Polarization of human gingival fibroblasts by Th1-, Th2-, Th17-, and Treg-derived cytokines.

BACKGROUND AND OBJECTIVE: The purpose of this study was to evaluate whether gingival fibroblasts (GFs) can be differently activated and polarized into distinct functional subtypes by T-helper (Th) cytokines. METHODS: Gingival fibroblasts were stimulated with interferon (IFN)-γ, interleukin (IL)-4, IL-17, and transforming growth factor (TGF)-ß, representative cytokines of Th1, Th2, Th17, and regulatory T cells, respectively, and the gene expression profiles were analyzed by microarray. Differentially expressed genes (DEGs) in GFs stimulated by 4 cytokines were screened, and a gene ontology (GO) analysis of the DEGs was conducted. To confirm the reliability of the microarray results, the DEGs that showed the largest differences compared with non-stimulated GFs were further analyzed by RT-PCR. To evaluate the effect of polarization on GFs responses to lipopolysaccharide (LPS), GFs stimulated by 4 cytokines were further stimulated with Escherichia coli LPS and mRNA levels of several genes were analyzed using RT-PCR. RESULTS: Differentially expressed genes by 4 Th cytokines were enriched in different GO terms, and the patterns of gene expression on GFs were shown functionally different. GFs stimulated with IFN-γ (GF(IFN-γ)) up-regulated the expression of chemokines (chemokine (C-X-C motif) ligand (CXCL)9, -10, -11, chemokine (C-C motif) ligand (CCL)8), molecules involved in antigen presentation, complement component 3 (C3), and other immune response-related molecules, whereas they down-regulated the expression of several types of collagen, extracellular matrix (ECM) components, and DNA replication and nuclear protein-related molecules. By contrast, GF(IL-4) up-regulated the expression of ECM components, cell adhesion molecules, and tissue development-related molecules and down-regulated the expression of chemokines (CXCL10 and CXCL8) and adaptive immune response-related molecules. GF(IL-17) up-regulated the expression of chemokines and other molecules for neutrophil infiltration and activation, the pro-inflammatory cytokine IL-6, and C3. GF(TGF-ß) up-regulated the expression of cell growth-related molecules, ECM components, several types of collagen, and cell adhesion molecules and down-regulated the expression of molecules related to complement activation and bacterial recognition. GFs stimulated by 4 cytokines responded differently to LPS. CONCLUSION: These results show that Th cytokines can polarize GFs into cells with functionally distinct features: immune-activating but tissue-destructive GF(IFN-γ), tissue-reparative, and immune-inhibiting GF(IL-4), highly pro-inflammatory GF(IL-17), and potent tissue-reparative GF(TGF-ß).

[An integrated approach to reducing hyperesthesia of teeth in patients with underlying somatic pathology]. / Kompleksnyi podkhod v reabilitatsii patsientov s giperesteziei tverdykh tkanei zubov na fone somaticheskikh zabolevanii.

THE AIM OF THE STUDY: Was to analyze the effectiveness of therapeutic and preventive measures aimed at reducing hyperesthesia of hard dental tissues in patients with background somatic diseases. MATERIALS AND METHODS: The study involved 113 patients with increased tooth sensitivity and treated in the gastroenterological and endocrinological departments of the S.M. Kirov City Clinical Hospital No.3¼ in Astrakhan in the period from 2018 to 2021 at the age of 26-43 years. The main group included 52 patients with confirmed diagnoses of gastric and duodenal ulcer, pancreatitis and type II diabetes mellitus who were treated for dental hyperesthesia with an integrated approach. The control group included 61 patients with periodontal disease without background somatic pathologies in whom hyperesthesia was treated by remineralizing therapy. The effectiveness of the treatment was determined in dynamics on the 10th and 40th days of treatment using OHI-S, PMA indices, dental hypersensitivity prevalence (DHP), dental hypersensitivity intensity (DHI), Dental Sensitivity Index (DSI), Efficacy of Dental Sensitivity Index (EDSI). In addition, the pH of saliva, the activity of lysozyme and S-IgA, and the levels of cytokines IL-1ß, IL-4, IL-6, and IL-8 were determined. RESULTS: The average value of OHI-S in the main group on the 10th day of treatment decreased from 2.25±0.12 (poor level of hygiene) to 1.47±0.09 (satisfactory level). The PMA index in the main group also tended to decrease from 32.1±1.44% (moderate degree of gingivitis) to 20.5±2.08% (mild degree) on the 10th day of treatment. The average values of DPH, DPI, EDSI and DSI in the main group had a noticeable decrease already on the 10th day from the start of treatment (from 12.3±1.66% to 2.1±1.22%; from 2.5±0.48 to 1.2±0.16; from 48.3±1.14% to 40.8±1.71%; from 42.1±2.07% to 20.8±1, 65% respectively). In the main group on the 10th and 40th day of treatment the pH values of non-stimulated and stimulated saliva stabilized (from 4.61±0.12 to 6.94±0.07 and from 5.47±0.21 to 7.42±0.24, respectively), the activity of lysozyme increased (from 45.97±1.46% to 55.19±0.96%) alongside with secretory IgA (from 0.17±0.02 to 0.33±0.21 mg/ml). Also, indicators of cytokines IL-1ß, IL-4, IL-6, IL-8 tended to improve. The analysis of the control group revealed persistent mean values that did not yield to significant changes either in the course of treatment. CONCLUSION: Thus, in patients of the main group, the results obtained indicate an improvement in the dental status and activation of cytokine regulation, providing a combination of active components of the mineral complex. In controls the method of remineralizing therapy for tooth hyperesthesia alleviated dental hypersensitivity, but without significant improvement of the laboratory results.

IFN-γ or IL-4 polarization impacts the response of gingival fibroblasts to oral bacteria.

BACKGROUND AND OBJECTIVE: We previously reported that gingival fibroblasts (GFs) can be polarized into functionally distinct subtypes, immune-activating but tissue-destructive or tissue-reparative, in response to T helper (Th1) and Th2 stimuli, respectively. The purpose of this study was to evaluate the effect of polarization on GFs responses to oral bacteria. METHODS: Unprimed (GF(-)) and IFN-γ (GF(IFN-γ)) or IL-4 primed (GF(IL-4)) GFs were stimulated with live Fusobacterium nucleatum or Porphyromonas gingivalis. The mRNA expression of IL-1ß, IL-4, LPS-recognizing components (Toll-like receptor (TLR) 4, CD14), molecules involved in antigen presentation (human leukocyte antigen (HLA)-ABC, HLA-DP, CD74, CD40), chemokines (C-X-C motif chemokine (CXCL)10, CXCL11, chemokine (C-C motif) ligand 20 (CCL20)), collagen type 1 alpha 1 (COL1A1), and matrix metalloproteinase (MMP)-1, and the protein levels of IL-1ß, CD14, CXCL11, CCL20, and COL1A1 accumulated in supernatants were analyzed using real-time PCR and ELISA. RESULTS: In response to oral bacteria, the GF(IFN-γ) significantly upregulated the expression of LPS-recognizing components, molecules involved in antigen presentation, CXCL10, and CXCL11, whereas the levels of IL-4 and COL1A1 were downregulated, compared with GF(-). The levels of IL-1ß, CCL20, and MMP-1 from GF(IFN-γ) were differently regulated between both bacteria; F. nucleatum was synergistically upregulated, but P. gingivalis was downregulated. The GF(IL-4) stimulated with both bacteria upregulated the levels of IL-4, whereas the levels of TLR4 and chemokines were downregulated, compared with GF(-). The regulation of IL-1ß, CD14, CXCL11, CCL20, and COL1A1 proteins showed a similar tendency with mRNA regulation. CONCLUSION: Polarization of GFs with IFN-γ or IL-4 affected the way that GFs responded to oral bacteria through up or downregulation of inflammatory responses and extracellular matrix control.

Salivary and Serum Interferon-Gamma/Interleukin-4 Ratio in Oral Lichen Planus Patients: A Systematic Review and Meta-Analysis.

Background and Objectives: Interferon-gamma (IFN-γ)/interleukin-4 (IL-4) ratio may indicate a change in the immune response with a potential pathological effect presented in oral lichen planus (OLP) patients. Herein, this meta-analysis evaluated the role of serum and salivary interferon-gamma/interleukin-4 ratio in the severity and development of OLP. Materials and Methods: The Scopus, Cochrane Library, PubMed, and Web of Science databases were systematically searched to retrieve the relevant studies published up from the database inception to March 2019. The crude mean difference (MD) and 95% confidence interval (CI) were calculated by RevMan 5.3 software using a random-effects model. A sensitivity analysis was performed on the results using the CMA 2.0 software. A total of 98 studies were retrieved from the databases, of which at last seven studies were included in this meta-analysis. Results: The findings showed that the pooled MDs of serum and salivary IFN-γ/IL-4 ratio were -0.22 (95% CI: -1.16, 0.72; p = 0.64) and 0.17 (95% CI: -1.50, 1.84; p = 0.84) in OLP patients compared to controls, respectively. In addition, the pooled MDs of serum and salivary IFN-γ/IL-4 ratio were -0.15 (95% CI: -0.53, 0.23; p = 0.43) and -0.39 (95% CI: -0.63, -0.15; p = 0.001) in patients with erythematous/ulcerative subtype compared to patients with reticular subtype, respectively. Conclusions: In conclusion, the results of meta-analysis demonstrated that serum and salivary IFN-γ/IL-4 ratio cannot play a major role in OLP development and severity.

Effects of new over-the-counter periodontal ointment-containing applicator with single-tuft brush on cytokine levels in gingival crevicular fluid during supportive periodontal therapy phase: a randomized double-blind clinical trial.

BACKGROUND AND OBJECTIVE: The biochemical effects of an over-the-counter (OTC) medication were studied, which consists of a single-tuft brush containing cetylpyridinium chloride as a bactericidal agent, dipotassium glycyrrhizate as an anti-inflammatory drug and allantoin as a promoter of cell proliferation and wound healing, for delivery to hardly brushed sites. MATERIAL AND METHODS: This randomized controlled double-blind study was performed in 61 subjects with chronic periodontitis in supportive periodontal therapy phase (test group: n = 27; placebo group: n = 28; dropout: n = 6). The OTC medication was self-applied twice a day for 12 wk to two molars with probing pocket depths of 4-6 mm. Biochemical indicators were evaluated at baseline and 12 wk using the suspension array system for eight cytokines and chemokines (interleukin [IL]-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-10, monocyte chemoattractant protein-1 and tumor necrosis factor [TNF]-α) in gingival crevicular fluid. RESULTS: The levels of IL-1ß, IL-6, IL-8 and TNF-α remained significantly lower in the test group compared to the placebo group. In the placebo group, when the probing pocket depth at baseline was 4 mm, IL-1ß increased, particularly in the second molar tooth, and the greatest increase was seen when PPD at baseline was 5-6 mm. In the test group, IL-1ß decreased markedly in cases with furcation involvement and low bleeding on probing at baseline. In both groups, IL-1ß, IL-6 and TNF-α were closely correlated with each other. CONCLUSION: This OTC medication is biochemically effective for steady chronic periodontitis in the supportive periodontal therapy phase.

Cytokine levels in plasma and gingival crevicular fluid in chronic periodontitis.

PURPOSE: To evaluate the Th1/Th2/Th17 cytokine levels in plasma and gingival crevicular fluid (GCF) from chronic periodontitis patients and healthy controls. METHODS: The concentration of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17, TNF, and IFN-γ were determined using a flow cytometric multiplex immunoassay (CBA), and was compared between the periodontitis group and the healthy group. Spearman rho coefficient was used to correlate cytokines in GCF in the periodontitis group and the healthy group, respectively. RESULTS: Comparisons of two groups of Th1/Th2/Th17 cytokine levels in plasma and GCF showed no statistically significant differences (P > 0.05), except Th17 (IL-17) level in plasma that was higher in the periodontitis group than the healthy group (P < 0.05). A stronger correlation between IL-17/IL-4 and IL-17/IL-10 was observed in periodontitis patients than in healthy controls.

Cytokine measurements in gingival crevicular fluid and periodontal ligament: Are they correlated?

INTRODUCTION: Mechanical stress can induce molecular changes in gingival crevicular fluid (GCF) and the periodontal ligament (PDL). It is still not clear whether changes in the PDL and GCF are linked. In this study, we aimed to analyze the expression of cytokines in GCF and PDL after mechanical stress. METHODS: Twenty-three healthy patients were included. The experimental group consisted of premolars subjected to a force of 0.980 N for 1, 3, 7, 14, 21, or 28 days. The contralateral teeth were the controls. GCF and PDL samples were collected at the same time points for analysis of cytokines using the cytometric bead array. RESULTS: Interleukin (IL)-6 (IL-6) production was significantly elevated in the PDL on day 1 after force application. Significantly strong positive correlations between GCF and PDL in experimental group were seen on days 3 (interferon-gamma), 7 (IL-10), 14 (IL-17A), and 28 (IL-17A, tumor necrosis factor-alpha), and significantly strong negative correlation were seen on days 14 (interferon-gamma) and 21 (IL-2, IL-10). CONCLUSIONS: Different patterns of IL-6 expression were seen in the PDL and GCF after mechanical stress. Despite occasional correlations between GCF and PDL, the molecular contributions of the PDL to the GCF changes could not be clearly defined by our model.

Influence of type 2 diabetes on local production of inflammatory molecules in adults with and without chronic periodontitis: a cross-sectional study.

BACKGROUND: Pathological changes in periodontal tissues are mediated by the interaction between microorganisms and the host immune-inflammatory response. Hyperglycemia may interfere with this process. The aim of this study was to compare the levels of 27 inflammatory molecules in the gingival crevicular fluid (GCF) of patients with type 2 diabetes, with and without chronic periodontitis, and of chronic periodontitis subjects without diabetes. A putative correlation between glycated haemoglobin (HbA1c) and levels of the inflammatory molecules was also investigated. METHODS: The study population comprised a total of 108 individuals, stratified into: 54 with type 2 diabetes and chronic periodontitis (DM + CP), 30 with chronic periodontitis (CP) and 24 with type 2 diabetes (DM). Participants were interviewed with the aid of structured questionnaire. Periodontal parameters (dental plaque, bleeding on probing and periodontal pocket depth) were recorded. The GCF levels of the 27 inflammatory molecules were measured using multiplex micro-bead immunoassay. A glycated haemoglobin (HbA1c) test was performed for patients with diabetes by boronate affinity chromatography. RESULTS: After adjustment for potential confounders, the DM + CP group had higher levels of IL-8 and MIP-1ß, and lower levels of TNF-α, IL-4, INF-γ, RANTES and IL-7 compared to the CP group. Moreover, the DM + CP group had lower levels of IL-6, IL-7 and G-CSF compared to the DM group. The DM group had higher levels of IL-10, VEGF, and G-CSF compared to the CP group. The levels of MIP-1α and FGF were lower in diabetes patients (regardless of their periodontal status) than in chronic periodontitis subjects without diabetes. Diabetes patients (DM + CP and DM) had higher Th-2/Th-1 ratio compared to the CP group. HbA1c correlated positively with the pro-inflammatory cytokines (Pearson correlation coefficient = 0.27, P value: 0.02). CONCLUSION: Type 2 diabetes and chronic periodontitis may influence the GCF levels of inflammatory molecules synergistically as well as independently. Type 2 diabetes was associated with high Th-2/Th-1 ratio, and modulated the local expression of molecules involved in the anti-inflammatory and healing processes.

Gingival crevicular fluid levels of interferon-γ, but not interleukin-4 or -33 or thymic stromal lymphopoietin, are increased in inflamed sites in patients with periodontal disease.

OBJECTIVE: To investigate the hypothesis that levels of interferon (IFN)-γ and interleukin (IL)-4, as well as the newer cytokines IL-33 and thymic stromal lymphopoietin (TSLP), in gingival crevicular fluid (GCF) samples differ from sites of patients at various clinical stages of periodontal disease and controls. BACKGROUND: Periodontal diseases result from the complex interplay between pathogenic bacteria and the host's immune responses. Several inflammatory mediators, such as IFN-γ and IL-4, have been detected in GCF samples in patients with periodontitis, but the results are mostly contradicting due to the lack of uniformity and collection of sites and methods of analysis. MATERIAL AND METHODS: GCF samples were collected from sites with different clinical characteristics (healthy, gingivitis and periodontitis sites) from periodontally healthy ( n = 14), plaque-induced gingivitis (n = 17) and chronic periodontitis (n = 11) subjects. The GCF samples were analyzed for the frequency of detection and levels of IFN-γ, IL-4, IL-33 and TSLP using a multiplex bead immunoassay. RESULTS: Inflamed sites in both patients with plaque-induced gingivitis and chronic periodontitis showed statistically significantly higher volume of GCF compared to non-inflamed sites in all patients. IFN-γ could be detected in about 50-70% of the samples analyzed and at significantly higher levels in sites with periodontitis compared to healthy sites in patients with chronic periodontitis (p = 0.035). We also show a statistically significant decrease of IFN-? in healthy sites of patients with chronic periodontitis as compared to gingivitis sites in patients with plaque-induced gingivitis (p = 0.047). Only some of the GCF samples showed detectable levels for IL-4 and TSLP, while IL-33 was below the detection level in all samples collected. CONCLUSIONS: These results suggest that IFN-γ levels in GCF depend on the clinical stage of the site and not on the disease stage of the patient, but need to be expanded to a greater number of subjects and additional analysis of corresponding gingival tissue biopsies for cytokine gene expression.

Natural killer T cells mediate alveolar bone resorption and a systemic inflammatory response in response to oral infection of mice with Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: T and B cells are known to be involved in the disease process of periodontitis. However, the role of natural killer T cells in the pathogenesis of periodontitis has not been clarified. MATERIALS AND METHODS: To examine the role of these cells, C57BL/6J (wild-type), CD1d(-/-) and α-galactosylceramide (αGC)-stimulated wild-type mice were orally infected with Porphyromonas gingivalis strain W83. RESULTS: Apart from CD1d(-/-) mice, the level of alveolar bone resorption was elevated by the infection and was further accelerated in αGC-stimulated mice. The infection induced elevated levels of serum amyloid A and P. gingivalis-specific IgG in the sera, although the degree of elevation was much smaller in the CD1d(-/-) mice. Infection-induced RANKL elevation was only observed in αGC-stimulated mice. Although the cytokines produced by splenocytes were mainly T-helper 1 type in wild-type mice, those in αGC-stimulated mice were predominantly T-helper 2 type. In the liver, the infection demonstrated no effect on the gene expression for interferon-γ, interleukin-4 and RANKL except αGC-stimulated mice in which the infection upregulated the gene expressions. CONCLUSION: This study is the first to show that natural killer T cells upregulated systemic and local inflammatory responses induced by oral infection with P. gingivalis, thereby contributing to the progression of alveolar bone resorption.

Immunological and microbiological findings after the application of two periodontal surgical techniques: a randomized, controlled clinical trial.

AIM: To examine microbiological and immunological alterations following two periodontal surgical techniques, over a 6-month period. MATERIALS AND METHODS: A total of 30 chronic periodontitis patients participated in the present randomized controlled clinical trial and were randomized in two groups. Modified Widman flap (MWF) was applied in the control group and apically positioned flap (APF), without intervention to the bone, in the experimental group. Gingival crevicular fluid samples and subgingival plaque samples from the operated sites were collected at baseline, 6th, 12th and 24th post-operative week. RESULTS: No major differences were noticed in immunological and microbiological profile of patients receiving either modified MWF or APF, for a period of 6 months. CONCLUSIONS: The choice of the periodontal surgical procedure does not seem to affect the immunological and the microbiological profile of patients with chronic periodontitis.

Photodynamic therapy in the treatment of class II furcation: a randomized controlled clinical trial.

AIM: To investigate the effect of photodynamic therapy (PDT) as adjunct to mechanical therapy in furcations. MATERIALS AND METHODS: A double-blind, parallel, randomized controlled clinical trial was conducted in subjects presenting class II furcations. The subjects were randomly allocated to a test (PDT; n = 16) or control group (non-activated laser/only photosensitizer; n = 21). At baseline, 3 and 6 months, clinical, microbiological and cytokine pattern evaluation was performed. Clinical attachment level was defined as the primary outcome variable. RESULTS: Clinical parameters improved after both therapies (p < 0.05) with no differences between groups at any time point (p > 0.05). At 6 months, real-time PCR evaluation showed a decrease in Porphyromonas gingivalis and Tannerella forsythia only in the PDT group (p < 0.05) with no inter-group differences. Regarding cytokines, IL-4 and IL-10 levels increased in both groups at 6 months. GM-CSF, IL-8, IL-1ß and IL-6 levels decreased only in the PDT group after 3 months (p < 0.05). At 3 months, inter-group analyses showed that GM-CSF, IFN-γ, IL-6 and IL-8 levels were lower in the PDT group. At 6 months, lower IL-1ß levels were also observed in the PDT group (p < 0.05). CONCLUSION: Photodynamic therapy did not promote clinical benefits for class II furcations; however, advantages in local levels of cytokines and a reduction in periodontopathogens were demonstrated.

Partial- and full-mouth scaling and root planing in type 2 diabetic subjects: a 12-mo follow-up of clinical parameters and levels of cytokines and osteoclastogenesis-related factors.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the effects of full-mouth scaling and root planing (FMSRP) and partial-mouth scaling and root planing (PMSRP), up to 12 mo after treatment, on clinical parameters, and levels of cytokines and osteoclastogenesis-related factors in type 2 diabetic subjects with chronic periodontitis. MATERIAL AND METHODS: Thirty-four subjects received FMSRP (n = 17) or PMSRP (n = 17) within 24 h or in multiple sessions, respectively. Clinical parameters and local levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-17, IL-23, IL-4, receptor activator of NF-ß ligand and osteoprotegerin were assessed at baseline, and 3, 6 and 12 mo after therapies. RESULTS: Clinical parameters improved after both therapies (p < 0.05), and no between-group differences were observed at any time-point (p > 0.05). Overall, there were no considerable differences in the local levels of the biomarkers studied between groups (p > 0.05). The IL-23 concentration and total amount of IFN-γ increased in the FMSRP group and decreased in the PMSRP group from baseline to 3 mo and from baseline to 6 mo, respectively (p < 0.05). CONCLUSION: Both PMSRP and FMSRP promoted benefits in clinical parameters and showed a similar modulation of cytokines and osteoclastogenesis-related factors at 12 mo in type 2 diabetic subjects.

Oral malodorous compound causes caspase-8 and -9 mediated programmed cell death in osteoblasts.

BACKGROUND AND OBJECTIVE: Hydrogen sulfide (H(2) S) is one of two volatile sulfur compounds that are known to be the main cause of oral malodor; the other is methyl mercaptan. Other known volatiles existing in mouth air do not contribute significantly to oral malodor originating in the oral cavity. Hydrogen sulfide is also known to be an etiological factor in periodontal disease. However, the effects of H(2) S on alveolar bone remain unclear. The objectives of this study were to determine the apoptotic effects of H(2) S on osteoblasts and to verify the apoptotic molecular pathways. MATERIAL AND METHODS: A clonal murine calvaria cell line was incubated with 50 ng/mL of H(2) S. To detect apoptosis, the cells were analysed by flow cytometry and ELISA. Mitochondrial membrane depolarization was assessed using flow cytometry as well. ELISA was used to evaluate the release of cytochrome c into the cytosol and to assess Fas ligand, p53, tumor necrosis factor α, interleukin IL1-α IL-ß, IL-2, IL-4, IL-10, interferon-γ, granulocyte-colony stimulating factor and granulocyte-macrophage colony stimulating factor. Caspase-3, -8 and -9 activities were estimated. Expression of BAX and Bcl-2 was assessed by real-time quantitative RT-PCR. DNA fragmentation was detected by single-cell gel electrophoresis. Fas receptors were evaluated by western blotting. RESULTS: After H(2) S incubation, apoptotic levels increased significantly in a time-dependent manner. Mitochondrial membrane depolarization, the release of cytochrome c, p53 and caspase-3, -8 and -9 and DNA fragmentation were all significantly greater. BAX gene activity was upregulated, whereas Bcl-2 remained low. Fas ligand/Fas receptor, tumor necrosis factor α and other cytokines were not increased to a significant degree. CONCLUSION: At less-than-pathological concentrations in gingival crevicular fluid, H(2) S induces apoptosis in osteoblasts. The molecular mechanisms underlying the apoptotic process include p53, a mitochondrial pathway and caspase-8 activation.

Does cytokine profiling of aspirate from jaw cysts and tumors have a role in diagnosis?

PURPOSE: The objective of the present study was twofold: first, to assess aspirates for use in cytokine profiling and second, to initiate pilot analyses to determine whether the cytokine profiling can serve as an aid in the diagnosis of jaw lesions. MATERIALS AND METHODS: The aspirates from 12 benign odontogenic cysts and tumors of the jaw were collected and randomized, and a formal incisional biopsy was performed to establish the tissue diagnosis. The biopsies revealed keratocystic odontogenic tumor, ameloblastoma, and dentigerous cyst. The cystic aspirate was analyzed using the Q-Plex Human Cytokine Screen to detect cytokine expression and determine the level of expression for each pathologic entity. An array of 16 cytokines was investigated, including interleukin (IL)-1α, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, interferon-γ, tumor necrosis factor (TNF)-α, and TNF-ß. Tables were developed to determine the ratio of expression for the candidate cytokine pairs that were differentially expressed among the 3 pathologic entities encountered. One-way analysis of variance was used to search for significant differences in the ratio of expression of the candidate pairs among the 3 entities. RESULTS: Cytokines expressed by the 3 distinct jaw lesions were detected in the aspirate without the need for tissue biopsy. Cytokine profiling of these entities is possible owing to differential expression of the various cytokines studied. The ratio of expression was significant (P < .05) for 15 pairs of cytokines: IL-5/IL-1α, IL-4/IL-2, IL-8/IL-4, TNF-ß/IL-6, IL-23/IL-6, TNF-α/IL-23, TNF-α/TNF-ß, TNF-α/IL-8, TNF-ß/IL-5, TNF-ß/TNF-α, TNF-ß/IL-13, IL-12/IL-23, IL-13/IL-15, IL-15/IL-2, and IL-6/IL-2. A comparison of the mean values indicated a "high/low" expression value for each lesion type for the 15 cytokine pairs. CONCLUSIONS: Cytokines, expressed by the 3 groups of jaw lesions, can be detected in the cystic aspirate, and a comparison of the ratio of the expression of the aspirates demonstrated a differential expression pattern of cytokines among the 3 groups. These ratios could assist in establishing a prompt and accurate diagnosis of lesions that might be difficult to discern clinically and radiographically. The use of a simple, minimally invasive aspiration procedure can help to establish an accurate diagnosis.

Cytokine expression in response to root canal infection in gnotobiotic mice.

AIM: To examine cytokine expression profiles during periapical lesion development in response to synergetic human pathogens in a gnotobiotic mouse model. METHODOLOGY: Human strains of Fusobacterium nucleatum and Peptostreptococcus prevotii were inoculated into the root canals of germ-free mice in either mono- or bi-association. Animals were killed 7 and 14 days after infection, and periapical tissues were collected. mRNA expression of the cytokines IFN-γ, TNF-α, Receptor activator of nuclear factor kappa-B ligand (RANKL), IL-10, IL-4 and transforming growth factor ß (TGF-ß) was assessed using real-time PCR. Levene's test was used to assess the equality of variance of the data, whereas a t-test for independent samples was used to evaluate the significance of the differences between groups (P < 0.05). RESULTS: The mRNA expression of IFN-γ and TNF-α was up-regulated by F. nucleatum during the acute (day 7) and chronic phase (day 14) of periapical lesion development. However, in bi-infection the expression of IFN-γ and TNF-α were effectively absent at both time-points. RANKL mRNA expression was down-regulated during dual infection at the chronic phase. As IL-4 expression was similar at both time-points, IL-4 does not appear to be involved in the periapical response to these bacterial strains. IL-10 was up-regulated during the chronic phase by mono-infection with either F. nucleatum or P. prevotii. Dual infection increased TGF-ß mRNA expression on day 7, which paralleled the decrease in IFN-γ and TNF-α mRNA levels at the same time-point. F. nucleatum increased TGF-ß mRNA expression during the chronic phase. CONCLUSION: Cytokine profiles depend on the nature of the bacterial challenge. Both TGF-ß and IL-10 appeared to be regulating the proinflammatory cytokine responses at both time-points of the periapical immune response.

T-helper-related cytokines in gingival crevicular fluid from adolescents with Down syndrome.

Subjects with Down syndrome have a high prevalence of periodontal disease. The aim was to investigate the level of Th1-, Th2- and Th17-related cytokines in the gingival crevicular fluid (GCF) of subjects with Down syndrome. Subjects with Down syndrome (n = 24) and controls (n = 29) with a mean age of 16.4 years were clinically examined with respect to periodontal probing depth (PD) and gingival inflammation in terms of bleeding on probing (BOP%). The controls were matched to subjects with Down syndrome regarding age and gingival inflammation (BOP%). All subjects answered a questionnaire regarding oral hygiene, medical history and socioeconomic background. GCF was collected and the concentration of the cytokines, IFN-γ, TNF-α, IL-1ß, IL-4, IL-6, IL-10, IL-12 and IL-17 were determined using Bio-Plex cytokine multiplex assays. The volume of GCF (microliters) was significantly higher in subjects with Down syndrome (P < 0.001) compared with controls. The mean concentrations (picogrammes per millilitre) of IL-1ß (P < 0.001), IL-4 (P = 0.002), IL-6 (P = 0.005), IL-10 (P = 0.001), IL-12 (P = 0.003), IFN-γ (P = 0.002), and TNF-α (P = 0.002) in GCF, respectively, were significantly higher in subjects with Down syndrome compared with controls. The regression line of the relationship between IFN-γ and IL-4 in GCF differed significantly (P = 0.006) in subjects with Down syndrome compared to controls. Subjects with Down syndrome demonstrated higher concentration of Th1-, Th2- and Th17-related cytokines with an altered relationship between Th1 cytokine, IFN-γ and Th2 cytokine, IL-4, in volume GCF compared to controls.

Cytokine profile in serum and gingival crevicular fluid of children with inflammatory bowel disease: A case-control study.

BACKGROUND: To evaluate the cytokine profile in gingival crevicular fluid (GCF) and serum of pediatric inflammatory bowel disease (IBD) patients and determine the cluster patterns of cytokines. METHODS: Fifty IBD patients and 21 systemically healthy children were enrolled in the study. The GCF samples were collected from the participants during periodontal examination and periodontal indices were recorded. Based on activity indexes and response to conventional treatment, patients with IBD were further categorized into subgroups as: remission, active disease, and treatment-resistant. Serum samples were obtained from IBD patients to determine serum levels of cytokines. The levels of pro- (interleukin (IL)-1ß, IL-12, IL-21, IL-22, IL-23, IL-17A, IL-17F) and anti-inflammatory (IL-4, IL-10) cytokines in serum and GCF were measured using Enzyme-linked Immunosorbent Assay (ELISA) kits. RESULTS: Among 50 IBD patients, 58% were in remission, 20% had active disease, and 22% were defined as treatment-resistant. The severity of gingival inflammation measured by the criteria of Löe had increasing trends in IBD patients with active disease and treatment resistance. GCF IL-1ß level was lower and GCF IL-4 and GCF IL-23 levels were higher in IBD patients compared to healthy controls. In the active disease group, more cytokine clusters occurred compared to the control group and other IBD subgroups, as explained by increased cytokine-cytokine interactions. CONCLUSIONS: Considering the increased complexity of cytokine interactions and the increased severity of gingival inflammation in patients with active disease, it can be concluded that disease activity might have an impact on gingival inflammation in pediatric patients with IBD.

Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis.

AIM: The aim of this study was to compare the expression of 22 chemokines and cytokines in gingival crevicular fluid (GCF) from smokers and non-smokers with periodontitis and periodontally healthy control subjects. MATERIALS AND METHODS: Forty subjects with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy control subjects participated in this study. Four diseased and two healthy sites were selected from each of the periodontitis subjects. GCF samples were collected and cytokines analysed utilizing a multiplexed immunoassay (Luminex(®) ). Statistical analyses employed non-parametric tests including the Mann-Whitney and Wilcoxon matched-pairs signed-rank tests. RESULTS: Compared with healthy control subjects, GCF in subjects with chronic periodontitis contained significantly higher amounts of interleukin (IL)-1α, IL-1ß, IL-6, IL-12(p40) (pro-inflammatory cytokines); IL-8, macrophage chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, regulated on activation normal T-cell expressed and secreted (RANTES) (chemokines); IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-15 [regulator of T-cells and natural killer (NK) cells]. Smokers displayed decreased amounts of pro-inflammatory cytokines [IL-1α, IL-6, IL-12(p40)], chemokines (IL-8, MCP-1, MIP-1, RANTES), and regulators of T-cells and NK cells (IL-7, IL-15). CONCLUSIONS: Periodontitis subjects had significantly elevated cytokine and chemokine profiles. Smokers exhibited a decrease in several pro-inflammatory cytokines and chemokines and certain regulators of T-cells and NK-cells. This reflects the immunosuppressant effects of smoking which may contribute to an enhanced susceptibility to periodontitis.

Distinct Th1, Th2 and Treg cytokines balance in chronic periapical granulomas and radicular cysts.

BACKGROUND: Periapical lesions are a host response that involves immune reaction to prevent dissemination of bacteria from an infected root canal. The purpose of this study was to evaluate the levels of nitric oxide (NO), IL-4, TGF-beta, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) in chronic periapical lesions and to determine their possible association with clinical and radiographic parameters. METHODS: Seventeen human radicular cysts and 30 periapical granulomas were used in this study. Cytokines and NO were assessed by enzyme-linked immunosorbent assay and by the Griess reaction respectively confirmed by immunohistochemical. RESULTS: TNF-alpha and IFN-gamma were detected in 10% of granulomas and in 41.2% and 70% of radicular cysts. IL-4 was reactive in 24% of cysts, and TGF-beta was positive in all samples. Patients with tenderness showed significantly higher levels of IFN-gamma and IL-4 (P < 0.05). Swelling was associated with high levels of TNF-alpha, IFN-gamma, and IL-4 (P < 0.05). Lesions presenting bone resorption were associated with high levels of NO (P < 0.05). CONCLUSIONS: Periapical granulomas display a regulatory environment characterized by high TGF-beta and low inflammatory cytokine levels, while radicular cysts has mist Th1 and Th2 inflammatory reaction with the presence of IFN-gamma, TNF-alpha, and IL-4.