concentrations of alpha 1-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]).

Topics: Adult; Aged; alpha 1-Antichymotrypsin; Bacterial Physiological Phenomena; Blotting, Western; Cathepsin G; Cathepsins; Collagenases; Dental Plaque; Enzyme Activation; Enzyme Precursors; Epithelium; Gingiva; Gingival Crevicular Fluid; Gingivitis; Humans; Immunoblotting; Immunoenzyme Techniques; Macrophages; Middle Aged; Monocytes; Neutrophils; Periodontitis; Periodontium; Serine Endopeptidases; Serine Proteinase Inhibitors; Spectrophotometry

Topics: Adult; Antibodies, Antineutrophil Cytoplasmic; Antibody Specificity; Antibody-Dependent Cell Cytotoxicity; Cathepsin G; Cathepsins; Cytoplasmic Granules; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique, Indirect; Humans; Lactoferrin; Leukocyte Elastase; Male; Middle Aged; Neutrophil Activation; Neutrophils; Periodontitis; Peroxidase; Serine Endopeptidases

Dental plaque is the major aetiological factor in periodontal diseases and contains several proteolytic enzymes. The origin of these proteinases is, however, poorly studied. This study was undertaken to characterize collagenase present in dental plaque of adult periodontitis patients. Vertebrate-type rather than bacterial-derived collagenase activity was detected in extracts of both supra- and subgingival dental plaque extracts of adult periodontitis patients. Dental plaque collagenase was found to exist predominantly in autoactive form. Dental plaque collagenase from periodontally healthy individuals existed in latent from. Latent dental plaque collagenase from periodontitis lesions could be activated by a 95 kD chymotrypsin-like proteinase from Treponema denticola and human leukocyte cathepsin G but not by human plasmin. Incubation of purified latent leukocyte collagenase with whole cells of Fusobacterium nucleatum, Eubacterium saburreum, Prevotella buccae and Porphyromonas gingivalis, however, did not result to the activation of the enzyme. Doxycycline in vitro inhibited dental plaque collagenase with an IC50-value of 20 microM. Dental plaque collagenase degraded more efficiently type I and II collagens than type III collagen. Western-blot analysis with specific anti-human neutrophil collagenase-antibody revealed that both in supra- and subgingival dental plaque extracts dental plaque collagenase had undergone proteolytic conversion from an 80 kD proform to a 58 kD active form which is associated with catalytic autoactivity as measured by functional collagenase assay. This reflects proteolytic activation of leukocyte collagenase in dental plaque probably by other proteases derived from potent periodontopathogenic bacteria such as T. denticola or other PMN proteases such as cathepsin G.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adult; Blotting, Western; Catalysis; Cathepsin G; Cathepsins; Collagen; Collagenases; Dental Plaque; Doxycycline; Endopeptidases; Enzyme Activation; Enzyme Precursors; Eubacterium; Fibrinolysin; Fusobacterium nucleatum; Gelatinases; Humans; Leukocytes; Matrix Metalloproteinase Inhibitors; Neutrophils; Periodontitis; Periodontium; Porphyromonas gingivalis; Prevotella; Serine Endopeptidases; Treponema

The levels of cathepsin G in gingival crevicular fluid (GCF) from chronic adult periodontitis patients and experimental gingivitis subjects were determined both by activity measurement using N-benzoyl-(DL)-phenylalanine-2-naphthyl ester as a substrate and by enzyme immunoassay using anti-human cathepsin G IgG. The activity level of cathepsin G in GCF of both periodontitis and experimental gingivitis has no significant correlation with all measured clinical parameters. Western immunoblotting using antibodies specific for cathepsin G or alpha 1-proteinase inhibitor revealed that the difficulty in demonstrating the association of its activity with the severity of these diseases was due largely to formation of the enzyme-inhibitor complexes. By contrast, statistically significant positive correlation was found between cathepsin G content in GCF of periodontitis, which was determined by enzyme immunoassay, and such clinical parameters as the GCF volume, the gingival index and probing depth. The increased cathepsin G content with increasing severity of periodontal inflammation was markedly diminished by the initial treatment. Although no significant activity was detectable in GCF of experimental gingivitis, a rapid increase of the immunoreactive cathepsin G was found in GCF at 3-5 d after refraining from oral hygiene measures, which rapidly decreased by 10 d. The progressively increased cathepsin G between 10th and 21st d rapidly decreased by cleaning of the teeth. The results indicate that cathepsin G is involved in the host's defensive mechanism against the invasion of etiologic microbes and/or the development of either periodontitis or gingivitis.

Topics: Adult; Affinity Labels; Aged; alpha 1-Antitrypsin; Blotting, Western; Cathepsin G; Cathepsins; Chronic Disease; Electrophoresis, Polyacrylamide Gel; Female; Gingival Crevicular Fluid; Gingivitis; Humans; Immunoglobulin G; Male; Middle Aged; Oral Hygiene; Periodontal Index; Periodontal Pocket; Periodontitis; Phenylalanine; Serine Endopeptidases

Five host-response indicators were measured by enzyme-linked immunosorbent assays on unstimulated whole saliva samples from 45 adults (19 male, 26 female). The participants were distributed among four dentate groups representing oral health (I), gingivitis (II), moderate periodontitis (III), and severe periodontitis (IV), and one group of edentulous volunteers (V). Levels of the host-response indicators varied widely, from zero, primarily with groups I and V, to relatively high values with groups II, III and IV. The levels ranged as follows: alpha 2-macroglobulin, 0-4941 ng/ml; alpha 1-antitrypsin, 2-2271 ng/ml; C-reactive protein, 0-472 pg/ml; cathepsin G, 0-6035 ng/ml; elastase, 0-164 ng/ml (free), 0-732 ng/ml (bound to alpha 1-antitrypsin), and 0-318 ng/ml (bound to alpha 2-macroglobulin). Statistical evaluation by planned contrasts showed that levels of host-response indicators for group I were significantly lower (except for alpha 1-antitrypsin) than for groups II, III, and IV. A trend analysis of groups I-IV showed that mean scores (again, except for alpha 1-antitrypsin) increased significantly in a positive, monotonic manner. Group V showed significantly lower values for elastase than in the other groups. The findings demonstrate that these factors can be detected in whole saliva and suggest that, except for alpha 1-antitrypsin, their levels are directly related to an individual's periodontal status.

Topics: Adult; Aged; Aged, 80 and over; alpha 1-Antitrypsin; alpha-Macroglobulins; C-Reactive Protein; Cathepsin G; Cathepsins; Enzyme-Linked Immunosorbent Assay; Female; Gingivitis; Humans; Male; Middle Aged; Mouth, Edentulous; Pancreatic Elastase; Periodontal Diseases; Periodontal Index; Periodontitis; Saliva; Salivary Proteins and Peptides; Serine Endopeptidases; Trypsin Inhibitors

The purpose of this study was to evaluate whether the reduced microbicidal activity of polymorphonuclear leukocytes (neutrophils) in patients with early-onset periodontitis is associated with a deficiency of nonoxidative microbicidal proteins. Neutrophils from 10 patients with early-onset periodontitis and 8 healthy control subjects were assessed for elastase isozymes 1 through 4, cathepsin G isozymes 1 through 4 and defensins (HNP-1, HNP-2 and HNP-3) using cationic and acid urea polyacrylamide gel electrophoresis. The results showed that both the total content and the relative distribution of elastase and cathepsin G isozymes was normal in neutrophils of patients with early-onset periodontitis. However, the HNP-3 content was significantly reduced in neutrophils from patients with generalized early-onset periodontitis. These findings indicate that the impaired microbicidal activities of neutrophils in patients with early-onset periodontitis does not appear to be based on an elastase or cathepsin G abnormality in neutrophils. Due to the high variability of HNP-1 + 2 and HNP-3 in neutrophils of control subjects, the reduced HNP-3 content in neutrophils probably plays a minor role in the pathogenesis of generalized early-onset periodontitis.

Topics: Adult; Aggressive Periodontitis; alpha-Defensins; Analysis of Variance; Blood Bactericidal Activity; Blood Proteins; Cathepsin G; Cathepsins; Defensins; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Female; Humans; Isoenzymes; Male; Neutrophils; Pancreatic Elastase; Periodontitis; Serine Endopeptidases

Topics: Adult; Cathepsin G; Cathepsins; Electrophoresis, Polyacrylamide Gel; Gingivitis; Humans; Neutrophils; Periodontitis; Serine Endopeptidases

Human neutrophil cathepsin G has been identified as a potent proteolytic activator of latent human neutrophil collagenase in vitro. In order to examine the role of cathepsin G in the activation mechanism of latent human neutrophil collagenase in vivo, gingival crevicular fluid was collected from periodontal pockets of patients with adult periodontitis and the relationship of cathepsin G to the proportion of endogenously active collagenase and total collagenase activity was determined. The changes in these parameters were monitored before and after periodontal therapy and compared to control values obtained for periodontal sites without clinical signs of inflammation or increased pocket depth. A significant decrease in cathepsin G and collagenase activity in gingival crevicular fluid collected from initially deep periodontal pockets was observed in response to scaling and root planing (P less than 0.025, Wilcoxon signed rank test). Also the proportion of endogenously active collagenase decreased (P less than 0.05). There was a significant correlation of cathepsin G and total collagenase activity. However, no correlation of cathepsin G activity and endogenously active collagenase was observed. The results indicate the existence of several distinct activation pathways for latent human neutrophil collagenase in vivo and suggest that, apart from cathepsin G, other proteolytic activation cascades and/or non-proteolytic activation pathways participate in the activation of latent human neutrophil collagenase in vivo.

Topics: Cathepsin G; Cathepsins; Collagenases; Dental Scaling; Gingival Crevicular Fluid; Humans; Pancreatic Elastase; Periodontal Pocket; Periodontitis; Root Planing; Serine Endopeptidases