Lysine-specific proteolytic activity responsible for forsythia detaching factor modification

ObjectivesThe objective of the present study was to clarify the lysine-specific proteolytic activity derived from periodontal pathogens responsible for Forsythia detaching factor (FDF) modification.DesignThe activity responsible for FDF modification in Tannerella forsythia and Porphyromonas gingivalis were evaluated by colorimetric assay using Ac-Arg-Ala-Lys-p-nitroaniline as a substrate. FDF modification in T. forsythia and P. gingivalis were evaluated by Western blotting using recombinant FDF (rFDF) as a substrate. Furthermore, the activity in GCF of 20 patients with periodontitis and 10 healthy subjects was also evaluated by colorimetric assay. Bacteria in subgingival plaque were detected using polymerase chain reaction.ResultsThe activity of both bacteria in colorimetric assay were 21.35 unit (P. gingivalis) and 3.61 unit (T. forsythia), respectively. Western blot analysis revealed that P. gingivalis was found to efficiently degrade rFDF and T. forsythia partially cleaved rFDF. The activity in GCF from patients with periodontitis (clinically healthy sites: CH, deep bleeding sites: DB and deep non-bleeding sites: DNB) was significantly higher than those from healthy subjects (healthy sites: H). Among the patients with periodontitis, the activity from CH was significantly lower than those from DB and DNB. T. forsythia was detected in 68.4% of DNB, in 78.4% of DB and in none of CH. P. gingivalis was detected in 63.2% of DNB, in 84.0% of DB and in 10.5% of CH. No bacterium was detected in healthy subjects.ConclusionThe lysine-specific proteolytic activity responsible for FDF modification correlates with the presence of major periodontal pathogens.     

Pro-inflammatory cytokine production from normal human fibroblasts is induced by Tannerella forsythia detaching factor

Background and Objective: Tannerella forsythia is a periodontal pathogen. Recently, we have reported that the cytopathic component of T. forsythia contains two distinct factors. One arrests the cell cycle at the G2 phase and the other, named forsythia detaching factor, detaches adhesion‐dependent immortalized human cells. In this study, we investigated the biological function of forsythia detaching factor using human normal fibroblasts. Material and Methods: A recombinant forsythia detaching factor, reported previously, was used. TIG‐3 cells, cultured in the absence or presence of forsythia detaching factor, were lysed and the supernatant was analyzed by western blotting with polyclonal forsythia detaching factor antibodies. The cells were subsequently fractionated to isolate the cytoplasmic, mitochondrial and remaining fractions. In order to measure the activity of mitochondria using nicotinamide adenine dinucleotide‐linked reductase, the water‐soluble tetrazolium method was used. The mitochondrial oxidative membrane potential was estimated by measuring the oxidization‐dependent fluorogenic conversion of dihydrotetramethylrosamine using flow cytometry. The concentration of interleukin‐8 in the culture supernatant was assayed using a Human IL‐8 ELISA kit. Results: Forsythia detaching factor‐treated cells detached from the substratum and aggregated from 3 to 24 h. Then, the detached cells resumed adhesion and proliferated after 48 h. The western blot analysis revealed that most forsythia detaching factor trans‐located into the mitochondrial fraction. Forsythia detaching factor suppressed the nicotinamide adenine dinucleotide‐linked reductase activity in a dose‐dependent manner and consequently increased the mitochondrial oxidative membrane potential. The production of interleukin‐8 was reinforced in forsythia detaching factor‐treated cells at 72 h through an increase of the mitochondrial oxidative membrane potential. Conclusion: The forsythia detaching factor might be involved in the virulence of T. forsythia through induction of the pro‐inflammatory cytokine interleukin‐8.     

Soluble Triggering Receptor Expressed on Myeloid Cells 1 (sTREM‐1) in Gingival Crevicular Fluid: Association With Clinical and Microbiologic Parameters

Background: Soluble triggering receptor expressed on myeloid cells 1 (sTREM‐1) belongs to the immunoglobulin superfamily and is involved in amplification of the inflammatory response to bacterial infection. This cross‐sectional study aims to investigate the levels of sTREM‐1 in gingival crevicular fluid (GCF) of individuals without periodontitis and with chronic periodontitis (CP) or generalized aggressive periodontitis (GAgP) and their association with the levels of key periodontal pathogens in subgingival plaque. Methods: GCF and subgingival plaque samples were obtained from healthy sites of participants without periodontitis (n = 20) and periodontitis sites of patients with CP (n = 22) and GAgP (n = 20). sTREM‐1 levels in GCF were measured by enzyme‐linked immunosorbent assay. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans levels in subgingival plaque were analyzed by quantitative real‐time polymerase chain reaction. Results: sTREM‐1 levels in GCF were higher in CP and GAgP than healthy sites by 3.6‐ and 4.4‐fold, respectively, with no significant differences between the two forms of periodontitis. Moreover, sTREM‐1 levels in GCF were positively correlated with site‐specific clinical periodontal parameters and levels of P. gingivalis, T. denticola, and T. forsythia, but not A. actinomycetemcomitans, in subgingival plaque. Conclusion: Increased GCF levels of sTREM‐1 at diseased sites and their positive correlation with clinical and microbiologic parameters strengthen the association of this inflammatory marker with periodontitis.     

Pathogenic potential of Tannerella forsythia enolase

Although enolases are cytosolic enzymes involved in the glycolytic pathway, they can also be secreted or expressed on the surface of a variety of eukaryotic cells and bacteria. Surface‐exposed enolases of eukaryotes and bacteria can function as plasminogen receptors. Furthermore, antibodies raised against bacterial enolases can react with host enolases, suggesting molecular mimicry between bacterial and host enzymes. In this study, we analyzed an enolase of the major periodontopathogen Tannerella forsythia, which is either secreted or present on the cell surface, via matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry and immunofluorescence, respectively. The T. forsythia enolase retained the enzymatic activity converting 2‐phosphoglycerate to phosphoenolpyruvate and showed plasminogen binding and activating ability, which resulted in the degradation of fibronectin secreted from human gingival fibroblasts. In addition, it induced proinflammatory cytokine production, including interleukin‐1β (IL‐1β), IL‐6, IL‐8, and tumour necrosis factor‐α (TNF‐a) in the human THP‐1 monocytic cell line. Taken together, our results demonstrate that T. forsythia enolase plays a role in pathogenesis in the host by plasminogen activation and proinflammatory cytokine induction, which has the potential to exaggerate inflammation in periodontitis.     

Effect of azithromycin, as an adjunct to nonsurgical periodontal treatment, on microbiological parameters and gingival crevicular fluid biomarkers in generalized aggressive periodontitis

Emingil G, Han B, Özdemir G, Tervahartiala T, Vural C, Atilla G, Baylas H, Sorsa T. The effect of azithromycin, as an adjunct to nonsurgical periodontal treatment, on microbiological parameters and gingival crevicular fluid biomarkers in generalized aggressive periodontitis. J Periodont Res 2012; 47: 729–739. © 2012 John Wiley & Sons A/S Background and Objective: To study the effectiveness of azithromycin in combination with nonsurgical periodontal therapy on clinical and microbiological parameters, and on the MMP‐8 and TIMP‐1 levels in gingival crevicular fluid, over a 6‐mo time‐period in patients with generalized aggressive periodontitis. Material and Methods: Thirty‐two patients with generalized aggressive periodontitis were included in this randomized, double‐blind, placebo‐controlled, parallel‐arm study. They were randomly assigned to azithromycin or placebo groups (500 mg once daily for 3 d). Probing depth, clinical attachment levels, presence of bleeding on probing and plaque were recorded. Gingival crevicular fluid samples were obtained from one single‐rooted tooth, while microbiological samples were obtained from two single‐rooted teeth, all with a probing depth of ≥ 6 mm. Microbiological parameters were analyzed by quantitative real‐time PCR for Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Fusobacterium nucleatum, Prevotella intermedia and total bacteria. Gingival crevicular fluid biomarkers were determined by immunofluorometric assay and ELISA. Results: All clinical parameters improved, and microbiological parameters and gingival crevicular fluid MMP‐8 levels significantly decreased, over the 6‐mo period (p < 0.05); both groups demonstrated similar improvements. The azithromycin group presented a higher percentage of deep pockets resolved (probing depth reduction of ≥ 3 mm from baseline) compared with the placebo group at 1 mo (p < 0.05). Conclusion: Adjunctive azithromycin therapy provides no additional benefit over nonsurgical periodontal treatment on clinical parameters, microbiological parameters and gingival crevicular fluid biochemical markers investigated in patients with generalized aggressive periodontitis.     

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.     

A comparison of antibody levels to Bacteroides gingivalis in serum and crevicular fluid from patients with untreated periodontitis

The levels of IgG antibody to Bacteroides gingivalis were measured in serum and sequential samples of crevicular fluid from healthy and diseased sites in patients with untreated periodontitis using ELISA. All subjects had detectable serum titres but there was a wide variation in titre between subjects. Moderate to strong correlations were found between serum and crevicular fluid levels of IgG. A statistically significant difference was observed between sequential samples of crevicular fluid. There was no difference in the level of specific IgG to B. gingivalis in crevicular fluid between healthy and diseased sites within the same individual.     

A pathogenic trace of Tannerella forsythia – shedding of soluble fully active tumor necrosis factor α from the macrophage surface by karilysin

Tannerella forsythia is implicated as a pathogen causing chronic and aggressive periodontitis. However, its virulence factors, including numerous putative proteases, are mostly uncharacterized. Karilysin is a newly described matrix metalloprotease‐like enzyme of T. forsythia. Since pathogen‐derived proteases may affect the host defense system via modulation of the cytokine network, the aim of this study was to determine the influence of karilysin on tumor necrosis factor‐α (TNF‐α). The results showed that karilysin cleaved the membrane form of TNF‐α on the surface of macrophages, and that this led to an increased concentration of soluble TNF‐α in the conditioned medium. Importantly, despite partial degradation of soluble TNF‐α by karilysin, the released cytokine retained its biological activity, inducing apoptosis and stimulating autocrine pathway of pro‐inflammatory gene expression. Notably, the observed effect required proteolytic activity by karilysin, since a catalytically inactive mutant of the enzyme did not affect TNF‐α secretion. The shedding was independent of the activity of ADAM17, a major endogenous TNF‐α converting enzyme. Karilysin‐dependent TNF‐α release from the cell surface is likely to occur in vivo because human plasma, the main constituent of gingival crevicular fluid, only slightly affected the sheddase activity of karilysin. Taken together, these results indicate that karilysin modulates the host immune response through regulation of TNF‐α secretion, and should therefore be considered as a new virulence factor of T. forsythia.     

Levels of serum immunoglobulin G specific to bacterial surface protein A of Tannerella forsythia are related to periodontal status

Background: Tannerella forsythia (Tf) is a Gram‐negative anaerobe implicated in the development of periodontal disease. Bacterial surface protein A (BspA) is a surface‐expressed and ‐secreted protein that is recognized as an important virulence factor of Tf. This study was undertaken to determine whether Tf BspA induces an antibody response in periodontal disease. We hypothesized that serum immunoglobulin (Ig)G antibody levels against BspA correlate with the disease of patients. Methods: Sera were obtained from 100 patients with cardiac disorders and periodontal disease and 73 patients who experienced myocardial infarction but were periodontally healthy. Sera samples were assayed for anti‐BspA antibody (total IgG and IgG subtypes) by enzyme‐linked immunosorbent assay (ELISA). Antibody levels were measured in ELISA units by using an arbitrary patient as a standard. Results: A negative correlation was found with BspA‐specific total IgG antibody titers and the severity of disease measured as the clinical attachment level (CAL) when healthy and diseased groups were analyzed separately (healthy group: [?0.23, correlation value] Student's t value [73 degrees of freedom] = 1.99; P = 0.05; diseased group: [?0.21] t [100 degrees of freedom] = 2.12; P = 0.03]). However, there was a positive correlation ([0.18 correlation value] Student's t value [173 degrees of freedom] = 2.39; P = 0.017) when healthy and diseased groups were combined. A strong positive correlation ([0.338 correlation value] Student's t value [173 degrees of freedom] = 4.69; P <0.0001) between the BspA‐specific IgG titers and periodontal probing depth was observed when healthy and disease groups were combined. Conclusions: Data demonstrated that antibodies to Tf BspA were elicited in patients with periodontal disease, and antibody levels were associated with the disease severity. Furthermore, data suggested that anti‐BspA IgG might have a protective function in periodontal disease by minimizing the loss of tooth attachment tissue.     

Periodontitis‐specific molecular signatures in gingival crevicular fluid

Xiang XM, Liu KZ, Man A, Ghiabi E, Cholakis A, Scott DA. Periodontitis‐specific molecular signatures in gingival crevicular fluid. J Periodont Res 2010; 45: 345–352. © 2010 John Wiley & Sons A/S Background and Objective: Periodontitis is currently diagnosed almost entirely on gross clinical manifestations that have been in situ for more than 50 years without significant improvement. The general objective of this study was, therefore, to evaluate whether mid‐infrared spectroscopy can be used to identify disease‐specific molecular alterations to the overall biochemical profile of tissues and body fluids. Material and Methods: A total of 190 gingival crevicular fluid samples were obtained from periodontitis (n = 64), gingivitis (n = 61) and normal sites (n = 65). Corresponding infrared absorption spectra of gingival crevicular fluid samples were acquired and processed, and the relative contributions of key functional groups in the infrared spectra were analysed. The qualitative assessment of clinical relevance of these gingival crevicular fluid spectra was interpreted with the multivariate statistical analysis‐linear discriminant analysis. Results: Using infrared spectroscopy, we have been able to identify four molecular signatures (representing vibrations in amide I, amide II/tyrosine rings and symmetric and asymmetric stretching vibrations of phosphodiester groups in DNA) in the gingival crevicular fluid of subjects with periodontitis or gingivitis and healthy control subjects that clearly demarcate healthy and diseased periodontal tissues. Furthermore, the diagnostic accuracy for distinction between periodontally healthy and periodontitis sites revealed by multivariate classification of gingival crevicular fluid spectra was 98.4% for a training set of samples and 93.1% for a validation set. Conclusion: We have established that mid‐infrared spectroscopy can be used to identify periodontitis‐specific molecular signatures in gingival crevicular fluid and to confirm clinical diagnoses. Future longitudinal studies will assess whether mid‐infrared spectroscopy represents a potential prognostic tool, recognized as key to advancement of periodontics.     

A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy

Thunell DH, Tymkiw KD, Johnson GK, Joly S, Burnell KK, Cavanaugh JE, Brogden KA, Guthmiller JM. A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01204.x. © 2009 John Wiley & Sons A/S Background and Objective: Cytokines and chemokines play an important role in the pathogenesis of periodontal diseases. The objective of this study was to quantitatively assess the effect of initial periodontal therapy on gingival crevicular fluid levels of a comprehensive panel of cytokines and chemokines, including several less extensively studied mediators. Material and Methods: Clinical examinations were performed and gingival crevicular fluid samples obtained from six subjects with generalized severe chronic periodontitis prior to initial periodontal therapy and at re‐evaluation (6–8 weeks). Four diseased and two healthy sites were sampled in each subject. Twenty‐two gingival crevicular fluid mediators were examined using a multiplex antibody capture and detection platform. Statistical analyses were performed by fitting mixed effects linear models to log‐transformed gingival crevicular fluid values. Results: Gingival crevicular fluid interleukin (IL)‐1α and IL‐1β were the only cytokines to differ in initially diseased vs. initially healthy sites. Following initial therapy, 13 of the 16 detectable cytokines and chemokines decreased significantly in diseased sites, including IL‐1α, IL‐1β, IL‐2, IL‐3, IL‐6, IL‐7, IL‐8, IL‐12 (p40), CCL5/regulated on activation, normally T cell expressed and secreted (RANTES), eotaxin, macrophage chemotactic protein‐1, macrophage inflammatory protein‐1α and interferon‐γ. At healthy sites, only three of the 16 mediators were significantly altered following therapy. Conclusion: This is the first study, to our knowledge, to evaluate such an extensive panel of gingival crevicular fluid mediators within the same sample prior to and following initial therapy. The results confirm that periodontal therapy effectively reduces pro‐inflammatory cytokines and chemokines, including less well‐described mediators that may be important in initiation and progression of periodontitis. The multiplex assay will prove useful for future gingival crevicular fluid studies.     

Crevicular Porphyromonas gingivalis‐specific immunoglobulin A levels in healthy and periodontitis‐affected Thai cohorts

Abstract Aim: Immunoglobulin A is a key humoral immune component involved in defense mechanisms against infections. Periodontitis, the chronic inflammatory disease causing periodontal destruction, adversely affects adults worldwide, including Thailand. As the development of periodontitis is partly mediated by immune components, levels of total and Porphyromonas gingivalis‐specific immunoglobulin A in gingival crevicular fluid of Thai cohorts were studied. Methods: Gingival crevicular fluid was collected from 24 patients with severe generalized chronic periodontitis and 22 healthy controls. The amount and concentration of total and Porphyromonas gingivalis‐specific immunoglobulin A in each gingival crevicular fluid sample were determined by enzyme‐linked immunosorbent assay. Results: The control group contained the highest concentrations of both types of gingival crevicular fluid–immunoglobulin A, but the lowest levels of these antibodies were found in the deep sites of the periodontitis group. Moreover, the concentrations of gingival crevicular fluid–immunoglobulin A and the degree of periodontitis severity appeared to have an inverse relationship. There was no significant difference in the amounts of gingival crevicular fluid–immunoglobulin A in the control and periodontitis groups. Conclusions: This study supports the hypothesis that high concentrations of specific gingival crevicular fluid–immunoglobulin A antibodies directed against Porphyromonas gingivalis, a potent periodontic microorganism, could retard periodontitis development.     

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.     

Gingival crevicular fluid and plasma levels of neuropeptide Substance-P in periodontal health, disease and after nonsurgical therapy

Background and Objective: The level of Substance‐P in gingival crevicular fluid has been found to correlate with clinical measures of periodontal disease. The present study was designed to assess the relationship between clinical parameters and levels of Substance‐P in the gingival crevicular fluid from inflamed gingiva, periodontitis sites and after treatment of periodontitis sites, and to correlate them to the Substance‐P levels of plasma. Material and Methods: Thirty, age‐ and gender‐matched subjects were divided into three groups (healthy, gingivitis and chronic periodontitis) based on modified gingival index scores and clinical attachment loss. A fourth group consisted of 10 subjects from the periodontitis group, 6–8 wk after initial therapy. Plasma and gingival crevicular fluid samples were collected and quantified for Substance‐P using an enzyme immunoassay. Results: The mean concentration of Substance‐P, both in gingival crevicular fluid and plasma, was observed to be highest in the periodontitis group (45.13 pg/mL in gingival crevicular fluid and 67.8 pg/mL in plasma) and lowest in the healthy group (6.07 pg/mL in gingival crevicular fluid and below the detection level in plasma). The mean Substance‐P concentration in the gingivitis group (11.42 pg/mL in gingival crevicular fluid and 38.8 pg/mL in plasma) and in the after‐treatment group (7.58 pg/mL in gingival crevicular fluid and 39.7 pg/mL in plasma) lay between the highest and lowest values. In all groups the gingival crevicular fluid levels showed a statistically significant positive correlation with that of plasma and clinical attachment loss. Conclusion: Substance‐P levels were highest in the gingival crevicular fluid from sites with periodontal destruction; however, periodontal treatment resulted in the reduction of Substance‐P levels. Gingival crevicular fluid and plasma Substance‐P levels showed a positive correlation in all of the groups.     

Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils

Hiroshima Y, Bando M, Inagaki Y, Mihara C, Kataoka M, Murata H, Shinohara Y, Nagata T, Kido J. Resistin in gingival crevicular fluid and induction of resistin release by Porphyromonas gingivalis lipopolysaccharide in human neutrophils. J Periodont Res 2012; 47: 554–562. © 2012 John Wiley & Sons A/S Background and Objective: Resistin is an adipocytokine that induces insulin resistance and is predominantly expressed in adipocytes and peripheral blood mononuclear cells. Resistin expression increases in inflammatory diseases as well as diabetes mellitus, and is upregulated by bacterial pathogens and proinflammatory cytokines. The aim of this study was to identify resistin in human gingival crevicular fluid, to compare the resistin levels in gingival crevicular fluid between subjects with and without periodontitis and diabetes mellitus and to investigate the regulation of resistin release from human neutrophils by Porphyromonas gingivalis lipopolysaccharide (P‐LPS). Material and Methods: Gingival crevicular fluid samples were collected from patients with chronic periodontitis (n = 24), patients with diabetes mellitus‐related periodontitis (n = 18) and healthy subjects (n = 21). Resistin in gingival crevicular fluid was determined using western blot analysis and an ELISA kit. The glycated hemoglobin (HbA_1c) value was obtained from patients with diabetes mellitus‐related periodontitis by a medical interview. Human neutrophils were cultured with P‐LPS (0–1000 ng/mL), or incubated with inhibitors of actin or microtubule polymerization in the absence or presence of P‐LPS. The medium and cellular fractions were used for determination of resistin by ELISA. Results: The resistin level in gingival crevicular fluid from patients with periodontitis or diabetes mellitus‐related periodontitis was significantly higher than that of healthy subjects. The resistin level in gingival crevicular fluid was correlated with gingival index score, but not blood HbA_1c value. The P‐LPS increased resistin release from human neutrophils, and its induction was decreased by actin polymerization inhibitors. Conclusion: We show, for the first time, the presence of resistin in gingival crevicular fluid. A high resistin level in gingival crevicular fluid samples from periodontitis patients may to some extent be related to P‐LPS‐induced resistin release from neutrophils.     

Investigation of a Novel Predictive Biomarker Profile for the Outcome of Periodontal Treatment

Background: An ability to predict the response to conventional non‐surgical treatment of a periodontal site would be advantageous. However, biomarkers or tests devised to achieve this have lacked sensitivity. The aim of this study is to assess the ability of a novel combination of biomarkers to predict treatment outcome of patients with chronic periodontitis. Methods: Gingival crevicular fluid (GCF) and subgingival plaque were collected from 77 patients at three representative sites, one healthy (probing depth [PD] ≤3 mm) and two diseased (PD ≥6 mm), at baseline and at 3 and 6 months after treatment. Patients received standard non‐surgical periodontal treatment at each time point as appropriate. The outcome measure was improvement in probing depth of ≥2 mm. Concentrations of active enzymes (matrix metalloproteinase [MMP]‐8, elastase, and sialidase) in GCF and subgingival plaque levels of Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum were analyzed for prediction of the outcome measure. Results: Using threshold values of MMP‐8 (94 ng/μL), elastase (33 ng/μL), sialidase (23 ng/μL), and levels of P. gingivalis (0.23%) and T. forsythia (0.35%), receiver operating characteristic curves analysis demonstrated that these biomarkers at baseline could differentiate healthy from diseased sites (sensitivity and specificity ≥77%). Furthermore, logistic regression showed that this combination of these biomarkers at baseline provided accurate predictions of treatment outcome (≥92%). Conclusion: The “fingerprint” of GCF enzymes and bacteria described here offers a way to predict the outcome of non‐surgical periodontal treatment on a site‐specific basis.     

Analysis of neutrophil‐derived antimicrobial peptides in gingival crevicular fluid suggests importance of cathelicidin LL‐37 in the innate immune response against periodontogenic bacteria

Introduction: During periodontitis, an innate immune response to bacterial challenge is primarily mediated by neutrophils. We compared neutrophilic content and the level of neutrophil‐derived antimicrobial peptides in gingival crevicular fluid (GCF) in two clinical forms of severe periodontitis. Methods: GCF was collected from 14 patients with aggressive periodontitis, 17 patients with chronic periodontitis, and nine healthy subjects. Samples were analyzed for periodontopathogen load using real‐time polymerase chain reactions. The amounts of myeloperoxidase and α‐defensins (HNP1–3) were determined by enzyme‐linked immunosorbent assay, and the level of cathelicidin (hCAP18/LL‐37) was assayed by Western blot. Results: Myeloperoxidase concentration was not correlated with levels of LL‐37 and HNP1–3 in samples from patients, compared to controls. The amount of HNP1–3 was twofold and fourfold higher in patients with aggressive and chronic periodontitis, respectively. Those with chronic disease had significantly elevated amounts of mature LL‐37. The increased concentration of both peptides in chronic periodontitis correlated with the load of Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Conclusion: The lack of a correlation between LL‐37, HNP1–3, and myeloperoxidase content suggests that neutrophils are not the sole source of these bactericidal peptides in the GCF of patients with periodontitis; and that other cells contribute to their local production. The bacterial proteases of P. gingivalis, T. forsythia, and T. denticola might degrade hCAP18/LL‐37, because the 11‐kDa cathelicidin‐derived fragment was present in GCF collected from pockets infected with these bacteria. Collectively, it appears that a local deficiency in LL‐37 can be considered as a supporting factor in the pathogenesis of severe cases of periodontitis.     

Tannerella forsythia‐produced methylglyoxal causes accumulation of advanced glycation endproducts to trigger cytokine secretion in human monocytes

The periodontal pathogen Tannerella forsythia has the unique ability to produce methylglyoxal (MGO), an electrophilic compound which can covalently modify amino acid side chains and generate inflammatory adducts known as advanced glycation endproducts (AGEs). In periodontitis, concentrations of MGO in gingival‐crevicular fluid are increased and are correlated with the T. forsythia load. However, the source of MGO and the extent to which MGO may contribute to periodontal inflammation has not been fully explored. In this study we identified a functional homolog of the enzyme methylglyoxal synthase (MgsA) involved in the production of MGO in T. forsythia. While wild‐type T.forsythia produced a significant amount of MGO in the medium, a mutant lacking this homolog produced little to no MGO. Furthermore, compared with the spent medium of the T. forsythia parental strain, the spent medium of the T. forsythia mgsA‐deletion strain induced significantly lower nuclear factor‐kappa B activity as well as proinflammogenic and pro‐osteoclastogenic cytokines from THP‐1 monocytes. The ability of T. forsythia to induce protein glycation endproducts via MGO was confirmed by an electrophoresis‐based collagen chain mobility shift assay. Together these data demonstrated that T. forsythia produces MGO, which may contribute to inflammation via the generation of AGEs and thus act as a potential virulence factor of the bacterium.     

Free soluble receptor activator of nuclear factor-kappab ligand in gingival crevicular fluid correlates with distinct pathogens in periodontitis patients

Aim: The aim of the experiment was to investigate the levels of free soluble receptor activator of nuclear factor‐κb ligand (sRANKL) in periodontal health and disease and their correlations to clinical parameters and important periodontal pathogens. Material and Methods: Chronic periodontitis (n=35) and periodontally healthy (n=38) subjects participated in the present study. Pocket depth, recession and bleeding index were recorded at a total of 221 sites. Subgingival plaque samples from these sites were analysed for the levels of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola. Gingival crevicular fluid samples were analysed with ELISA for levels of free sRANKL. Comparisons between groups were performed by applying non‐parametric tests (Mann–Whitney) and correlations among parameters were sought for with Spearman's r coefficient. Results: Mean levels of free sRANKL were higher in periodontitis subjects and correlated significantly with mean counts of T. denticola on the subject level and P. gingivalis, T. denticola on the site level (Spearman's r coefficient, p<0.05), but not with clinical parameters. No correlations were found between the levels of free sRANKL and investigated parameters in periodontally healthy individuals. No effect of smoking was found on investigated parameters and correlations (univariate analysis of variance and pairwise comparisons). Conclusions: Findings from the present study suggest a correlation of levels of sRANKL with important pathogens in periodontitis patients.     

Levels of specific immunoglobulin G to Porphyromonas gingivalis in gingival crevicular fluid are related to site disease status

Titers of immunoglobulin G (IgG) directed against Porphyromonas gingivalis in gingival crevicular fluid of 40 periodontitis patients were measured at three sites in each patient (healthy, gingivitis and periodontitis) by enzyme-linked immunosorbent assay. When paired analyses were performed using Wilcoxon signed-rank tests, periodontitis sites were found to have lower median titers than gingivitis sites. Both systemic and locally-produced antibodies contribute to the overall gingival crevicular fluid antibody profile. Albumin, in contrast, is derived only from serum, and thus this protein serves as an indicator of serum contribution to gingival crevicular fluid. Correction was therefore made for systemic input to the gingival crevicular fluid IgG profile by expressing the results per unit of albumin. When this was done, periodontitis sites were also found to have significantly lower antibody levels than gingivitis sites. These findings suggest that a failure of local antibody production or reduction in quantities, by, for example, degradation by bacterial proteases, may contribute to the change from a gingivitis to a periodontitis lesion.