Analysis of proteins in human gingival crevicular fluid by mass spectrometry

Kido J, Bando M, Hiroshima Y, Iwasaka H, Yamada K, Ohgami N, Nambu T, Kataoka M, Yamamoto T, Shinohara Y, Sagawa I, Nagata T. Analysis of proteins in human gingival crevicular fluid by mass spectrometry. J Periodont Res 2012; 47: 488–499. © 2012 John Wiley & Sons A/S Background and Objective: Gingival crevicular fluid is a bodily fluid transuded from periodontal tissues into the gingival crevice and periodontal pocket, and contains many species of components. Proteins in gingival crevicular fluid have been studied as markers for periodontal diseases. Mass spectrometric analysis is used for the analyses of proteins, lipids, saccharides and metals, and expected as an approach for disease diagnosis. For better analysis of the protein components in gingival crevicular fluid, we investigated proteins in gingival crevicular fluid samples from the healthy gingival crevice and periodontal pocket using mass spectrometry. Material and Methods: Gingival crevicular fluid samples were collected from subjects who gave their informed consent and were periodontally healthy or had diseased pockets. These samples were electrophoretically separated, and each fraction on the gels was analysed by nano liquid chromatography coupled with tandem mass spectrometry. Antimicrobial peptides detected in gingival crevicular fluid were confirmed by western blotting. Results: One hundred and four proteins were detected in gingival crevicular fluid samples from both healthy sites and sites of periodontitis; 64 proteins were contained only in gingival crevicular fluid from healthy sites and 63 proteins were observed only in gingival crevicular fluid from periodontitis sites. These proteins were blood‐, cytoskeleton‐, immunity‐, inflammation‐ and lipid‐related proteins and enzymes. Some proteins, including ceruloplasmin, glycogen phosphorylase, glutathione S‐transferase, phosphoglycerate mutase, psoriasin, S100A11 and resistin, were identified for the first time in gingival crevicular fluid. Antimicrobial peptides, such as lactoferrin, α1‐antitrypsin, lipocalin, S100A7, S100A8, S100A9 and cathelicidin, were observed by mass spectrometry and western blotting. Conclusion: Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.     

Vascular endothelial growth factor in gingival tissues and crevicular fluids of diabetic and healthy periodontal patients

BACKGROUND: Periodontal disease is one of the major oral problems encountered in patients with diabetes mellitus (DM). Vascular changes, neutrophil dysfunction, altered collagen synthesis, and genetic predisposition observed in DM may contribute to periodontitis; and the vascular alterations observed in such patients may depend on vascular endothelial growth factor (VEGF) actions. Few reports are available about the mechanism of neovascularization and the angiogenic factors that contribute to the periodontal pathology and the role of VEGF in periodontal diseases. The aim of this study is to compare VEGF expression in healthy and periodontally diseased tissues with gingival crevice fluid (GCF) of healthy persons and diabetic patients. METHODS: Gingival tissue and GCF samples were collected from sites of periodontitis in 10 healthy subjects and in 10 type 2 diabetic patients, and from the sites of healthy gingiva within the same groups. Therefore, each patient became his/her own control. Additionally, 10 people without any systemic or periodontal diseases were enrolled, forming a negative control group. Thus, a total of 50 tissue and 50 GCF samples were provided. RESULTS: No VEGF staining was observed in the negative control group or in the systemically healthy people's healthy tissue samples, whereas four samples of diabetic patients showed positive staining (P < 0.05). However, VEGF was revealed in two tissue samples of periodontal sites of systemically healthy people and in six samples of the diabetic patients (P > 0.05). In all test groups, GCF VEGF levels were higher in periodontal sites (P < 0.05) than in healthy sites. CONCLUSION: The results of this study showed that VEGF is increased in all periodontal tissues of both groups and in the healthy sites of diabetic patients. Additionally, GCF VEGF values increased in periodontal sites of all test groups.     

Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLC

BACKGROUND: The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromatography quadropole mass spectrometry. AIMS: To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease. METHODS: GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms. RESULTS: Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied. CONCLUSIONS: The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.     

Detection of herpetic viruses in gingival crevicular fluid of patients suffering from periodontal diseases: prevalence and effect of treatment

Background/Aim:  Although the role of bacteria in the etiology of periodontitis is well established, it has been suggested that herpetic viruses could contribute to the initiation and progression of this disease. The aim of this study was to determine the prevalence of human cytomegalovirus (HCMV), Epstein–Barr virus (EBV) and herpes simplex virus (HSV) in gingival crevicular fluid (GCF) samples obtained from periodontally healthy, gingivitis and periodontitis patients. In addition, the effect of periodontal treatment (scaling and root planing) on the persistence of herpetic viruses was evaluated in a sub-group of patients suffering from chronic periodontitis.
Methods:  The presence of viruses in GCF samples was assessed by a nested PCR amplification technique. The persistence of viruses in periodontal sites was evaluated following a scaling and root planing therapy.
Results:  A statistically significant higher prevalence of HCMV was observed in periodontitis patients as compared to healthy control subjects (35 vs. 8%, respectively; P  = 0.0377). A trend for a higher prevalence of HSV was also noted in the periodontitis group, in comparison with healthy control subjects. In addition, a higher prevalence of HCMV was associated with deep periodontal pockets in subjects suffering from periodontitis. In the sub-group of periodontitis patients, periodontal therapy resulted in the elimination (HCMV and EBV) or reduction (HSV) of the herpetic viruses.
Conclusions:  This study showed that the prevalence of HCMV and HSV viruses in GCF is higher in patients suffering from periodontitis compared to periodontally healthy subjects, and that the prevalence of HCMV is higher in deep periodontal pockets. It also brought evidences that periodontal therapy may be associated with virus elimination in diseased sites.     

Comparative Analysis of Calcium‐Binding Myeloid‐Related Protein‐8/14 in Saliva and Serum of Patients With Periodontitis and Healthy Individuals

Background: This study aims to investigate calcium‐binding myeloid‐related protein (MRP)‐8/14 in the saliva and serum of individuals with periodontitis and periodontally healthy individuals for the assessment of its role in the pathogenesis and clinical diagnosis of periodontitis. Methods: This cross‐sectional study includes 56 patients with periodontitis and 44 periodontally healthy individuals. Saliva and serum were collected for the detection of MRP‐8/14 and calcium levels. Periodontopathic bacteria were determined by polymerase chain reaction in saliva. Correlations between salivary and serum MRP‐8/14 levels and clinical parameters, bacteria, and calcium were analyzed with Pearson correlation in a multiple regression model. MRP‐8/14 levels were documented with receiver operating characteristic (ROC) curves. Results: Compared with healthy individuals, MRP‐8/14 levels were significantly higher in both the saliva and serum of patients with periodontitis, but calcium was increased only in saliva. A high diagnostic potential of salivary MRP‐8/14 was detected for periodontitis (ROC = 0.86). Salivary MRP‐8/14 levels correlated significantly with the presence of the periodontopathogen Treponema denticola, as well as with the clinical parameters of periodontitis. Conclusion: MRP‐8/14 in saliva might be a potential diagnostic parameter for periodontal disease.     

The in vivo expression of membrane-bound CD14 in periodontal health and disease

BACKGROUND: Membrane-bound CD14 (mCD14) is a myeloid differentiation antigen expressed on monocytes/macrophages and neutrophils. It is a key molecule responsible for the innate recognition of bacteria by host cells and functions as an important receptor for bacterial lipopolysaccharide. This study investigated the in vivo expression profile and levels of mCD14 in healthy and diseased gingival tissues. METHODS: Gingival biopsies were obtained from 24 patients with chronic periodontitis, including 22 periodontal pocket tissues, 13 clinically healthy tissues, and 18 inflamed connective tissues (i.e., granulation tissues). Gingival biopsies from seven periodontally healthy subjects were used as controls. mCD14 was detected by immunohistochemistry. RESULTS: mCD14 was detected in 21 of 22 periodontal pocket tissues and all other categories of tissues. The mCD14-positive cells were mainly confined to the gingival epithelium-connective tissue interface. The expression levels in periodontally healthy subjects were significantly higher than in the patients. Within the patients, clinically healthy tissues showed greater levels of mCD14 than periodontal pocket tissues and granulation tissues. CONCLUSIONS: mCD14 was commonly expressed in both healthy and diseased gingival tissues and was predominantly confined to the epithelium-connective tissue interface. The positive relationship observed between mCD14 expression levels and periodontal health may imply that mCD14 is associated with favorable host responses to bacterial challenge and contributes to maintaining periodontal homeostasis.     

Calcitonin gene-related peptide in gingival crevicular fluid in periodontal health and disease

The aims of the present study were to investigate whether calcitonin gene-related peptide (CGRP) was present in gingival crevicular fluid in both periodontal health and disease and to study the relationship with periodontal inflammation. Gingival crevicular fluid (GCF) was collected from a healthy, a gingivitis and a periodontitis site in 18 subjects with periodontitis and from a healthy site in 19 subjects without periodontitis. The volume of GCF was measured and each sample subsequently analysed for CGRP by radioimmunoassay. In subjects with periodontitis, CGRP immunoreactivity (CGRP-IR) was not detected in any periodontitis sites, nor in 67% of gingivitis and 28% of periodontally-healthy sites. The total amount of CGRP-IR was significantly elevated in periodontally healthy (p=0.0015) and gingivitis (p=0.027) compared with periodontitis sites. CGRP-IR was present in 89% of the healthy sites sampled in control subjects at comparable levels to those in healthy sites in periodontitis subjects. It is concluded that in periodontal inflammation, particularly in deep pockets, constituents of GCF process and degrade CGRP.     

Comparison of CCL28, interleukin‐8, interleukin‐1β and tumor necrosis factor‐alpha in subjects with gingivitis,chronic periodontitis and generalized aggressive periodontitis

Background and Objective: Cytokines produced by various cells are strong local mediators of inflammation. Mucosa‐associated epithelial chemokine (CCL28), interleukin‐8 (IL‐8), interleukin‐1beta (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α) are major cytokines that play important roles in the periodontal inflammatory process. In this study we aimed to compare the levels of CCL28, IL‐8, IL‐1β and TNF‐α in the gingival crevicular fluid of both periodontally healthy subjects and in subjects diagnosed with gingivitis, chronic periodontitis and generalized aggressive periodontitis. Material and Methods: A total of 84 subjects participated in the study: 21 subjects had gingivitis, 21 subjects had chronic periodontitis, 21 subjects had generalized aggressive periodontitis and 21 were periodontally healthy. The levels of CCL28, IL‐8, IL‐1β and TNF‐α were analyzed using enzyme‐linked immune sorbent assay (ELISA). Results: The total levels of CCL28 and IL‐8 in the gingival crevicular fluid of the generalized aggressive periodontitis group (324.74 ± 42.62 pg/30 s, 487.62 ± 49.21 pg/30 s) were significantly higher than those of the chronic periodontitis group (268.81 ± 28.64 pg/30 s, 423.65 ± 35.24 pg/30 s), the gingivitis group (146.35 ± 17.46 pg/30 s, 310.24 ± 48.20 pg/30 s) and the periodontally healthy group (92.46 ± 22.04 pg/30 s, 148.41 ± 24.64 pg/30 s). Similarly, the total levels of IL‐1β and TNF‐α in the generalized aggressive periodontitis group (110.23 ± 9.20 pg/30 s, 1284.46 ± 86.32 pg/30 s) were significantly higher than those in the chronic periodontitis group (423.65 ± 35.24 pg/30 s, 82.64 ± 9.12 pg/30 s), the gingivitis group (52.10 ± 7.15 pg/30 s, 824.24 ± 44.68 pg/30 s) and the periodontally healthy group (36.44 ± 8.86 pg/30 s, 628.26 ± 34.61 pg/30 s). Conclusion: CCL28, IL‐8, IL‐1β and TNF‐α may play key roles in the host response to inflammation in periodontal diseases. As the severity of periodontal diseases increases, destruction of periodontal tissues also increases. Inflammation is one among many factors that trigger periodontal tissue destruction. Identification of the mediators that influence the development and progression of inflammation in periodontal diseases may be very important in understanding the prognoses of periodontal diseases.     

Periodontal disease in mothers indicates risk in their children

Introduction.  It is well established that severe periodontitis clusters in families, but there are no data about the relationship between mothers with chronic periodontitis and their children's periodontal status.
Objective.  To evaluate a risk for periodontal diseases in children of periodontally diseased and healthy mothers.
Methods.  Four study groups were included: (I) 20 female patients with untreated generalized severe chronic periodontitis, (II) their children (34), (III) 13 periodontally healthy mothers and (IV) their children (13). Material was collected from years 2004–2006. The clinical examination included registration of visible plaque index, modified gingival index and, bleeding sites on probing. Periodontal microbiological samples were obtained from all study subjects and the isolates were identified according to morphology and biochemical profiles; similar interfamilial pathogens were compared by PCR-technique.
Results.  The children of diseased mothers more frequently had periodontal diseases, especially gingivitis. In addition, clinical parameters of gingival inflammation were more expressed and oral hygiene was worse in this group of children. VPI and VPI% of the diseased and healthy mothers differed significantly. The most common oral pathogens were P. intermedia/nigrescens and A. actinomycetemcomitans . The children of healthy mothers harboured pathogens less frequently than the children of diseased mothers. The sharing of P. intermedia/nigrescens was more frequent (5 families) than A. actinomycetemcomitans (2 families).
Conclusion.  Maternal indicators, such as periodontitis, hygiene habits, and periodontal microflora are risk factors for childhood periodontal diseases, and might be predictive of future childhood and adolescent periodontitis.     

Identification of intracellular oral species within human crevicular epithelial cells from subjects with chronic periodontitis by fluorescence in situ hybridization

BACKGROUND AND OBJECTIVE: Interactions between oral bacteria and gingival epithelial cells play an important role in the pathogenesis of periodontal diseases. This study used in situ hybridization with 16 rRNA probes and confocal microscopy to detect the periodontal pathogens Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythia, and Treponema denticola within epithelial cells from periodontal pockets, gingival crevice, and buccal mucosa collected from subjects with chronic periodontitis (n = 14) and good periodontal health (n = 8). MATERIAL AND METHODS: Each green fluorescent species-specific and universal probe was hybridized with all 58 epithelial samples from the 22 patients. The samples were observed by confocal microscopy to confirm the intracellular localization of oral species of bacteria. The mean frequency of detection and number of intracellular bacteria per epithelial cell were computed for each sample. RESULTS: The frequency of cells with internalized bacteria was higher in samples from the gingival crevice than in samples from the oral mucosa. Epithelial cells from all subjects harbored intracellular bacteria; however, patients with periodontitis presented significantly higher counts of bacteria per cell than periodontally healthy individuals (p < 0.05). Periodontal pathogens showed a trend to be detected in higher numbers in epithelial cells from periodontitis patients. In particular, T. forsythia and T. denticola were significantly more prevalent in periodontal pocket cells than healthy sulci and buccal cell samples in the periodontitis group (p < 0.05). CONCLUSION: Those findings indicate that crevicular and buccal cells present internalized bacteria, regardless of periodontal status. However, higher bacterial loads are detected in cells from subjects with periodontitis.     

Identification of bacterial species on or in crevicular epithelial cells from healthy and periodontally diseased patients using DNA-DNA hybridization

The purpose of this investigation was to identify bacterial species present on or in crevicular epithelial cells in healthy and diseased sites using DNA probes. In order to achieve this aim, further improvements were made in the separation of unattached bacteria from those adherent to epithelial cells isolated from the human gingival crevice or periodontal pocket. Then the DNA probes were used to determine the prevalence of detectable DNA from 15 microbial species on or in crevicular epithelial cells. One sample was taken from a single subgingival site in each of 51 individuals ranging in age from 19 to 45 years. Samples were taken from 27 sites of clinically healthy subjects and 24 samples were taken from subjects having periodontally diseased sites. DNA-DNA hybridization indicated that a majority of epithelial cells from healthy sites (63%) were in contact with or harbored Streptococcus oralis. On the other hand, species such as Bacteroides forsythus, Prevotella intermedia, Capnocytophaga ochracea and Campylobacter rectus were more frequently detected in elevated numbers in periodontally diseased sites. Cluster analysis of the microbial profiles generally aggregated subjects with and without periodontitis into separate cluster groups. The cluster patterns suggest the possibility that microbial complexes will be, in part, determined by the receptors available on the epithelial cells.     

Effect of inflammation,smoking and stress on gingival crevicular fluid cytokine level

BACKGROUND: Recent studies have shown that cytokines are pivotal to the pathogenesis of periodontal diseases and may be used as markers in diagnosis. AIM: The aim of the present study was to determine the levels of interleukin (IL)-1beta, IL-4, IL-6 and IL-8 in gingival crevicular fluid of periodontally healthy and diseased individuals and to study their association to smoking, stress and clinical periodontal parameters. MATERIAL AND METHODS: A total of 80 patients were included in the study : 20 patients with early onset or aggressive periodontitis (EOP), 20 with chronic adult periodontitis (AP), 20 with gingivitis (G) and 20 patients with healthy periodontium (H). GCF was collected by means of Durapore strips, from four sites per patient, randomly selected in each quadrant. The contents of IL- 1beta, IL-4, IL-6 and IL-8 were measured in 320 samples by use of commercially available sandwich enzyme-linked immunoadsorbent assays. RESULTS: In periodontally diseased subjects the total amounts of IL-1beta, IL-6 and IL-8 were significantly elevated as compared to healthy subjects, whereas IL-4 showed an inverse relationship to periodontal status and higher amounts were found in the healthy group. The amounts of all four cytokines were positively correlated with probing depths. IL-4, IL-6 and IL-8 were significantly correlated to smoking while stress was associated with IL-1beta, IL-6 and IL-8 levels. CONCLUSIONS: The present data suggest that crevicular IL-1beta, IL-6 and IL-8 reflect the activity of periodontal destruction, whereas IL-4 shows an inverse correlation to it. The enhanced production of inflammatory cytokines in the presence of smoking and stress may have clinical consequences.     

HHV-6, HHV-7, HHV-8 in gingival biopsies from chronic adult periodontitis patients. A case-control study

BACKGROUND: Recent reports have suggested that various herpesviruses may be involved in the occurrence and progression of different forms of periodontal disease. OBJECTIVE: The objective of the present study was to investigate the presence of the novel herpesviruses HHV-6, HHV-7 and HHV-8 in gingival biopsies from patients affected by chronic adult periodontitis. As control, gingival biopsies from periodontally healthy subjects were analysed. MATERIALS AND METHODS: Gingival biopsies were harvested from 23 volunteers: 13 patients affected by chronic adult periodontitis (CAP) and 10 periodontally healthy subjects. Each CAP patient contributed two biopsies involving the epithelium and connective tissue facing the sulcus/periodontal pockets: one biopsy from a site having a probing pocket depth (PPD) > or =5 mm and presenting with bleeding upon probing (affected site) at the time of biopsy collection, and the other biopsy from a site with PPD< or =3 mm and without bleeding on probing (nonaffected site). After DNA extraction, nested PCR was used in herpesvirus identification. RESULTS: HHV-6 DNA sequences were detected in one non-affected site (8%) and no affected sites (0%) of CAP patients. One biopsy (10%) in healthy subjects revealed HHV-6 positivity. Tissue specimens in 10/13 CAP patients (77%) and 7/10 healthy subjects (70%) contained HHV-7 DNA. HHV-7 prevalence in affected and nonaffected sites of CAP patients was 77% and 54%, respectively. HHV-8 was detected in 7.7% of CAP patients and 0% of healthy subjects. CONCLUSIONS: Gingival tissue may act as a reservoir for HHV-7. A high prevalence of HHV-7 was detected in both periodontally diseased and healthy individuals. The prevalence of HHV-6 and -8 was similarly low in both groups. Our data do not support an association of investigated herpesvirus species with destructive periodontal disease.     

Radioimmunoassay quantification of adrenomedullin in human gingival crevicular fluid

OBJECTIVE: To investigate whether adrenomedullin (ADM), a multifunctional peptide with key roles in host antimicrobial defence and inflammation, was present and quantifiable in human gingival crevicular fluid (GCF) and to study its relationship with periodontal health and disease. DESIGN: GCF samples (30s) were collected using perio-paper strips from one diseased site in 21 subjects with periodontal disease and one healthy site from 19 control subjects with no evidence of periodontal disease. Samples were analysed by radioimmunoassay using a specific anti-human ADM antibody. RESULTS: Measurable adrenomedullin-like immunoreactivity (ADM-LI) was present in all the GCF samples collected. ADM-LI was significantly higher in periodontitis sites (mean 493.6 pg) than in control healthy sites (mean 248.5 pg), p = 0.0016. CONCLUSION: It is concluded that ADM is present in GCF at levels at which it could have an antibacterial role in the gingival crevice and modulate the pathophysiology of periodontal inflammation.     

Human gingival crevicular fluid contains MRP8 (S100A8) and MRP14 (S100A9), two calcium-binding proteins of the S100 family

Human gingival crevicular fluid contains unidentified proteins which might play a role as markers in periodontal diseases. Therefore, low-molecular-weight proteins found in human gingival crevicular fluid (GCF), but absent from serum, were identified in the present study by means of two-dimensional electrophoresis (2-D PAGE) analysis. GCF, serum, and whole saliva were collected from periodontitis and healthy subjects, as well as from edentulous and newborn subjects. Protein samples were separated by two-dimensional polyacrylamide gel electrophoresis, stained with silver, and compared with reference protein maps in the SWISS-2D PAGE database. In GCF and saliva from periodontitis patients and healthy subjects, four dominant low-molecular-mass (from 8 to 14 kDa) acidic spots were observed. They were not found in serum and were less visible in saliva from edentulous and newborn subjects. From N-terminal amino acid sequencing, the two 2-D protein spots of 8 kDa and isoelectric points between 6.5 and 7.0 were both identified as protein MRP8 (SI00A8), a member of the S100 family of calcium-binding proteins. Using peptide mass fingerprinting and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), we identified the other two protein spots, with mass of 14 kDa and isoelectric points between 5.5 and 6.0, as protein MRP14 (S100A9), also belonging to the S100 family. The presence of MRP8 and MRP14 in GCF was confirmed by Western blot, with monoclonal antibodies. The two polypeptides, MRP8 and MRP14, identified in GCF represent the major difference between the 2-D PAGE patterns of serum and GCF, and we hypothesize that they may play an important role in the gingival sulcus and could represent possible markers for periodontal diseases.     

Levels of specific immunoglobulin G to the forsythia detaching factor of Tannerella forsythia in gingival crevicular fluid are related to the periodontal status

Onishi H, Arakawa S, Nakajima T, Izumi Y. Levels of specific immunoglobulin G to the forsythia detaching factor of Tannerella forsythia in gingival crevicular fluid are related to the periodontal status. J Periodont Res 2010; 45: 672–680. © 2010 John Wiley & Sons A/S Background and Objective: Forsythia detaching factor (FDF) is a putative virulence factor of Tannerella forsythia that induces detachment of adherent cells and interleukin‐8 production in human fibroblasts. The objective of the present study was to clarify the relationship between anti‐FDF IgG levels in gingival crevicular fluid and the clinical status in patients with periodontitis and in healthy subjects. Material and Methods: Gingival crevicular fluid and subgingival plaque samples were obtained from both the diseased and healthy sites of 37 patients with periodontitis and from 30 healthy subjects. Anti‐FDF IgG levels were evaluated, and both the fdf gene and T. forsythia 16S ribosomal RNA (rRNA) were detected using the PCR. Results: Anti‐FDF IgG levels (of both diseased and healthy sites) of patients with periodontitis were significantly higher than those of healthy subjects. Among the patients with periodontitis, anti‐FDF IgG levels of healthy sites were significantly higher than those of diseased sites and the levels showed negative correlations with probing pocket depth and clinical attachment level. Among the patients with periodontitis, T. forsythia 16S rRNA was detected in 18 of 37 diseased sites and in 5 of 29 healthy sites, and the fdf gene was detected in 19 of 37 diseased sites and in 7 of 29 healthy sites. By contrast, no healthy subjects were positive for T. forsythia 16S rRNA or the fdf gene. Conclusion: These data suggest that anti‐FDF IgG levels in gingival crevicular fluid are related to the periodontal status.     

Correlations between pentraxin 3 or cytokine levels in gingival crevicular fluid and clinical parameters of chronic periodontitis

The gingival crevicular fluid (GCF) contains various biomarkers, such as interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IL-10, among others. These cytokines have been reported to correlate with gingival inflammation and periodontal status. Therefore, the analysis of GCF may be useful for the diagnosis of periodontal status. Pentraxin 3 (PTX3) is the first identified long pentraxin, and is released by several cell types in response to proinflammatory signals. The aim of this study was to determine the levels of IL-1β, IL-6, IL-8, TNF-α, IL-10 and PTX3 in GCF from diseased and healthy sites in patients with chronic periodontitis. Cross-sectional clinical data were obtained from 50 patients with chronic periodontitis. GCF samples were collected with paper strips from one periodontal diseased site and one periodontally healthy site per subject. The levels of IL-1β, IL-6, IL-8, IL-10 and TNF-α were determined using a multiplexed bead immunoassay, and the PTX3 level was measured using an enzyme-linked immunosorbent assay. Mean clinical parameters were significantly higher at diseased sites (P < 0.01) as compared to healthy sites, and the mean levels of PTX3, IL-1β, IL-6, IL-8, IL-10 and TNF-α were higher in diseased sites (P < 0.01) than in healthy sites. There were strong correlations between PTX3 or IL-1β and periodontal status. These results suggest that GCF PTX3 levels might be useful as a diagnostic marker for periodontal disease.     

Significance of thrombomodulin release from gingival epithelial cells in periodontitis patients

Background and Objective:  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.
Material and methods:  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.
Results:  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.
Conclusion:  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.     

Ⅱ型糖尿病合并牙周病患者龈沟液中细胞因子/趋化因子的表达水平

目的 探讨Ⅱ型糖尿病合并牙周病患者与单纯牙周病患者龈沟液（gingival crevicular fluid,GCF）中细胞因子/趋化因子的表达水平。 方法 选取伴Ⅱ型糖尿病的牙周病患者52例,单纯牙周病患者40例,用Luminex FLEXMAP3D仪和Human Cytokine/Chemokine试剂盒检测GCF中14种细胞因子/趋化因子的表达水平。 结果 牙周病部位：嗜酸性粒细胞趋化因子、巨噬细胞炎症蛋白-1α、粒细胞-巨噬细胞集落刺激、白介素-6、肿瘤坏死因子-α和白介素-12的浓度,糖尿病组受试者高于非糖尿病组受试者（P<0.0035）。 结论 糖尿病可影响牙周病部位细胞因子/趋化因子的表达,糖尿病可能是牙周病的促进因素。     

Tissue inhibitors of metalloproteinases level and collagenase activity in gingival crevicular fluid: the relevance to periodontal diseases

OBJECTIVES: To provide an overall assessment of levels of tissue inhibitors of metalloproteinases (TIMPs), collagenase activities, and of immuno-reactivities for matrix metalloproteinases (MMP)-1 and -8 in gingival crevicular fluid (GCF) obtained from healthy subjects, and gingivitis and periodontitis patients, and to analyse the relationships between periodontal tissue destruction and the GCF components in periodontal diseases by principal component analysis. MATERIALS AND METHODS: GCF was sampled with sterile paper strips from 10 gingivitis and 11 periodontitis patients. Ten volunteers served as clinically healthy controls. TIMP-1 and -2 protein amounts in GCF were measured by ELISA, and active and APMA-activatable collagenase activities were determined by functional assays using image-analysis after SDS-PAGE. RESULTS: GCF TIMP-1 level and both active and latent collagenase activities were significantly higher in the diseased groups than in the healthy group. TIMP-2 was detectable in only 29% of all subjects (mean: 2.06 ng). Western blot analysis showed that MMP-8 was the major interstitial collagenase in the GCF of the diseased groups. Principal component analysis using clinical parameters and the GCF components has indicated components one to three account for 87% of total variation when evaluating the relevance of their measurements to periodontal diseases. CONCLUSIONS: We conducted the functional and immunological characterization of MMPs and TIMPs in the GCF of periodontally diseased patients. Principal component analysis indicated components one to three explaining 87% of total variation, and further suggested that higher collagenase activity (especially in active collagenase) would be an important marker in evaluating the pathogenesis of periodontitis. Consequently, these observations may have significant therapeutic and diagnostic implications.