Proteome data set of human gingival crevicular fluid from healthy periodontium sites by multidimensional protein separation and mass spectrometry

Carneiro LG, Venuleo C, Oppenheim FG, Salih E. Proteome data set of human gingival crevicular fluid from healthy periodontium sites by multidimensional protein separation and mass spectrometry. J Periodont Res 2012; 47: 248–262. © 2011 John Wiley & Sons A/S Background and Objective: Gingival crevicular fluid has been of major interest for many decades as a valuable body fluid that may serve as a source of biomarkers for both periodontal and systemic diseases. Owing to its very small sample size, submicroliter volumes, identification of its protein composition by classical biochemical methods has been limited. The advent of highly sensitive mass spectrometric technology has permitted large‐scale identification of protein components of many biological samples. This technology has been employed to identify the protein composition of gingival crevicular fluid from inflamed and periodontal sites. In this report, we present a proteome data set of gingival crevicular fluid from healthy periodontium sites. Material and Methods: A combination of a periopaper collection method with application of multidimensional protein separation and mass spectrometric technology led to a large‐scale documentation of the proteome of gingival crevicular fluid from healthy periodontium sites. Results: The approaches used have culminated in identification of 199 proteins in gingival crevicular fluid of periodontally healthy sites. The present gingival crevicular fluid proteome from healthy sites was compared and contrasted with those proteomes of gingival crevicular fluid from inflamed and periodontal sites, as well as serum. The cross‐correlation of the gingival crevicular fluid and plasma proteomes permitted dissociation of the 199 identified gingival crevicular fluid proteins into 105 proteins (57%) that can be identified in plasma and 94 proteins (43%) that are distinct and unique to the gingival crevicular fluid microenvironment. Such analysis also revealed distinctions in protein functional categories between serum proteins and those specific to the gingival crevicular fluid microenvironment. Conclusion: Firstly, the data presented herein provide the proteome of gingival crevicular fluid from periodontally healthy sites through establishment of innovative analytical approaches for effective analysis of gingival crevicular fluid from periopapers both at the level of complete elusion and with removal of abundant albumin, which restricts identification of low‐abundant proteins. Secondly, it adds significantly to the knowledge of gingival crevicular fluid composition and highlights new groups of proteins specific to the gingival crevicular fluid microenvironment.     

Effect of scaling and root planing on interleukin-1β, interleukin-8 and MMP-8 levels in gingival crevicular fluid from chronic periodontitis patients

Konopka ?, Pietrzak A, Brzezińska‐B?aszczyk E. Effect of scaling and root planing on interleukin‐1β, interleukin‐8 and MMP‐8 levels in gingival crevicular fluid from chronic periodontitis patients. J Periodont Res 2012; 47: 681–688. © 2012 John Wiley & Sons A/S Background and Objective: There are few data concerning the effect of scaling and root planing on the levels of immune and inflammatory mediators in gingival crevicular fluid from patients with chronic periodontitis. Therefore, in this study the influence of scaling and root planing was determined on amounts of interleukin (IL)‐1β, IL‐8 and MMP‐8 in gingival crevicular fluid from patients with chronic periodontitis, in relation to clinical parameters. Material and Methods: A total of 51 patients were enrolled in this study. The study population consisted of 30 patients with generalized advanced chronic periodontitis, while 21 periodontally healthy subjects were recruited for the control group. The clinical parameters included approximal plaque index, gingival index, pocket depth and clinical attachment loss. The amounts of IL‐1β, IL‐8 and MMP‐8 in gingival crevicular fluid were measured by ELISA. Periodontal parameters as well as gingival crevicular fluid humoral factor amounts were evaluated in the control group and in chronic periodontitis patients at baseline and at 1 and 4 wk after scaling and root planing treatment. Results: At baseline, there were significant differences between control subjects and chronic periodontitis patients in terms of clinical attachment loss, pocket depth, gingival index (p < 0.001) and approximal plaque index (p < 0.01). The amounts of IL‐1β, MMP‐8 (p < 0.001) and IL‐8 (p < 0.01) in gingival crevicular fluid were significantly lower in healthy subjects than in chronic periodontitis patients. Scaling and root planing led to improvement in all examined clinical parameters, apart from clinical attachment loss. Periodontal treatment also resulted in a significant decrease in the amounts of IL‐1β, IL‐8 and MMP‐8 in comparison to baseline, especially 4 wk after scaling and root planing (p < 0.001); however, the amounts of these humoral factors were still higher than those in control group. Conclusion: Our observations indicated that short‐term nonsurgical therapy resulted in a significant improvement in periodontal indices and in a marked decrease of IL‐1β, IL‐8 and MMP‐8 gingival crevicular fluid levels. Nevertheless, no significant correlations were found between clinical parameters and amounts of humoral factors after therapy.     

Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease

Da? A, F?rat ET, Kadiro?lu AK, Kale E, Y?lmaz ME. Significance of elevated gingival crevicular fluid tumor necrosis factor‐α and interleukin‐8 levels in chronic hemodialysis patients with periodontal disease. J Periodont Res 2010; 45: 445–450. © 2010 John Wiley & Sons A/S Background and Objective: The prevalence of chronic renal disease in industrialized countries is increasing, and chronic renal disease and periodontitis can have significant, reciprocal effects. The aim of this study was to evaluate the associations between specific clinical parameters and the levels of tumor necrosis factor‐α (TNF‐α) and interleukin‐8 (IL‐8) in the gingival crevicular fluid of hemodialysis (HD) patients with periodontal disease. Material and Methods: Forty‐three HD patients and 43 systemically healthy subjects were enrolled in this study. Plaque index (PI), gingival index (GI) and pocket depth were used to determine periodontal status. Venous blood samples were obtained from each patient in the morning before the dialysis session and analyzed to determine the levels of inflammatory, biochemical and hematological parameters. Gingival crevicular fluid was collected from all subjects, and the levels of TNF‐α and IL‐8 were determined in the gingival crevicular fluid samples. Results: The following results were obtained from HD patients and controls: TNF‐α (pg/mL), 31.40 ± 1.46 and 3.06 ± 0.15 (p < 0.001); IL‐8 (pg/mL), 90.98 ± 94.03 and 35.05 ± 16.86 (p < 0.001); PI, 1.69 ± 1.02 and 0.04 ± 0.02 (p < 0.001); GI, 0.82 ± 0.06 and 0.04 ± 0.02 (p < 0.001); and pocket depth, 2.23 ± 0.63 and 1.51 ± 0.05 (p < 0.001), respectively. In addition, there were positive correlations between TNF‐α and PI (r = 0.642, p < 0.001), between TNF‐α and GI (r = 0.565, p < 0.001), between TNF‐α and pocket depth (r = 0.522, p < 0.001), between IL‐8 and PI (r = 0.402, p = 0.002), between IL‐8 and GI (r = 0.396, p = 0.002), and between IL‐8 and pocket depth (r = 0.326, p = 0.012). There were negative correlations between albumin and PI (r = ?0.491, p < 0.001), albumin and GI (r = ?0.406, p < 0.001), albumin and pocket depth (r = ?0.464, p < 0.001) and albumin and CRP (r = ?0.467, p = 0.002) and between the gingival crevicular fluid levels of TNF‐α and IL‐8, TNF‐α and hemoglobin (r = ?0.745, p < 0.001; r = ?0.285, p < 0.05) (respectively). Conclusion: The levels of TNF‐α and IL‐8 in gingival crevicular fluid were significantly higher in HD patients than in controls. There were strong, positive correlations between clinical periodontal parameters and the levels of inflammatory cytokines in gingival crevicular fluid from the HD patients.     

Expression of cytokines in the gingival crevicular fluid and serum from patients with inflammatory bowel disease and untreated chronic periodontitis

Figueredo CM, Brito F, Barros FC, Menegat JSB, Pedreira RR, Fischer RG, Gustafsson A. Expression of cytokines in the gingival crevicular fluid and serum from patients with inflammatory bowel disease and untreated chronic periodontitis. J Periodont Res 2011; 46: 141–146.© 2010 John Wiley & Sons A/S Background and Objective: Previous studies have reported an increased prevalence/severity of chronic periodontitis in patients with inflammatory bowel disease. However, the pathogenesis of periodontal lesions in such patients has not been characterized. The aim of this pilot study was to characterize the pattern of expression of cytokines in the gingival crevicular fluid and serum from patients with untreated chronic periodontitis and Crohn’s disease, ulcerative colitis and systemically healthy controls. Material and Methods: Fifteen patients with Crohn’s disease, 15 patients with ulcerative colitis and 15 controls participated in the study. All subjects had been diagnosed with untreated chronic periodontitis. The clinical parameters evaluated were clinical attachment loss, bleeding on probing and percentage of plaque. The gingival crevicular fluid was sampled from four shallow and four deep periodontal sites of each patient. The concentrations of the cytokines interleukin (IL)‐1β, IL‐4, IL‐6, IL‐10, IL‐12p40, IL‐12p70, interferon‐γ and tumor necrosis factor‐α were measured using a commercially available Lincoplex kit and the concentration of IL‐18 was measured using an ELISA. Results: Multiple comparisons analysis showed that clinical attachment loss, bleeding on probing, percentage of plaque and volume of gingival crevicular fluid were similar across the groups. The concentration of IL‐4 in the gingival crevicular fluid differed significantly between groups in shallow sites (p = 0.046), with higher values found for the controls. In serum, the concentration of IL‐18 was also significantly different between groups, with lower values found for controls (p = 0.018). Conclusion: This study showed a higher concentration of IL‐18 in serum, but not in the gingival crevicular fluid, from periodontitis patients with Crohn’s disease or ulcerative colitis compared with controls. The expression of cytokines was similar in the gingival crevicular fluid from patients with untreated chronic periodontitis who also had Crohn’s disease or ulcerative colitis and in systemically healthy controls with untreated chronic periodontitis.     

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

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

Interleukin-4, a T-helper 2 cell cytokine, is associated with the remission of periodontal disease

Background and Objectives: Interleukin‐4 (IL‐4), secreted mainly by T‐helper 2 cells, is a key cytokine for the growth and proliferation of B lymphocytes. Previous studies have proved that IL‐4 has an anti‐inflammatory effect owing to its efficient inhibition of the production of proinflammatory cytokines such as tumour necrosis factor‐α (TNF‐α), IL‐1α, IL‐1β, IL‐6 and IL‐8 by monocytes/macrophages. The aim of the present study was to assess the relation between clinical parameters and concentrations of IL‐4 within gingival crevicular fluid from inflamed gingiva and periodontitis sites and, subsequently, after treatment of the periodontitis sites. Material and Methods: A total of 60 subjects were divided into three groups based on gingival index (GI), pocket probing depth and clinical attachment loss (CAL): healthy (group 1), gingivitis (group 2) and chronic periodontitis (group 3). A fourth group (group 4) consisted of 20 subjects from group 3, 6–8 weeks after treatment (i.e. scaling and root planing). Gingival crevicular fluid samples collected from each patient were quantified for IL‐4 using the enzymatic immunometric assay. Results: The highest mean concentration of IL‐4 was obtained for group 1 (99.39 ± 49.33 pg/mL) and the lowest mean concentration of IL‐4 was obtained for group 3 (15.78 ± 21.92 pg/mL). The mean IL‐4 concentrations for group 2 (64.34 ± 39.56 pg/mL) and group 4 (68.92 ± 42.85 pg/mL) were intermediate between the levels in healthy subjects and periodontitis subjects. Conclusion: The mean concentration of IL‐4 decreased from periodontal health to disease. Thus, we suggest that type 2 helper T cell cytokine, as represented by IL‐4, was associated with the remission or improvement of periodontal disease.     

Influence of smoking on interleukin-1beta level, oxidant status and antioxidant status in gingival crevicular fluid from chronic periodontitis patients before and after periodontal treatment

Toker H, Akp?nar A, Ayd?n H, Poyraz O. Influence of smoking on interleukin‐1beta level, oxidant status and antioxidant status in gingival crevicular fluid from chronic periodontitis patients before and after periodontal treatment. J Periodont Res 2012; 47: 572–577. © 2012 John Wiley & Sons A/S Background and Objective: The aim of this study was to evaluate the impact of smoking on the relationship between interleukin‐1 (IL‐1β) and oxidation in patients with periodontitis and response to nonsurgical periodontal therapy. Material and Methods: Data were obtained from 30 patients with generalized chronic periodontitis (15 smokers and 15 nonsmokers) and from 10 periodontally healthy controls. IL‐1β level, total oxidant status (TOS) and total antioxidant status (TAS) were recorded in gingival crevicular fluid. Probing depth, clinical attachment level, gingival and plaque indices and bleeding on probing were also measured. The gingival crevicular fluid and clinical parameters were recorded at baseline and 6 wk after periodontal treatment. Results: The study showed statistically significant improvement of clinical parameters in both smokers and nonsmokers after periodontal treatment. Moreover, the baseline IL‐1β levels were significantly higher in smokers compared with nonsmokers (p < 0.05). After periodontal treatment, the IL‐1β levels were significantly reduced in both smokers and nonsmokers (p < 0.05). There were no significant differences in TOS and TAS between periodontitis patients and healthy controls at baseline and 6 wk after periodontal treatment. The level of IL‐1β in gingival crevicular fluid was positively correlated with TOS in both smokers and nonsmokers. Conclusions: Periodontal treatment improved the clinical parameters in both smokers and nonsmokers. The results confirm that periodontal therapy has an effect on IL‐1β levels in gingival crevicular fluid, but not on TOS and TAS.     

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.     

TNF-alpha TGF-beta2 and IL-1beta levels in gingival and peri-implant crevicular fluid before and after de novo plaque accumulation

Aims: The aim of this split‐mouth study was to investigate levels of tumour necrosis factor alpha (TNF‐α), transforming growth factor beta (TGF‐β2) and interleukin‐1 beta (IL‐1β) in gingival crevicular fluid (GCF) and peri‐implant crevicular fluid (PICF) after a 21‐day‐period of de novo plaque accumulation in the same patient. Material and Methods: In 25 patients, samples of GCF and PICF were collected in the sulcus of the tooth and of the implant after professional hygiene. After the no‐hygiene phase (21 days), second samples of GCF and PICF were taken. Third samples were collected after 69 days of re‐establishment oral hygiene techniques. The crevicular fluids were used to determine the volume and the levels of TNF‐α, TGF‐β2 and IL‐1β. Results: The volume of the crevicular fluids increased significantly after 21 days of plaque accumulation around teeth and implants and decreased significantly by 69 days. TNF‐α and TGF‐β2 did not change significantly among the three different samples. A significant increase of IL‐1β was observed after plaque accumulation around the teeth GCF, whereas in the PICF the increase was not statistically significant. Conclusions: These data suggest that increased volumes of GCF and PICF could be useful markers of early inflammation in gingival and peri‐implant tissues. In the presence of de novo plaque, implants showed lower, and nearly significant, levels of IL‐1β compared with teeth.     

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.     

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.     

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.     

Levels of interleukin-21 in patients with untreated chronic periodontitis

Background: A growing body of evidence suggested that interleukin (IL)‐21 enhances the effector phase during T‐cell responses. The aim of our study is to determine the levels of IL‐21 in periodontal sites from patients with chronic periodontitis and controls. Methods: The population studied consisted of 34 patients (15 with chronic periodontitis and 19 healthy patients). Twenty samples (10 gingival crevicular fluid [GCF] and 10 gingival biopsies) were collected from each group before the patients with periodontitis received periodontal treatment. Total protein concentrations were measured in all samples; the presence of IL‐21 was confirmed by immunohistochemistry and Western blot, and IL‐21 levels were quantified through an enzyme‐linked immunosorbent assay. Statistical analyses were performed using statistical software. Data were expressed as patient means ± SDs or medians (interquartile ranges) by using the χ², Student t, and Mann‐Whitney U tests. Results: GCF IL‐21 was mainly detected in patients with chronic periodontitis (P <0.05). Levels of IL‐21 in gingival tissues were significantly higher in patients with chronic periodontitis compared to healthy individuals (P <0.05). The Western blot and immunohistochemical staining confirmed the presence of IL‐21 in periodontal tissues and GCF. Conclusion: IL‐21 was highly expressed in patients with chronic periodontitis, especially in gingival biopsies; therefore, IL‐21 might play a role in the T‐cell response.     

Proinflammatory and Anti‐Inflammatory Cytokines in Gingival Crevicular Fluid and Serum of Patients With Rheumatoid Arthritis and Patients With Chronic Periodontitis

Background: The aim of this study is to evaluate proinflammatory and anti‐inflammatory cytokine levels in gingival crevicular fluid (GCF) and serum of rheumatoid arthritis (RA) and chronic periodontitis (CP) patients to assess whether cytokine profiles distinguish patients with RA and patients with CP while using healthy patients as background controls. Methods: A total of 49 patients, 17 patients with RA (three males and 14 females; mean age: 47.82 ± 10.74 years), 16 patients with CP (10 males and six females; mean age: 44.00 ± 7.00 years), and 16 controls (eight males and eight females; mean age: 28.06 ± 6.18 years) were enrolled. Patients with RA were under the supervision of rheumatologists; 15 of the patients with RA were being treated with methotrexate–sulfasalazine combined therapy, and two of the patients were being treated with leflunomid therapy. Periodontal parameters (plaque index, gingival index, probing depth, and clinical attachment level) were recorded. Interleukin (IL)‐1β, IL‐4, IL‐10, and tumor necrosis factor‐α (TNF‐α) were determined in GCF and IL‐1β and IL‐10 in serum by enzyme‐linked immunosorbent assay. Results: There were significant differences found among RA, CP, and control groups for all periodontal parameters (P <0.05). The total amount and concentration of GCF IL‐1 β, IL‐4, IL‐10, and TNF‐α were similar in RA and CP patients (P >0.05). Although the total amount and concentration of serum IL‐10 was not significantly different among the groups (P >0.05), serum IL‐1β was significantly lower in the RA group compared to CP patients and controls and was higher in GCF of the RA group compared to the CP group. Conclusions: Although clinical periodontal disease parameters indicated more severe periodontal disease in CP compared to RA patients, immunologic evaluation did not reveal consistent results regarding proinflammatory and anti‐inflammatory cytokine levels. This might be a result of the use of non‐steroidal anti‐inflammatory drugs and rheumatoid agents by patients with RA.     

Full-mouth profile of active MMP-8 in periodontitis patients

Kraft‐Neumärker M, Lorenz K, Koch R, Hoffmann T, Mäntylä P, Sorsa T, Netuschil L. Full‐mouth profile of active MMP‐8 in periodontitis patients. J Periodont Res 2012; 47: 121–128. © 2011 John Wiley & Sons A/S Background and Objective: MMP‐8 in gingival crevicular fluid is considered as a protease with high destructive potential because of its ability to degrade collagen in periodontitis‐affected patients. The aim of this study was to investigate whether there was a relationship between clinical diagnostic parameters and the concentration of active MMP‐8 (aMMP‐8) in gingival crevicular fluid in a site‐level full‐mouth analysis. Based on these data, the prognostic value of aMMP‐8 levels in relation to pocket depth may be evaluated. Material and Methods: Clinical measurements of pocket depth, bleeding on probing (BOP), plaque index (PlI) and gingival index (GI), as well as samples of gingival crevicular fluid, were obtained from four sites of each tooth of nine healthy female patients with chronic generalized periodontitis. The aMMP‐8 concentration in gingival crevicular fluid was quantified by ELISA using specific monoclonal antibodies. Multiple linear regression models for the single measures of aMMP‐8 and pocket depth were calculated with GI and BOP as additional variables. Results: Between 92 and 112 recordings were obtained for each parameter in each patient. Mean values of between 31.5 and 88.8% were calculated for pocket depths of ≥ 4 mm. Mean pocket depths ranged from 3.11 to 4.73 mm, the mean BOP values ranged from 34.0 to 96.7% and the mean full‐mouth gingival crevicular fluid aMMP‐8 concentration ranged from 3.2 to 23.7 ng/mL. Conclusion: In this sample of female periodontitis patients, a broad range of intra‐individual and interindividual aMMP‐8 values was found. Although the explained variance was rather weak, a statistically significant relationship between aMMP‐8 and pocket depth was proven.     

Repeatability of gingival crevicular fluid collection and quantification,as determined through its alkaline phosphatase activity: implications for diagnostic use

Background and Objective: In spite of four decades of studies on gingival crevicular fluid, no data have been reported on the repeatability of gingival crevicular fluid collection and the subsequent quantification procedures. The present study reports, for the first time, on the repeatability and method error of gingival crevicular fluid collection and quantification, as determined through its alkaline phosphatase (ALP) activity. Diagnostic considerations are then explored. Material and Methods: Twenty‐seven healthy subjects (17 women and 10 men; mean age ± SD, 21.2 ± 4.8 years) with optimal periodontal status were enrolled according to a blind prospective design. The gingival crevicular fluid was collected at baseline, and after 1 d, 1 wk and 3 mo. At each clinical session, two consecutive rounds of gingival crevicular fluid collection were made from each of the four maxillary incisors, allowing the recovery of resting and flow gingival crevicular fluid. The total ALP activities were determined spectrophotometrically, and repeatability and method errors for the resting, flow and overall (resting + flow) gingival crevicular fluid ALP activities were calculated, relative to the corresponding baseline levels. Results: No significant differences were seen over time, although the flow gingival crevicular fluid ALP activity was generally lower than that for the resting gingival crevicular fluid. The method errors ranged from 40 to 58%, with the flow and overall gingival crevicular fluid activities showing the highest and lowest errors, respectively. Conclusion: Reliable use of the gingival crevicular fluid ALP collection and quantification, both in research and diagnosis on an individual basis, should take into account relevant errors, and variations are to be considered as true only above relevant thresholds.     

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.     

Is Interleukin‐17 Involved in the Interaction Between Polycystic Ovary Syndrome and Gingival Inflammation?

Background: Polycystic ovarian syndrome (PCOS) is a hormonal disorder of females of reproductive age that impacts their oral and systemic health. The aim of this study is to evaluate interleukin‐17A (IL‐17A), IL‐17F, IL‐17A/F, and IL‐17E (IL‐25) levels in gingival crevicular fluid (GCF), saliva, and serum of non‐obese females with PCOS and with either a clinically healthy periodontium or gingivitis. Methods: Thirty‐one females with PCOS, 30 females with PCOS and gingivitis, and 12 systemically and periodontally healthy females participated in the study. Clinical periodontal measurements, body mass index, and Ferriman‐Gallwey score (FGS) (a measure of hirsutism in females) were recorded. Circulating levels of sex hormones, cortisol, and insulin were also determined. Levels of IL‐17 cytokines were measured by enzyme‐linked immunosorbent assay. Results: The general linear model multivariate analysis, adjusting for age or plaque index, showed that the two groups with PCOS had higher concentrations of IL‐17A, IL‐17F, and IL‐17A/F in serum and higher levels of IL‐17A and IL‐17F in GCF and saliva but lower serum IL‐17E than systemically healthy females. Levels of IL‐17E were lowest in females with PCOS and gingivitis who also had the highest FGS. Serum IL‐17A and IL‐17F levels correlated positively with FGS and periodontal probing depth (all ρ >0.33; P <0.005). Serum IL‐17E showed the reverse relationship and also correlated negatively with IL‐17A (ρ >?0.28; P <0.05). Conclusions: IL‐17 levels are altered in non‐obese females with PCOS and may influence gingival inflammation. Additional studies are warranted to clarify the relationship between PCOS and gingivitis.     

Host β-globin gene fragments in crevicular fluid as a biomarker in periodontal health and disease

Thaweboon B, Laohapand P, Amornchat C, Matsuyama J, Sato T, Nunez PP, Uematsu H, Hoshino E. Host β‐globin gene fragments in crevicular fluid as a biomarker in periodontal health and disease. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2008.01197.x. © 2009 John Wiley & Sons A/S Background and Objective: Leukocytes and epithelium are the first line of defense in preventing bacterial invasion into periodontium. Some of these cells die in gingival crevicular fluid, whereupon their DNA is spilled out. The present study was designed to investigate the profile of host β‐globin gene fragments in the gingival crevicular fluid of various periodontal conditions. Material and Methods: Gingival crevicular fluid from 40 teeth with chronic periodontitis, 30 with gingivitis and 22 that were clinically healthy were centrifuged (3000 g , 10 min). The supernatant (cell‐free gingival crevicular fluid) was centrifuged again (13,000 g , 10 min), resulting in the pellet and the supernatant as debris and debris‐free fractions, respectively. Specific primers for amplifying 110 bp, 536 bp and 2 kb amplicons of human β‐globin gene were used to investigate host DNA by quantitative and qualitative polymerase chain reaction. Results: The periodontitis group showed the largest amount of host β‐globin gene fragments, while the healthy group had the lowest. In the debris and debris‐free fractions, the 536 bp and 2 kb amplicons were more often detected in the periodontitis group than in the other groups. Interestingly, the presence of 2 kb amplicon in the debris fraction could be used to discriminate periodontitis from gingivitis and healthy groups because we found it in 85% of periodontitis samples but only in 13% of gingivitis samples, and it was absent in the healthy group. Conclusion: This study shows the different DNA profiles of cell‐free gingival crevicular fluid in periodontal health and disease. It suggests that the quantity and quality of host DNA are dependent on the disease conditions. Therefore, the β‐globin gene fragments in cell‐free gingival crevicular fluid may be a potential biomarker of periodontal disease progression.