OBJECTIVEThis study aimed to investigate the difference in salivary protease expression in patients with chronic periodontitis and normal individuals.

METHODSThe stimulating saliva in patients with chronic periodontitis and normal individuals were collected. Protein chip technology was adapted to analyze salivary protease spectrum.

RESULTSAmong the 34 proteases in the chip, disintegrin and metalloproteinase (ADAM)8, matrix metalloproteinase (MMP)-8, MMP-12, neprilysin/CD10, and uridylyl phosphate adenosine/urokinase showed a significantly increased concentration in the saliva of chronic periodontitis patients compared with those in the saliva of normal individuals (P<0.01). By contrast, the concentrations of ADAM9, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, ADAMTS13, cathepsin B, E, L, V, X/Z/P, kallikrein 6, 7, 11, 13, MMP-9, proteinase 3, presenilin-1, and proprotein convertase 9 sharply decreased (P<0.05).

CONCLUSIONSThe results demonstrated that protease spectrum in the saliva of chronic periodontitis patients and normal individuals significantly differed. Analysis of salivary protease spectrum is a potential clinical method to examine, diagnose, and monitor chronic periodontitis.

Chronic Periodontitis ; Humans ; Matrix Metalloproteinase 8 ; Matrix Metalloproteinase 9 ; Saliva

OBJECTIVETo evaluate the release of matrix metalloproteinase-8 (MMP-8) and apoptosis rate of polymorphonuclear leukocytes (PMNs) after PMNs was triggered by Enterococcus faecalis (E. faecalis) in vitro.

METHODSThe activated E. faecalis suspension was prepared and added to PMNs suspension as experiment group. As a positive control, phorbol myristate acetate (PMA) was used. As negative control, PMNs suspension was incubated with PBS. The release of MMP-8 was measured at 0, 20, 60, 120 min by ELISA method. E. faecalis lysate acted on PMNs as experiment group, PMNs suspension was incubated with PBS as negative control, samples in two groups were incubated at 37 degrees C for 2, 5, 10, 15 h. The apoptosis rate of PMNs was tested by Flow Cytometry.

RESULTSAt 0 min, there was no significant difference of MMP-8 release in the experiment group and positive control (P>0.01); whereas at 60, 120 min, E. faecalis induced a significant lower MMP-8 release compared with the positive control (P<0.01). The apoptosis rate of PMNs in both groups increased along with time, and apoptotic rate in experiment group was higher than that in the control group at 2, 5, 10, 15 h (P<0.01).

CONCLUSIONAfter E. faecalis act on PMNs, no significant release of MMP-8 from PMNs was observed. E. faecalis don't induce PMNs apoptosis delay.

Apoptosis ; Enterococcus faecalis ; Humans ; Matrix Metalloproteinase 8 ; Neutrophils ; Tetradecanoylphorbol Acetate

BACKGROUND: Bronchial asthma is a chronic inflammatory disease of the airways characterized by inflammatory cell infiltrations, which require extracellular matrix (ECM) breakdown and inflammatory cell migration. Airway remodeling with ECM deposition is another characteristic of asthma and reflect imbalance of collagen homeostasis. Collagen homeostasis is regulated by balance of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). OBJECTIVE: We performed this study to evaluate the clinical significance and the role of MMPs and TIMPs in induced sputum of patients with symptomatic asthma. METHODS: We measured the concentrations of MMP-9 and its tissue inhibitor, TIMP-1, in induced sputum of 16 symptomatic asthmatics, 6 active smokers, and 5 healthy control subjects. RESULTS: Concentrations of MMP-9 and TIMP-1 were greater in patients with asthma than in control subjects. The molar ratio between MMP-9 and TIMP-1 was significantly lower in asthmatics than in control subjects (p<0.05). In asthmatics, MMP-9 concentrations were correlated with the number of total inflammatory cells, neutrophils and eosinophils (Rho=0.618, Rho=0.545 and Rho=0.384, p<0.01, p<0.01 and p=0.058, respectively). The concentrations of MMP-9 was negatively correlated with FEV1 (Rho=-0.467, p<0.05) and positively correlated with the levels of sputum ECP and IL-8 (Rho=0.595 and Rho=0.769, p<0.01 and p<0.01, respectively). CONCLUSION: MMP-9 may be involved in active inflammatory processes in symptomatic chronic airway disease, and the lower ratio of MMP-9/TIMP-1 in asthmatics suggests that MMP-9/TIMP-1 imbalance may be involved in airway remodeling.
Airway Remodeling ; Asthma ; Cell Movement ; Collagen ; Eosinophils ; Extracellular Matrix ; Homeostasis ; Humans ; Interleukin-8 ; Matrix Metalloproteinase 1* ; Matrix Metalloproteinases ; Metalloproteases ; Molar ; Neutrophils ; Sputum* ; Tissue Inhibitor of Metalloproteinase-1

This in vitro study monitored MMP-8 production on PMN by stimulated with the following three groups; Sonicated extracts of E. faecalis (SEF), SEF treated with Ca(OH)2 (12.5mg/ml) for 7 days, and lipopolysaccharides (LPS) of E. coli. The level of MMP-8 in each group was immediately measured by ELISA. The data were analyzed with Kruskal-Wallis test and Mann-Whitney U test. In the SEF group, the level of production of MMP-8 was higher than the negative control group in low concentration (0.05microg/ml) of SEF (p < 0.05), but it decreased with an increase in the concentration of SEF (p < 0.05). In the case of SEF treated with Ca(OH)2, all of the MMP levels were higher than negative control group (p < 0.05), but no statistical difference was found among the different SEF concentrations (p > 0.05). All of the levels in E. coli LPS were increased with increasing concentrations (p < 0.05). According to this study we could summarize as follows: 1. MMP-8 was expressed at low level in untreated PMN group and the levels of MMP-8 were upregulated in PMN stimulated by E. coli LPS groups. 2. In the SEF groups, the level of production of MMP-8 decreased with an increase in the concentration of SEF (p < 0.05). So E. faecalis may have suppressive effect on the production of MMP-8 by PMN. 3. In the case of SEF treated with Ca(OH)2, all of the MMP levels at different SEF concentrations were higher than untreated PMN group (p < 0.05), but no statistical difference was found among the different SEF concentrations (p > 0.05).
Calcium Hydroxide ; Enterococcus faecalis* ; Enterococcus* ; Enzyme-Linked Immunosorbent Assay ; Humans* ; Lipopolysaccharides ; Matrix Metalloproteinase 8* ; Neutrophils*

OBJECTIVE: There is no data on which is more important for the intensity of intra-amniotic inflammation (IAI) between total grade or involved anatomical region in acute histologic chorioamnionitis (acute-HCA) of preterm-gestations. The objective of current study is to examine this issue. METHODS: The intensity of IAI was measured by amniotic fluid (AF) white blood cell (WBC) count and matrix metalloproteinase-8 (MMP-8) concentration in 225 singleton preterm-gestations (<36 weeks) who had acute-HCA including chorio-decidua involvement and delivered within 5 days of amniocentesis. Acute-HCA was defined in the presence of acute inflammatory changes in each anatomical region (i.e., chorio-decidua, amnion or chorionic plate). Patients were divided into 6 groups according to total grade (i.e., 1-6) and the presence or absence of chorio-decidua restriction (i.e., chorio-decidua restriction vs. extension beyond chorio-decidua) of acute-HCA. RESULTS: There was no significant difference in a median AF WBC and MMP-8 between the two groups (group-1, cases with total grade 1 vs. group-2, cases with total grade 2) among cases with chorio-decidua restriction (each for P>0.05) and between the four groups (group-3, cases with total grade 2 vs. group-4, cases with total grade 3 vs. group-5, cases with total grade 4 vs. group-6, cases with total grade 5-6) among cases with extension beyond chorio-decidua (each for P>0.05). However, group-3 (cases with extension beyond chorio-decidua) had a significantly higher median AF WBC and MMP-8 than group-2 (cases with chorio-decidua restriction) among cases with total grade 2 (each for P<0.05). CONCLUSION: Involved anatomical region is more important than total grade for the intensity of IAI in acute-HCA of preterm-gestations.
Amniocentesis ; Amnion ; Amniotic Fluid ; Chorioamnionitis ; Chorion ; Female ; Humans ; Inflammation ; Leukocytes ; Matrix Metalloproteinase 8 ; Pregnancy

OBJECTIVETo investigate the pathogenicity of matrix metalloproteinase 8, 9 (MMP-8, MMP-9) regulations of polymorphonuclear leukocytes (PMNs) by challenge of Porphyromonas gingivalis (P. gingivalis) with different fimA genotypes.

METHODSThe studies mainly adopt the isopycnic sedimentation separation to separate the PMNs from human peripheral blood. P. gingivalis ATCC 33277 (type I), WCSP 115 (type II), WCSP 1.5 (type III), W83 (type IV), WCSP 559 (type IV) were assessed for their inductions of MMP-8, MMP-9 expression in PMNs. MMP-8, MMP-9 protein levels in culture supernatant were determined by ELISA at different time intervals (5 min, 30 min, 1 h, 2 h) following continuous co-culture of bacteria with PMNs.

RESULTSMMP-8 and MMP-9 protein levels produced by PMNs co-culture with the I fimA-IV fimA P. gingivalis were significantly stronger than unsimulated group. The velocity and quantity of MMP-8 produced by PMNs co-culture with the II fimA P. gingivalis and IV fimA P. gingivalis were more than III fimA, IVfimA P. gingivalis. The MMP-9 protein levels produced by PMNs co-culture with the I fimA, II fimA, IV fimA P. gingivalis was significantly stronger than III fimA and IV fimA P. gingivalis.

CONCLUSIONII fimA and IV fimA P. gingivalis have stronger pathogenicity relatively, which indicate that fimA genotype is associated with pathogenesis of P. gingivalis.

Coculture Techniques ; Fimbriae Proteins ; Genotype ; Humans ; Matrix Metalloproteinase 8 ; Neutrophils ; Porphyromonas gingivalis

OBJECTIVETo evaluate the anti-matrix metalloproteinase (MMP) activity of a novel crosslinking quaternary ammonium methacrylates, 2-methacryloxylethyl dodecylmethyl ammonium bromide (MAE-DB).

METHODSThe effects of MAE-DB at different concentrations (1%, 3%, 5%, 7%) on soluble matrix metalloproteinase-8 (MMP-8) were investigated using fluorescent assay kit. Readings were taken every 20 min for 3 h. The 1, 10-phenanthroline provided by the assay kit served as control group. Demineralized dentin beams were randomly divided into three groups (n = 50) and immersed in different solutions: artificial saliva, MAE-DB incorporated artificial saliva and chlorhexidine incorporated artificial saliva. After temperature cycling, the changes of ultimate tensile strength were measured to determine the effect of MAE-DB on the activity of matrix-bound endogenous matrix metalloproteinases. The morphology of dentin collagen fibrils in the three groups was observed via transmission electron microscopy (TEM).

RESULTSMAE-DB could effectively inhibit the activity of soluble MMP-8. The inhibition percentage of 3% MAE-DB was 99.53% after 1 h, and it was significantly higher than that of 1, 10-phenanthroline (95.71%, P < 0.05). After temperature cycling, the ultimate tensile strengths of MAE-DB groups were significantly higher than those of the artificial saliva groups and the chlorhexidine groups (P < 0.05). TEM micrographs of MAE-DB group revealed that the microstructure of the collagen fibrillar was intact, while the fibrillar in the artificial saliva group was disrupted, indicating a protective function of MAE-DB on dentin collagen.

CONCLUSIONSMAE-DB can inhibit the activity of MMP and protect dentin collagen from enzyme degradation.

Ammonium Compounds ; pharmacology ; Chlorhexidine ; pharmacology ; Dentin ; drug effects ; Matrix Metalloproteinase 8 ; Methacrylates ; chemistry

OBJECTIVETo elucidate the relationship between collagen degradation and cervical ripening by detecting dynamic expressions of matrix metalloproteinases 2 (MMP-2) and 8 (MMP-8) in rat cervix.

METHODSPF rats were divided into 5 groups (n=6), namely non-pregnancy estrus interval group, gestational days 10, 16, and 19 groups, and immediately postpartum group. The wet weight of the cervix was measured and HE staining was used to display the general structure of the cervix. VG staining was applied to visualize the collagen fibers and muscular fibers. Immunohistochemistry was performed to observe the expressions of MMP-2 and MMP-8 in the cervix.

RESULTSHE staining showed that the rat uterine cervix consisted mainly of fibroblasts and fibrous connective tissues. A small quantity of neutrophils could be seen in the cervix stroma of the rats immediately after immediately parturition, but not at the other time points. The wet weight of the antepartum cervix had increased, and a more obvious increase was seen in the wet weight of the cervix immediately after parturition. The collagen fibers of the cervix consisted of collagen fibers and smooth muscle fibers, and their proportions showed no significant variation at the time points around the parturition. Immediately after parturition, the collagen fibers and muscular fibers in the cervix became loosened as compared with that before parturition. MMP-2 expression was found in the cervical stroma but not in the squamous epithelium in nonpregnancy, term pregnancy, and immediately after parturition; the smooth muscle cells, vascular wall, and stromal fibroblasts showed positive expression of MMP-2. Enhanced intensity of MMP-2 staining was seen in term pregnancy and postpartum group in comparison with that in the other groups. MMP-8 expression was observed in the cervix of rats immediately after parturition, with scattered neutrophils positive for MMP-8 spotted in the stroma of the ripened cervix. MMP-8 expression was not detected in the other groups.

CONCLUSIONRipened cervical fibrous tissue becomes loose and broken, and cervical ripening is accompanied by infiltration of neutrophils from exogenous vessels. These changes are particularly evident after parturition. MMP-2 and MMP-8 cooperate to degrade the cervical fibers, leading to cervical softening and expansion.

Animals ; Cervical Ripening ; metabolism ; Cervix Uteri ; enzymology ; Female ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 8 ; metabolism ; Pregnancy ; Rats ; Rats, Sprague-Dawley

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L1), 6 h (group L2), 12 h (group L3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L1, L2, L3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L1, L2, L3) were also significantly higher than that of control group (P<0.01). Moreover, among group L1, L2 and L3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.
Endotoxins ; Lung/pathology ; Matrix Metalloproteinase 8/*metabolism ; Random Allocation ; Rats, Sprague-Dawley ; Respiratory Distress Syndrome, Adult/chemically

BACKGROUND: Excessive extracellular matrix (ECM) deposition by airway inflammation is presumed to play an important role in the pathogenesis of worsening airflow obstruction (Ed- acceptable three-word noun) seen during acute exacerbations of chronic bronchitis. Although many proteases can cleave ECM molecules, matrix metalloproteinases (MMPs) and their inhibitors are likely to be the physiologically relevant mediators of ECM degradation. OBJECTIVES: The purpose of this study was to demonstrate that antibiotic treatment can change airway MMPs and TIMP-1 concentrations/levels by controlling airway inflammation in acute exacerbation of chronic bronchitis. METHOD: We studied 40 patients, all of whom had an acute exacerbation of chronic bronchitis. The patients were treated with two different antibiotics, moxifloxacin and clarithromycin, in a double-blind manner for 7 days. Sputum samples were induced and collected before and after antibiotic therapy. We measured the sputum concentration of MMP-1,-9, TIMP-1, IL-8 and secretory leukocyte proteinase inhibitor (SLPI) in sputum supernatants by ELISA method. RESULTS: There was no difference after antibiotic treatment in the sputum concentrations of MMP-1,-9, TIMP-1, IL-8 and SLPI between the patients treated with moxifloxacin and those treated with clarithromycin. But the sputum concentrations of TIMP-1, and SLPI, and the TIMP-1/MMP-1 ratio were significantly reduced by the antibiotic therapy. There were significant positive correlations between sputum TIMP-1 levels and IL-8 levels (p<0.01, r=0.751), and between the sputum TIMP-1/MMP-1 ratio and IL-8 levels (p<0.01, r=0.752). The sputum SLPI levels were significantly elevated by antibiotic treatment and were negatively correlated with sputum TIMP-1 levels (p<0.01, r=-0.496) and TIMP-1/MMP-1 levels (p<0.01, r=-0.456). CONCLUSION: The study shows that the worsening of airway inflammation in acute exacerbation of chronic bronchitis is associated with an imbalance between the concentrations/levels of TIMP-1 and MMPs. Antibiotic treatment can prevent progression of airway narrowing in acute exacerbation of chronic bronchitis by modulation of the protease and anti-protease imbalance.
Anti-Bacterial Agents ; Bronchitis, Chronic* ; Clarithromycin ; Enzyme-Linked Immunosorbent Assay ; Extracellular Matrix ; Humans ; Inflammation ; Interleukin-8 ; Leukocytes ; Matrix Metalloproteinase 1* ; Matrix Metalloproteinases* ; Peptide Hydrolases ; Sputum* ; Tissue Inhibitor of Metalloproteinase-1