快速进展性牙周炎患者龈沟液中的弹性蛋白酶活性

目的探讨中性多形核白细胞与快速进展性牙周炎的关系。方法检测２例快速进展性牙周炎患者共１０２个位点（其中２８个位点进行治疗前后的对比）的龈沟液弹性蛋白酶活性,将人多形核白细胞弹性蛋白酶特异底物———Ｓ２４８４与龈沟液反应,测吸光度值,以反映龈沟液弹性蛋白酶活性。结果快速进展性牙周炎龈沟液中的弹性蛋白酶活性［（０．６３±０．３８）Ａｂｓ／位点］明显高于健康对照组［（０．０７±０．０５）Ａｂｓ／位点］,差异有显著性;弹性蛋白酶活性的高低与龈沟液体积、探诊深度、附着丧失和出血指数呈正相关关系;治疗后龈沟液体积和各临床指数显著降低,弹性蛋白酶活性也从治疗前的（０．７３±０．３６）Ａｂｓ／位点下降为（０．１±０．１７）Ａｂｓ／位点,差异有显著性。结论快速进展性牙周炎患者的中性多形核白细胞并不是趋化反应不足,而是过度浸润与释放溶酶体酶,起协同致炎作用。     

Collagenase activity in gingival crevicular fluid of patients with juvenile periodontitis

The nature and origin of collagenases in gingival crevicular fluid of juvenile periodontitis patients was investigated. Gingival crevicular fluid collected from deep untreated periodontal pockets of juvenile periodontitis patients was found to contain only vertebrate collagenase (EC 3.4.24.7) activity that cleaved soluble type-I and -III collagens into 3/4 and 1/4 length fragments, as analyzed by SDS-polyacrylamide gel electrophoresis. Type II collagen was degraded at a markedly slower rate. This substrate specificity is indicative of collagenases produced by fibroblasts, epithelial cells and macrophages. We have previously found that collagenase in gingival crevicular fluid of adult periodontics patients appears to be mainly derived from polymorphonuclear leukocytes (PMN). The reasons for the apparent difference in collagenase source between the groups were investigated. We examined whether the pathogen characteristic for juvenile periodontitis, Actinobacillus actinomycetemcomitans, can release collagenase from normal human PMNs. All 10 A. actinomycetemcomitans strains tested, freshly isolated from the subgingival plaque of juvenile periodontitis patients, caused release of collagenase from PMNs in vitro. These results suggest that the lack of normally functioning PMNs in the periodontium of juvenile periodontitis patients may result in a colonization of bacteria that activate the resident periodontal cells to produce increased amounts of collagenase.     

Determination of serum antibody against Bacteroides gingivalis from rapidly progressive periodontitis and juvenile periodontitis patients

Bacteroides gingivitis (Bg) is one of the major pathogens associated with periodontitis. This is the first report in China on serum antibody level against Bg from patients with rapidly progressive periodontitis (RPP) and juvenile periodontitis (JP) using ELISA method. 21 RPP patients, 20 JP patients and 30 healthy subjects (H) were involved in this study. The results showed that the ratio of positive antibody response was 100% in RPP, 80% in JP and 30% in control group. The antibody response in both RPP and JP groups were significantly greater than that in healthy group (P less than 0.001). The difference between RPP and JP groups were also statistically significant (P less than 0.05).     

Protein carbonyl levels in serum,saliva and gingival crevicular fluid in patients with chronic and aggressive periodontitis

Objective

This study aims at evaluating the degree of protein carbonyl (PC) levels in serum, gingival crevicular fluid (GCF) and saliva in patients who suffer from chronic periodontitis (CP) and generalized aggressive periodontitis (GAP).

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.     

Lipid peroxidation levels and total oxidant status in serum, saliva and gingival crevicular fluid in patients with chronic periodontitis

OBJECTIVES: Increased levels of reactive oxygen species lead to oxidative stress. Recent data suggest increased lipid peroxidation (LPO) levels and oxidative stress in periodontitis. Malondialdehyde (MDA), a significant LPO product, increases in oxidative stress. In this study, MDA levels and total oxidant status (TOS) in serum, saliva and gingival crevicular fluid (GCF) were investigated in patients with chronic periodontitis (CP). MATERIALS AND METHODS: Thirty-six CP patients and 28 periodontally healthy controls were included in the study. Following clinical measurements and samplings, MDA and TOS levels were measured by high-performance liquid chromatography and a novel automatic colorimetric method, respectively. RESULTS: While the saliva and GCF MDA levels, and serum, saliva and GCF TOS values were significantly higher in the CP group than the control group (p<0.05), no significant difference in serum MDA levels was found (p>0.05). Strong positive correlations were observed between periodontal parameters and MDA and TOS levels (p<0.05). CONCLUSIONS: The results revealed that LPO significantly increased locally in the periodontal pocket/oral environment, while TOS displayed both systemic and local increases in periodontitis. The findings suggest that increased LPO and TOS may play an important role in the pathology of periodontitis, and are closely related to the clinical periodontal status.     

Peroxidases, Iactoferrin and lysozyme in peripheral blood neutrophils, gingival crevicular fluid and whole saliva of patients with localized juvenile periodontitis

OBJECTIVE: The aim of this study was to examine the longitudinal association of selected non-immune antimicrobial host factors (peroxidases, lysozyme and lactoferrin) to the localized juvenile periodontitis (LJP) disease status.
MATERIALS AND METHODS: Peroxidases, lysozyme and lactoferrin were quantitated from seven patients with LJP before and after periodontal therapy. Analyses were performed from simultaneously collected samples of peripheral blood polymorphonuclear leukocytes (PMNs), gingival crevicular fluid (GCF from diseased sites) and paraffin-stimulated whole saliva. Similar assays were done also from seven periodontally healthy controls.
RESULTS During untreated phase of LIP myeloperoxid-ase, lysozyme and lactoferrin concentrations were remarkably elevated in peripheral blood PMNs, also reflected in their high concentrations in GCF. All these values normalised with respect to healthy controls during the periodontal therapy. No similar longitudinal changes were seen in whole saliva but during therapy salivary per-oxidase concentrations declined below the control values, in accordance with our previous observations in parotid saliva samples of LJP patients.
CONCLUSIONS: In LJP the concentrations of lysozyme, lactoferrin and myeloperoxidase are significantly elevated in peripheral blood PMNs, also reflected in GCF. During periodontal therapy these values decline and approach those observed in healthy controls. No similar changes are seen in stimulated whole saliva.     

牙周炎患者非刺激性全唾液、龈沟液及血清中CD147的表达及意义

目的研究发现CD147可能参与调节牙周疾病的进展过程,因而评估牙周炎患者经牙周基础治疗前后和健康者的非刺激性全唾液、龈沟液及血清中CD147水平,探讨其与牙周炎的相关性以及作为牙周炎诊断及预后标志物的可能性。方法酶联免疫吸附试验(ELISA)检测20例牙周炎患者治疗前、后及20名健康人非刺激性全唾液、龈沟液及血清中CD147的水平,并记录牙周袋探诊深度(PD)、附着丧失(AL)和出血指数(BI)。结果经牙周基础治疗6周后,牙周炎患者的临床指标除AL外BI及PD均低于治疗前(P<0.05);治疗后非刺激性全唾液、龈沟液及血清中CD147水平明显降低,与治疗前相比差异具有统计学意义(P<0.05);除血清外,牙周炎患者治疗前、后的非刺激性全唾液和龈沟液中CD147水平仍高于健康对照组,其差异有统计学意义(P<0.05)。结论牙周炎患者、健康者的非刺激性全唾液及龈沟液中均有CD147表达,并随牙周炎症减轻CD147表达降低。     

Matrix metalloproteinases and their inhibitors in gingival crevicular fluid and saliva of periodontitis patients

Abstract Matrix metalloproteinases (MMPs) and serine proteinases seem to be related to tissue destruction in periodontitis. The presence of MMPs in gingival crevicular fluid (GCF) and saliva, however, has not been studied comprehensively with the enzyme-linked immunosorbent assay (ELISA)-technique, We therefore examined the levels of MMP-1,-3.-8 and -9, and their endogenous inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP-1). in GCF and saliva of patients with adult periodontitiss (AP) and localized juvenile periodontitis (LJP). Elevated levels of MMP-1 were detected in LJP GCF compared to AP and control GCF. Elevated levels of TIMP-1 were also detected in LJP GCF in comparison to AP and control GCF Higher MMP-8 levels were detected in AP GCF compared to LJP and control GCF. The relative low levels of MMP-3 were present in all studied GCF samples. Elevated levels of MMP-8 were further detected in saliva of AP compared to LJP and the controls. Both MMP-1 and TIMP-1 were detected in all studied saliva samples, but not significant differences were detected between the studied groups. Our ELISA-results confirm that (i) PMN MMP-8 and MMP-9 are the main collagenase and gelatinase in AP GCF, whereas GCF collagenase in LJP seems to be of the MMP-1-type; (ii) only low levels of TIMP-1. endogenous MMP-inhibitor. are present in AP GCF. which emphasises the importance of doxycycline as a possible adjunctive drug in the treatment of AP patients; (iii) tests based on specific antibodies against PMN MMPs. especially MMP-8, might serve as a reliable method of measuring and monitoring enzyme levels in GCF from different periodontitis patients.     

Protein carbonyl levels in serum and gingival crevicular fluid in patients with chronic periodontitis

OBJECTIVE: Evidence reveals the role of reactive oxygen species (ROS) in many pathologies including periodontitis. Protein carbonylation is the most widely used biomarker for oxidative damage to proteins, and reflects cellular damage induced by ROS. In this study protein carbonyl (PC) levels in serum and gingival crevicular fluid (GCF) in patients with chronic periodontitis (CP) was evaluated. MATERIALS AND METHODS: Thirty-three patients with CP and 24 healthy controls were included in the study. Following the clinical measurements and samplings, total protein levels in serum and GCF were determined by Bradford method, and serum and GCF PC levels were measured by modified Levine method. RESULTS: PC levels in serum and GCF were significantly higher in the CP group compared to the control group (p<0.05). In all subjects, serum and GCF PC levels showed statistically significant positive correlations with all clinical parameters (p<0.05). CONCLUSIONS: The results suggest that both systemic and local/periodontal protein carbonylation increase in CP compared to health and that elevated levels of PCs may be a sign of oxidative damage in periodontitis and correlate well with the periodontal status.     

Increased interleukin-18 in gingival crevicular fluid from periodontitis patients

INTRODUCTION: This study aimed to measure the levels of interleukin-18 (IL-18) in inflamed shallow sites and inflamed deep sites in patients with periodontitis and to compare the data with results from inflamed shallow sites in patients with gingivitis. A secondary aim was to examine the composition of the subgingival microbiota in the sampled sites. METHODS: Gingival crevicular fluid was collected from five gingivitis sites and five periodontitis sites from 18 patients with chronic periodontitis, and from five gingivitis sites from 15 patients with gingivitis. Samples from each site category were pooled and IL-18 levels were measured using an enzyme-linked immunosorbent assay. The subgingival microbiota was analyzed by checkerboard DNA-DNA hybridization. RESULTS: All clinical parameters and gingival crevicular fluid volumes were higher in periodontitis sites compared with gingivitis sites from patients with periodontitis and gingivitis. The total amount of IL-18 was higher in periodontitis sites than gingivitis sites in both periodontitis (P = 0.018) and gingivitis (P = 0.002) patients and was higher in gingivitis sites from periodontitis patients than in those from gingivitis patients (P = 0.015). There were higher levels of Tannerella forsythia, Porphyromonas gingivalis, and Treponema denticola (red complex species) in periodontitis sites compared with gingivitis sites in both the periodontitis and gingivitis patients (P < 0.001). CONCLUSION: Levels of IL-18 were higher in patients with chronic periodontitis compared with patients with gingivitis, even at sites with similar pocket depths. The presence of similar levels of red complex species in gingivitis sites from periodontitis patients and from gingivitis patients suggested that the higher levels of IL-18 were not associated with a different microbial challenge.     

Physical activity, inflammatory biomarkers in gingival crevicular fluid and periodontitis

Aims: To examine the associations of physical activity with interleukin 1- β (IL-1 β ), C-reactive protein (CRP) and periodontitis and to investigate whether any relationship between physical activity and inflammatory mediators differs between periodontitis cases and non-cases.
Material and Methods: In this population-based case control study of Australians aged 18+ years, dentists conducted oral epidemiologic examinations identifying cases with moderate or severe periodontitis and periodontally healthy controls. Gingival crevicular fluid samples collected during examinations were analysed for inflammatory biomarkers. Subject-completed questionnaires assessed leisure-time physical activity. Exposure odds ratios (ORs) were estimated in multivariable logistic regression models adjusting for periodontitis risk indicators.
Results: Of 751 subjects (359 cases, 392 controls), those meeting a prescribed threshold for leisure-time physical activity had lower adjusted odds of elevated IL-1 β : OR=0.69, (95% CI=0.50–0.94) and detectable CRP: OR=0.70 (0.50–0.98) than less active adults. Physical activity was not associated with periodontitis: OR=1.14 (0.80–1.62). Periodontitis modified the association between levels of physical activity and detectable CRP. Increasing quartiles of physically activity were associated with decreasing probability of detectable CRP, but the effect was limited to periodontitis cases and was not apparent among non-cases.
Conclusion: Leisure-time physical activity may protect against an excessive inflammatory response in periodontitis.     

牙周炎患者龈沟液中IL—1活性检测

目的：通过对牙周炎龈沟液（ｇｉｎｇｉｖａｌｃｒｅｖｉｃｕｌａｒｆｌｕｉｄ，ＧＣＦ）中ＩＬ－１活性进行检测，初步探讨ＩＬ－１在牙周炎症中的作用。方法：以成人牙周炎为研究对象，通过细胞检测法对患牙ＧＣＦ中ＩＬ－１活性进行检测。结果：在患牙ＧＣＦ中可测到具有较高活性的ＩＬ－１，而在健康ＧＣＦ中未能测到。结论：牙周炎时局部有活性ＩＬ－１渗出，ＩＬ－１是参与牙周炎症反应的重要细胞因子。     

The gingival crevicular fluid Interleukin-1β and tumour necrosis factor-α levels in patients with rapidly progressive periodontitis

Cytokines are believed to play an important role in the pathogenesis of periodontal diseases. In the present study, gingival crevicular fluid (GCF) levels of two important cytokines, interleukin 1-/3 (IL-1β) and tumour necrosis factor-α (TNF-α) and, in addition, serum IL-1β levels, were determined in patients with severe and rapid periodontal breakdown by use of ELISA. While IL-1β was detected in all of the GCF samples studied, TNF-α could only be detected in about half the samples. The mean GCF IL-1β level was 38.45 ± 13.99 pg/mL, and the mean TNF-α level was 3.20 ± 1.39 pg/mL, respectively. The GCF IL-1β levels also presented a strong positive correlation with the mean pocket depths. Although weak, both of the cytokines also presented correlations with the presence of bleeding on probing. Additionally GCF samples contained increased IL-1β levels when compared with the serum samples suggesting local production mechanisms. The findings of the present study suggest that these cytokines may be involved in the pathogenesis of periodontal diseases (IL-1β being more significant), and also may help in defining the active phase of periodontal breakdown.     

Single-dose concentrations of tinidazole in gingival crevicular fluid, serum, and gingival tissue in adults with periodontitis

Previous studies have shown that metronidazole is effective in the treatment of subgingival microflora associated with destructive periodontitis. The aim of this study was to determine whether tinidazole, a close analogue of metronidazole, would reach sufficient concentrations in serum, gingival crevicular fluid, and gingival tissue, to inhibit putative periodontopathic bacteria. Ten adult patients with moderate to advanced periodontitis took a single 2-g dose of tinidazole orally. Samples were assayed by high-performance liquid chromatography. The concentrations of tinidazole in serum and GCF were in a similar range (3.2-46.5 micrograms/mL). Tinidazole was not detected in the GCF in three of the patients. The drug was found in gingival tissue obtained at two h (0.17 +/- 0.14 micrograms/mg) and six h (0.15 +/- 0.18 micrograms/mg) after oral administration. The mean concentration of tinidazole in serum at 24 h (13 +/- 3.0 micrograms/mL) is greater than the minimum inhibitory concentration for anaerobic bacteria as reported by others. The present data suggest that a single 2-g oral dose of tinidazole may lead to the presence of potentially bactericidal levels of tinidazole for up to 24 h in the periodontal pockets of some patients with periodontitis.     

Profiling gingival crevicular fluid from smoking and non-smoking chronic periodontitis patients

BACKGROUND: Smoking is a major risk factor for the development and progression of chronic periodontitis (CP). Gingival crevicular fluid (GCF) derived from these patients contains many proteins that could serve as important diagnostic indicators. A protein chip technology called Surface Enhanced Laser Desorption/Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS) has recently been developed to facilitate protein profiling of complex biological mixtures. The hypothesis to be tested was SELDI-TOF-MS could distinguish between GCF of CP patients by smoking status and pocket depth. METHODS: GCF samples collected before treatment from sixteen CP patients (eight smokers and eight non-smokers) at both shallow and deep sites were evaluated by SELDI-TOF-MS. Spectral fingerprints were constructed for both cohorts and analyzed by a two-way ANOVA according to smoking status and pocket depth. Significance threshold was set at p < 0.05. Mean molecular weight (MW) peaks and intensities were also analyzed. RESULTS: The spectral fingerprints were significantly different between the two cohorts when analyzed by ANOVA according to smoking status (p < 0.0001) but not pocket depth (p = 0.9876). Also, the mean intensity of many individual MW peaks were determined to be significantly different between the two cohorts. Several peaks ranging in MW from 11-14 kDa were only detected in the GCF obtained from smokers. CONCLUSIONS: This study has demonstrated that profiling of GCF by SELDI-TOF-MS can distinguish between CP smokers and non-smokers. Moreover, over-expressed proteins in GCF from smokers may serve as biomarkers for this high risk patient population.     

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

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

Reaction of human sera from juvenile periodontitis, rapidly progressive periodontitis, and adult periodontitis patients with selected periodontopathogens

The levels of serum antibody reactive to selected periodontopathogens were determined in 182 clinically characterized patients: 35 healthy control, 50 juvenile periodontitis, 42 adult periodontitis and 55 rapidly progressive periodontitis. Reactive antibody levels were determined using an enzyme-linked immunosorbent assay with whole cell preparations of Bacteroides gingivalis, Capnocytophaga (Bacteroides) ochraceus, Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans (Y-4) serving as antigens. Increased reactivity to B. gingivalis and F. nucleatum was observed in all three disease groups studied while antibody reactive to A. actinomycetemcomitans was increased in juvenile and rapidly progressive periodontitis. Antibody levels reactive to C. ochraceus in healthy subjects did not differ from those observed in any disease patient groups. Possible implications in the etiology and progression of the diseases coupled with environmental changes which occur in the econiche of the periodontal pocket are described.