Specificity of Antimicrobial Peptide LL‐37 to Neutralize Periodontopathogenic Lipopolysaccharide Activity in Human Oral Fibroblasts

Background: The antimicrobial peptide LL‐37 is known to have a potent lipopolysaccharide (LPS)‐neutralizing activity in various cell types. Because of observed heterogeneity within periodontopathogenic LPS, the authors hypothesized that LL‐37 had specificity to neutralize such LPS activity. The present study, therefore, aims to investigate the LPS‐neutralizing activity of LL‐37 to various periodontopathogenic LPS in interleukin‐8 (IL‐8) production after challenging them in human oral fibroblasts. Methods: Human periodontal ligament fibroblasts (PDLF) and gingival fibroblasts (GF) were cultured from biopsies of periodontal ligament and gingival tissues. After cell confluence in 24‐well plates, LPS (10 μg/mL) from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans were added with or without LL‐37 (10 μg/mL). After 18 hours, the supernatant was collected and analyzed in IL‐8 production by enzyme‐linked immunosorbent assay. Results: All periodontopathogenic LPS statistically significantly induced IL‐8 production in both PDLF and GF (P <0.01). After neutralization with LL‐37, both PDLF and GF showed a statistically significant reduction in IL‐8 production compared with LPS‐treated groups without LL‐37 (P <0.01), and the percentage of reduction in IL‐8 production in PDLF appeared to be higher than in GF. In addition, the percentage of reduction in IL‐8 production varied considerably according to each periodontopathogenic LPS. Conclusions: The antimicrobial peptide LL‐37 had an ability to suppress periodontopathogenic LPS‐induced IL‐8 production in both PDLF and GF. Its LPS‐neutralizing activity revealed specificity to periodontopathogenic LPS and seemed to be dependent on the heterogeneity within LPS between different genera.     

Effect of nonsurgical periodontal therapy on serum and gingival crevicular fluid cytokine levels during pregnancy and postpartum

Background and Objective: A low‐grade systemic inflammatory status originating from periodontal infection has been proposed to explain the association between periodontal disease and systemic conditions, including adverse obstetric outcomes. The aim of this study was to evaluate the effect of periodontal therapy during pregnancy on the gingival crevicular fluid and serum levels of six cytokines associated with periodontal disease and preterm birth. Material and Methods: A subsample of 60 women (18–35 years of age) up to 20 gestational weeks, previously enrolled in a larger randomized clinical trial, was recruited for the present study. Participants were randomly allocated to receive either comprehensive nonsurgical periodontal therapy before 24 gestational weeks (n = 30, test group) or only one appointment for supragingival calculus removal (n = 30, control group). Clinical data, and samples of blood and gingival crevicular fluid, were collected at baseline, at 26–28 gestational weeks and 30 d after delivery. The levels of interleukin (IL)‐1β, IL‐6, IL‐8, IL‐10, IL‐12p70 and tumor necrosis factor‐α were analyzed by flow cytometry. Results: After treatment, a major reduction in periodontal inflammation was observed in the test group, with bleeding on probing decreasing from 49.62% of sites to 11.66% of sites (p < 0.001). Periodontal therapy significantly reduced the levels of IL‐1β and IL‐8 in gingival crevicular fluid (p < 0.001). However, no significant effect of therapy was observed on serum cytokine levels. After delivery, the levels of IL‐1β in the gingival crevicular fluid of the test group were significantly lower than were those in the control group (p < 0.001), but there were no significant differences between test and control groups regarding serum cytokine levels. Conclusion: Although periodontal therapy during pregnancy successfully reduced periodontal inflammation and gingival crevicular fluid cytokine levels, it did not have a significant impact on serum biomarkers.     

Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts

Background and Objective: Short‐chain fatty acids, such as butyric acid and propionic acid, are metabolic by‐products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. Material and Methods: Human GFs were exposed to various concentrations of butyrate (0.5–16 mm ) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide‐staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2’,7’‐dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT‐PCR using specific primers or to western blotting using specific antibodies, respectively. Results: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4–16 mm ) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm ) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT‐PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. Conclusion: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle‐related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.     

Tumor Necrosis Factor‐α and Interleukin (IL)‐1β, IL‐6, and IL‐8 Impair In Vitro Migration and Induce Apoptosis of Gingival Fibroblasts and Epithelial Cells,Delaying Wound Healing

Background: Multiple factors affect oral mucosal healing, such as the persistence of an inflammatory reaction. The present study evaluates effects of tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, IL‐6, and IL‐8 on epithelial cells (ECs) and human gingival fibroblasts (GFs) in vitro. Methods: GFs and ECs were seeded in 96‐well plates (1 × 10⁴ cells/well) in plain culture medium (Dulbecco’s modified Eagle’s medium [DMEM]) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum, and incubated for 24 hours. Both cell lines were exposed for 24 hours to the following cytokines: 1) TNF‐α (100 ng/mL); 2) IL‐1β (1 ng/mL); 3) IL‐6 (10 ng/mL); and 4) IL‐8 (10 ng/mL). All cytokines were diluted in serum‐free DMEM. Control cultures were exposed only to serum‐free DMEM. Effects of exposure to inflammatory cytokines were determined by means of: 1) apoptosis (anexin V); 2) cell migration (wound healing assay); 3) inflammatory cytokine synthesis (enzyme‐linked immunosorbent assay). Data were analyzed by Kruskal–Wallis and Mann–Whitney U tests (α = 0.05). Results: Increased apoptosis rates were noted when cells were exposed to inflammatory cytokines, except ECs exposed to IL‐1β. Cell migration was negatively affected by all inflammatory cytokines for both cell lines. ECs and GFs exposed to IL‐6 and IL‐8 significantly increased synthesis of TNF‐α and IL‐1β. Conclusions: Demonstrated results indicate negative effects of tested inflammatory cytokines on ECs and GFs, inducing apoptosis and impairing cell migration. These results can justify delayed oral mucosa healing in the presence of inflammatory reaction.     

Salivary Biomarkers Associated With Gingivitis and Response to Therapy

Background: Salivary biomarkers are potentially important for determining the presence, risk, and progression of periodontal disease. However, clinical translation of biomarker technology from lab to chairside requires studies that identify biomarkers associated with the transitional phase between health and periodontal disease (i.e., gingivitis). Methods: Eighty participants (40 with gingivitis, 40 healthy) provided saliva at baseline and 7 to 30 days later. An additional sample was collected from gingivitis participants 10 to 30 days after dental prophylaxis. Clinical parameters of gingival disease were recorded at baseline and the final visit. Salivary concentrations of interleukin (IL)‐1β, IL‐6, matrix metalloproteinase (MMP)‐8, macrophage inflammatory protein (MIP)‐1α, and prostaglandin E₂ (PGE₂) were measured. Results: Clinical features of health and gingivitis were stable at both baseline visits. Participants with gingivitis demonstrated significantly higher bleeding on probing (BOP), plaque index (PI), and gingival index (GI) (P ≤0.002) and a significant drop in BOP, PI, and GI post‐treatment (P ≤0.001). Concentrations of MIP‐1α and PGE₂ were significantly higher (2.8 times) in the gingivitis group than the healthy group (P ≤0.02). After dental prophylaxis, mean biomarker concentrations did not decrease significantly from baseline in the gingivitis group, although concentrations of IL‐1β, IL‐6, and MMP‐8 approached healthy levels, whereas MIP‐1α and PGE₂ concentrations remained significantly higher than in the healthy group (P ≤0.04). Odds ratio analyses showed that PGE₂ concentrations, alone and in combination with MIP‐1α, readily discriminated gingivitis from health. Conclusions: Salivary PGE₂ and MIP‐1α discriminate gingivitis from health, and patients with gingivitis who return to clinical health continue to produce inflammatory mediators for weeks after dental prophylaxis.     

Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis

Scheres N, Laine ML, de Vries TJ, Everts V, van Winkelhoff AJ. Gingival and periodontal ligament fibroblasts differ in their inflammatory response to viable Porphyromonas gingivalis. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01229.x © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Porphyromonas gingivalis is an oral pathogen strongly associated with destruction of the tooth‐supporting tissues in human periodontitis. Gingival fibroblasts (GF) and periodontal ligament fibroblasts (PDLF) are functionally different cell types in the periodontium that can participate in the host immune response in periodontitis. This study aimed to investigate the effects of viable P. gingivalis on the expression of genes associated with inflammation and bone degradation by these fibroblast subsets. Material and Methods: Primary human GF and PDLF from six healthy donors were challenged in vitro with viable P. gingivalis W83 for 6 h. Gene expression of inflammatory cytokines in GF and PDLF was analyzed using real‐time PCR, and protein expression was analyzed using ELISA. Results: Viable P. gingivalis induced a strong in vitro inflammatory response in both GF and PDLF. We found increased gene expression of interleukin (IL)‐1β, IL‐6, IL‐8, tumor necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cell expressed and secreted (RANTES). Macrophage colony‐stimulating factor was induced and the expression of osteoprotegerin was decreased in GF, but not in PDLF. In nonchallenged cells, a higher level of expression of IL‐6 was observed in GF than in PDLF. Between individual donors there was large heterogeneity in responsiveness to P. gingivalis. Also, in each individual, either GF or PDLF was more responsive to P. gingivalis. Conclusion: Considerable heterogeneity in responsiveness to P. gingivalis exists both between GF and PDLF and between individuals, which may be crucial determinants for the susceptibility to develop periodontitis.     

The relationship of gingival crevicular fluid short chain carboxylic acid concentration to gingival inflammation

Abstract Short-chain carboxylic acids (SCCA; C≤5: e.g., lactic acid, propionic acid, butyric acid) are metabolic by-products of bacterial metabolism which accumulate in the gingival crevice, and exhibit significant biological activity, including the ability to alter gene expression. It has been hypothesized that among the activities of SCCAs are their ability to contribute to gingival inflammation. This concept complements the notion that specific periodontal pathogens are the causative agents of gingival inflammation. To begin testing these 2 hypotheses, we examined the relationship between SCCA concentrations, specific putative periodontal pathogens, and gingival inflammation in medically healthy periodontally diseased subjects. We reasoned that if SCCAs and/or specific periodontal pathogens were causative gingival inflammatory agents, gingival inflammation should increase with increasing concentration of the inflammatory mediator. We also recognized that other clinical variables needed to be controlled for, and an objective quantitative assessment of gingival inflammation used. To accomplish these tasks, sites within subjects were stratified by location and pocket depth, and the following quantified: bacteria] presence; SCCA concentration: and gingival inflammation. The results indicated that gingival inflammation directly and significantly correlated with SCCA concentrations in the maxillary and mandibular molars, incisors and canines (all r≥0.47; all p≤ 0.015; too few bicuspids were available for complete analysis). The relationship between gingival inflammation and SCCA concentration was best described by a natural log relationship. Gingival inflammation did not, however, correlate positively with either the total number of specific putative periodontal pathogens, or the sum of subsets of these pathogens (?0.31 ≤r≤ 0.39; 0.08 ≤p 0.75) for any of the locations. Finally, the SCCA concentration did not correlate with the level of individual or groups of pathogens. These data, together with historical work and other preliminary data, support the hypothesis that SCCA, rather than specific putative periodontal pathogens, may be a causative agent in gingival inflammation. This work may, in part, begin to explain the apparent lack of a direct relationship between current gingival inflammation and the prediction of bacterially mediated periodontal attachment loss.     

Effect of periodontal treatment on IL-1beta, IL-1ra, and IL-10 levels in gingival crevicular fluid in patients with aggressive periodontitis

Aim: The aim of this study was to examine the effect of phase I periodontal treatment on the levels of interleukin (IL)‐1β, IL‐1ra, and IL‐10 in gingival crevicular fluid (GCF) in patients with generalized aggressive periodontitis (G‐AgP). Material and Methods: Data were obtained from 15 patients with aggressive periodontitis and 15 healthy controls. GCF was collected from at least four pre‐selected sites (one shallow, at least two moderate, or at least one deep pockets) in patients with G‐AgP. In the healthy group, GCF samples were collected from one site. The cytokine levels were determined by an enzyme‐linked immunosorbent assay. Probing depth, clinical attachment level (CAL), gingival and plaque indices, and bleeding on probing were measured. The GCF sampling and clinical measurements were recorded at baseline and 6 weeks later after periodontal treatment. Results: IL‐1β levels were significantly higher at the moderate and deep pocket sites compared with the shallow sites (p<0.05). After periodontal therapy, IL‐1β levels were significantly reduced in the moderate and deep pocket sites (p<0.05). IL‐1ra levels at baseline of the moderate and deep pocket sites were significantly lower than the control sites (p<0.05). IL‐10 levels were similar in all pockets and did not change after periodontal therapy. Conclusions: The periodontal treatment improves the clinical parameters in G‐AgP, and this improvement is evident in deep pocket sites for pocket depth and CAL values. These results confirm that IL‐1β is effective for evaluating the periodontal inflammation and can thus be used as a laboratory tool for assessing the activity of periodontal disease.     

Interleukin-11, interleukin-1beta, interleukin-12 and the pathogenesis of inflammatory periodontal diseases

Objectives: The balance between pro‐inflammatory and anti‐inflammatory cytokines may be crucial for determining the immunopathology of gingivitis (G) and periodontitis. This study aimed to analyse interleukin‐1β (IL‐1β), IL‐11 and IL‐12 levels in gingival crevicular fluid (GCF) of patients with G and chronic periodontitis (CP). Material and Methods: Fourty subjects including 12 CP, 14 G and 14 controls (C) were enrolled. GCF samples were collected from six maxillary sites per patient and analysed for IL‐1β, IL‐11 and IL‐12 by an enzyme‐linked immunosorbent assay. Results: Significantly lower concentrations of IL‐11 were detected in CP compared with both G and C groups (p<0.05). The CP group had a significantly higher total amount of IL‐12 and IL‐1β compared with the C group (p<0.05). The IL‐11:IL‐1β cytokine ratio was higher in both G and C groups compared with the CP group. The IL‐11:IL‐1β ratio became progressively lower with increasing probing depth (p<0.01). Conclusions: Our data showed that IL‐11 levels are significantly decreased in GCF from sites with periodontitis compared with G and healthy sites. Because of the possible preventive effect of IL‐11 on inflammation, IL‐11 may be an important factor in the therapeutic modulation of periodontal disease.     

The influence of academic stress on gingival inflammation

Abstract: Objective: The aim of the present study was to investigate the effects of academic stress on periodontal health, in relation to inflammatory markers in gingival crevicular fluid (GCF) and cortisol in saliva. Materials and methods: The study included 20 healthy dental hygienists (females: mean age 29.3 ± 8.5 SD) and was conducted during a major exam period and 4 weeks later after the exams. A clinical examination was performed and GCF was collected from four sites in each subject on these two occasions. Interleukin (IL)‐1β, IL‐4, IL‐6, IL‐10 levels were determined using Luminex 100 and cortisol amounts by radioimmunoassay (RIA ¹²⁵I). Students registered their perceived stress on a visual analogue scale (VAS). Significance of the findings was determined using paired t‐test, Wilcoxon‐matched pair and Spearman’s rank correlations. Results: Students had higher amounts of dental plaque (P < 0.007) and gingival inflammation (P < 0.001) during the exam period compared with after the exams. The amounts of IL‐6 and IL‐10 in GCF were significantly increased during the time of examinations. The median level of cortisol in saliva was also significantly raised during the exam period compared with after the exams, 20.52 nmol/l (range: 11.91–27.34) and 16.41 nmol/l (range: 10.91–24.17) respectively, P < 001. The results from the VAS registration revealed a significant difference (P < 001) between the two occasions. Conclusion: Academic stress appears to affect periodontal health, shown by more plaque accumulation, gingival inflammation and increased amounts of IL‐6, IL‐10 in GCF and cortisol in saliva.     

Interleukin‐1 β, interleukin‐12 and interleukin‐18 levels in gingival fluid and serum of patients with gingivitis and periodontitis

Introduction: Cytokines are of major importance in periodontal disease progression. Interleukin‐12 (IL‐12) stimulates interferon‐γ production by T helper type 1 (Th1) cells while IL‐18 induces Th1 responses when present with IL‐12 but Th2 responses in the absence of IL‐12. IL‐1β has been correlated with periodontal disease destruction. This study determined the local concentrations of these cytokines in sites of gingivitis and periodontitis. Methods: Gingival crevicular fluid was collected from two sites in each of 10 gingivitis patients and from two gingivitis sites and two periodontitis sites from each of 10 periodontitis patients. Serum samples were also collected. IL‐1β, biologically active IL‐12 p70, the IL‐12 p40 subunit and IL‐18 concentrations were determined by enzyme‐linked immunoabsorbent assay. Results: IL‐1β and IL‐18 concentrations were higher in the gingival crevicular fluid from periodontitis patients than in that from gingivitis patients; IL‐18 concentrations were higher than those of IL‐1β. Very little IL‐12, either p40 or p70, was detected in the gingival crevicular fluid samples. In the serum, very low levels of cytokines were found. The level of serum IL‐12 p40, however, was higher than in the fluid from periodontitis sites of periodontitis patients. Conclusion: The local production of IL‐1β and IL‐18 in the gingival crevicular fluid increased with increasing inflammation and IL‐18 was the predominant cytokine at both gingivitis and periodontitis sites. Very little IL‐12 was detected with levels decreasing with increasing inflammation. These results suggest that there is an association between severity of periodontal disease and levels of IL‐1, IL‐12 and IL‐18.     

Level of Interleukin‐35 in Gingival Crevicular Fluid,Saliva, and Plasma in Periodontal Disease and Health

Background: A novel member of the interleukin (IL)‐12 family, IL‐35 is an important inhibitory cytokine released by regulatory T cells. The aim of this study is to evaluate gingival crevicular fluid (GCF), saliva, and plasma levels of IL‐35 in periodontal disease and health. Methods: Samples of GCF, whole saliva, and plasma were obtained from systemically healthy, non‐smoking individuals with gingivitis (n = 20) or chronic periodontitis (CP) (n = 20) and periodontally healthy individuals (n = 20). Full‐mouth clinical periodontal measurements, including probing depth (PD), bleeding on probing, gingival index, and plaque index (PI), were also recorded. Enzyme‐linked immunosorbent assay was used to determine IL‐35 levels in the samples. Data were tested statistically by analysis of variance and Pearson rank correlation test. Results: All clinical parameters were significantly higher in the CP group than the healthy and gingivitis groups (P <0.001). The GCF total amount of IL‐35 was significantly higher in the CP group than the other groups (P = 0.04), whereas the GCF concentration of IL‐35 was significantly higher in the healthy group than the other groups (P = 0.002). There were significant differences among the study groups in terms of salivary IL‐35 level (P <0.001), with the highest level observed in the healthy group and the lowest in the CP group. There was no statistical difference between groups in plasma levels of IL‐35 (P >0.05). There was a positive correlation between GCF total amount of IL‐35 and PD (r = 0.338, P = 0.03) and PI (r = 0.374, P = 0.005) parameters. Conclusions: IL‐35 could have an important role in suppressing periodontal inflammation and maintaining periodontal health. Additional studies are required to evaluate its role in periodontal diseases.     

Bacterial and salivary biomarkers predict the gingival inflammatory profile

Background: The aim of this human investigation is to explore the relationship of gingivitis with salivary biomarkers, periodontal pathogens, and interleukin (IL)‐1 polymorphism after a transient inflammatory burden. Methods: Thirty healthy human participants were randomized by IL‐1 genotype status to control for potential influences of this particular single nucleotide polymorphism on the inflammatory profile. Oral hygiene practices ceased for 21 days to induce gingivitis (induction), after which home care was reinstated until 35 days (resolution). Clinical parameters included plaque (PI) and gingival (GI) indices and papillary bleeding score (PBS). Levels and proportions of 40 subgingival bacteria were determined using checkerboard DNA‐DNA hybridization. Saliva was analyzed using a multiplex protein array for 30 biomarkers associated with host defense, inflammation, tissue destruction, and angiogenesis. Results: Mean PI, GI, and PBS values were significantly increased during induction and decreased during resolution as measured at 35 days (P <0.01), although no differences were observed between IL‐1 groups. Participants were stratified as either “high” or “low” responders based on inflammatory response (high: GI >1.5; low: GI ≤1.5). Baseline levels of salivary IL‐6 and IL‐8 demonstrated the highest ability to discriminate between high and low responders (area under the curve [AUC] of 0.81 and 0.72, respectively). Salivary biomarkers, matrix metalloproteinases (MMPs), and bacterial biofilm were combined to generate receiver operating characteristic curves. High levels of IL‐6 and MMP‐1 at baseline demonstrated the strongest ability to predict high responders (AUC of 0.89; odds ratio of 17.0; 95% confidence interval, 1.7 to 171.7). Conclusion: In this proof‐of‐concept investigation, we identified specific biomarker and microbial signatures that are associated with gingival inflammation ( ClinicalTrials.gov number NCT00980525).     

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.     

Cimetidine Reduces Interleukin‐6, Matrix Metalloproteinases‐1 and ‐9 Immunoexpression in the Gingival Mucosa of Rat Molars With Induced Periodontal Disease

Background: Histamine seems to act, via H₂ receptor, on inflammatory processes by stimulating interleukin (IL)‐6 and matrix metalloproteinase (MMP) release. As cimetidine is an H₂ receptor antagonist, the authors hypothesize that this antiulcer drug reduces IL‐6, MMP‐1, and MMP‐9 immunoexpression in gingiva with induced periodontal disease (PD). To confirm a possible modulatory role of IL‐6 on MMPs, the relationship between IL‐6/MMP‐1 and IL‐6/MMP‐9 immunoexpression was evaluated. Methods: Forty‐six male rats were distributed into the cimetidine group (CimG: received daily intraperitoneal injections of 100 mg/kg of body weight of cimetidine) or the saline group (SG). PD was induced by cotton ligature around the maxillary left first molars (PDSG and PDCimG). The right molars were used as controls (SG and CimG). After 7, 15, 30, and 50 days, maxillary fragments were processed for paraffin embedding or for transmission electron microscopy. Tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in the alveolar process surface and number of IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells in the gingival mucosa were quantified. Statistical analyses were performed (P ≤0.05). Results: In PDSG and PDCimG, gingival mucosa exhibited few collagen fibers among numerous inflammatory cells. In PDCimG, the number of TRAP‐positive osteoclasts and IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells was significantly lower than in PDSG at all periods. A positive correlation between IL‐6/MMP‐1 and IL‐6/MMP‐9 was detected in PDSG and PDCimG. Conclusion: Cimetidine decreases bone loss through reduction of osteoclast number and induces reduction of IL‐6, MMP‐1, and MMP‐9 immunoexpression, reinforcing the idea that the beneficial effect of cimetidine in PD may be due to reduction of IL‐6 immunolabeling in the inflamed gingival mucosa.     

Gingival Crevicular Fluid,Serum Levels of Receptor Activator of Nuclear Factor‐κB Ligand,Osteoprotegerin, and Interleukin‐17 in Patients With Rheumatoid Arthritis and Osteoporosis and With Periodontal Disease

Background: This study is performed to evaluate gingival crevicular fluid (GCF) and serum levels of soluble receptor activator of nuclear factor‐κB ligand (sRANKL), interleukin (IL)‐17A, IL‐17E, IL‐17F, IL‐17A/F, and osteoprotegerin (OPG) in women with rheumatoid arthritis (RA), osteoporosis (OPR), and those who are systemically healthy (SH), all with periodontal disease. Methods: GCF and serum samples were obtained before any periodontal intervention from 17 women with RA, 19 with OPR, and 13 who were SH with periodontitis. Full‐mouth clinical periodontal measurements were recorded. sRANKL, OPG, and IL‐17 levels were determined by enzyme‐linked immunosorbent assay. Results: Clinical periodontal measurements were similar in the three study groups. Although the total amounts of GCF albumin, OPG, IL‐17A, and IL‐17A/F were similar in the study groups, there were statistically significant differences in GCF concentrations of sRANKL, OPG, IL‐17A, IL‐17E, IL‐17F, and IL‐17A/F. The sRANKL/OPG ratios were significantly higher in the RA group than in the OPR and SH groups (P <0.05). Serum sRANKL, sRANKL/OPG, and IL‐17A/IL‐17E ratios were significantly higher, whereas OPG concentrations were significantly lower in the RA group compared to other groups (P <0.05). Serum IL‐17A concentrations were significantly higher in the RA and OPR groups than in the SH group (P <0.05). Conclusion: Increased inflammatory mediator levels in patients with RA, despite the long‐term use of various anti‐inflammatory drugs, suggest that these patients may have a propensity to overproduce these inflammatory mediators.