Human interleukin-1 beta and interleukin-1 receptor antagonist secretion and velocity of tooth movement

The cytokines interleukin-1 beta (IL-1 beta) and IL-1 receptor antagonist (IL-1RA) probably play a part in orthodontic tooth movement. Here, the force magnitudes and the area of force application in the compressed periodontal ligament (PDL) were controlled and the velocity of tooth movement correlated with concentrations of IL-1 beta and IL-1RA in the gingival crevicular fluid (GCF). Seven individuals undergoing orthodontic treatment involving maxillary first premolar extractions and distal movement (bodily retraction) of the maxillary canines participated in the 84-day study. For each participant, continuous retraction forces were applied so that they received equivalent PDL stresses of 13 kPa for one canine and 4 kPa for the other. GCF cytokine concentrations from experimental and control teeth were expressed relative to total protein in the GCF and compared using an 'Activity Index' (AI)=Experimental (IL-1 beta/IL-1RA)/Control (IL-1 beta/IL-1RA). The results showed that the velocity of tooth movement in an individual was related to their AI. The correlation between AI and tooth movement was stronger from the distal (R(d)=0.78) than from the mesial (R(m)=0.65) of retracted teeth. The results demonstrate that equivalent force systems produce individual differences in cytokine production, which correlate with interindividual differences in the velocity of canine retraction.     

Gingival crevicular interleukin-1 and interleukin-1 receptor antagonist levels in periodontally healthy and diseased sites

Interleukin-l (IL-1) molecules, IL-lα and IL-lβ are cytokines involved in the acute-phase response against infection and in the pathogenesis of periodontal destruction. Administration of exogenous IL-1 receptor antagonist (IL-1ra) is effective in reducing the inflammatory reactions mediated by IL-1. However, the relationship between these three naturally occurring IL-1 molecules and periodontal diseases has been poorly characterized. We investigated the correlation of gingival crevicular IL-1 molecules and the clinical status of patients with different severities of periodontitis. IL-lα, IL-1β, IL-1ra and the total IL-1/IL-1ra ratio (IL-1 activity index; IL-1AI) were measured in 75 gingival crevicular fluid (GCF) samples from non-inflamed gingiva sites in 2 healthy subjects and diseased sites in 7 patients with several types of periodontitis. IL-lα, IL-1bT and IL-1ra were measured by specific non-cross-reactive enzyme linked immunosorbent assay. The probing depth, gingival index and alveolar bone loss of each site was recorded at the time of GCF sampling. The total amount of IL-lα, IL-1β and the IL-1AI, but not total IL-1ra, were found to be correlated with alveolar bone loss score. Three IL-1 molecules were also measured in the gingival tissue of patients with periodontitis. A similar progressive decrease of the IL-1AI was detected in gingival tissue with periodontitis. These results suggest that the amounts of both crevicular IL-1 and IL-1AI are closely associated with periodontal disease severity.     

Cytokine levels in gingival crevicular fluid of erupting primary teeth correlated with systemic disturbances accompanying teething

PURPOSE: The aim of this study was to investigate whether there are increased levels of the inflammatory cytokines IL-1beta, IL-8, and TNFalpha in the gingival crevicular fluid (GCF) of erupting primary teeth. This increase could explain such clinical manifestations as fever, diarrhea, increased crying, and sleeping and eating disturbances that occur at this time. METHODS: Sixteen healthy children aged 5 to 14 months (mean=9.8 months) were examined twice a week over 5 months. Gingival crevicular fluid samples were taken from erupting teeth. As a control, GCF was collected from the same teeth 1 month later. Cytokine production was measured by ELISA. Signs and clinical symptoms were listed. Pearson correlation coefficients were used in the comparisons described below. A paired t test was used to analyze the same variable at different times. RESULTS: Fifty teeth of the 16 children were studied. GCF samples were collected from 21 of these teeth. Statistically significant differences (P<.05) were found with regard to the occurrence of fever, behavioral problems, and coughing during the teething period and the control period. During the control period, 72% of the children did not exhibit any clinical manifestations, whereas during the teething period only 22% of the children did not exhibit any clinical manifestations. The study revealed high levels of inflammatory cytokines during the teething period, with a statistically significant difference in TNFalpha levels (P<.05) between the teething period and the control period. Correlations were found between cytokine levels and some of the clinical symptoms of teething: IL-1beta and TNFalpha were correlated with fever and sleep disturbances; IL-beta and IL-8 were correlated with gastrointestinal disturbances; IL-1beta was correlated with appetite disturbances. CONCLUSIONS: Cytonkines appear in the GCF of erupting prmary teeth. The cytokine levels are correlated to some symptoms of teething.     

Gingival crevicular fluid IL-8: correlation with local IL-1β levels and patient estrogen status

Gingival crevicular fluid (GCF) IL-8 and IL-1,1β levels were determined by sandwich enzyme-linked immunosorbent assays. Associations between IL-8 and IL-1β GCF levels, and between these cytokines and patient estrogen status were evaluated. IL-8 and IL-1β were detected more frequently and in higher amounts/30 s GCF sample in estrogen-deficient patients than in estrogensufficient patients. IL-8 and IL-1β GCF levels were significantly correlated. These lindings suggest that GCF IL-8 levels are associated with patient estrogen status and local IL-1β concentrations.     

The gingival crevicular fluid Interleukin-1β and tumour necrosis factor-α levels in patients with rapidly progressive periodontitis

Cytokines are believed to play an important role in the pathogenesis of periodontal diseases. In the present study, gingival crevicular fluid (GCF) levels of two important cytokines, interleukin 1-/3 (IL-1β) and tumour necrosis factor-α (TNF-α) and, in addition, serum IL-1β levels, were determined in patients with severe and rapid periodontal breakdown by use of ELISA. While IL-1β was detected in all of the GCF samples studied, TNF-α could only be detected in about half the samples. The mean GCF IL-1β level was 38.45 ± 13.99 pg/mL, and the mean TNF-α level was 3.20 ± 1.39 pg/mL, respectively. The GCF IL-1β levels also presented a strong positive correlation with the mean pocket depths. Although weak, both of the cytokines also presented correlations with the presence of bleeding on probing. Additionally GCF samples contained increased IL-1β levels when compared with the serum samples suggesting local production mechanisms. The findings of the present study suggest that these cytokines may be involved in the pathogenesis of periodontal diseases (IL-1β being more significant), and also may help in defining the active phase of periodontal breakdown.     

Levels of interleukin-1 beta, -8, and -10 and RANTES in gingival crevicular fluid and cell populations in adult periodontitis patients and the effect of periodontal treatment

BACKGROUND: Various cytokines have been identified at sites of chronic inflammation such as periodontitis. Cytokines are synthesized in response to bacteria and their products, inducing and maintaining an inflammatory response in the periodontium. The purpose of the present study was to investigate the involvement of interleukin-1 beta (IL-1 beta), IL-8, and IL-10 and RANTES (regulated on activation, normally T cell expressed and secreted) and the cell populations associated with the immune response in destructive periodontitis, as well as the effect of periodontal therapy on cytokine levels in gingival crevicular fluid (GCF). METHODS: Data were obtained from 12 patients with moderate to advanced periodontitis and 6 healthy controls. Patients presenting at least 2 sites with > or =2 mm clinical attachment loss were included in the destructive periodontitis group. After monitoring for 4 months, only 6 patients showed destructive periodontitis and GCF samples and soft tissues biopsies were collected from these patients. GCF samples and biopsies were collected both from active (12 CGF samples and 6 biopsies) and inactive (12 CGF samples and 6 biopsies) sites. The comparison with healthy controls was carried out by collecting GCF samples from 6 healthy volunteers (12 samples) and biopsies during the surgical removal of wisdom teeth. In periodontal patients, clinical data and GCF samples were obtained prior to periodontal treatment (72 samples) and 2 months after periodontal therapy (72 samples). GCF was collected using a paper strip; eluted and enzyme-linked immunoabsorbent assays (ELISA) were performed to determine cytokine levels. The inflammatory infiltrate was analyzed by immunohistochemistry of gingival biopsy samples with monoclonal antibodies against CD3, CD8, CD4, CD11c, and CD19 antigens. RESULTS: Cellular components of the inflammatory infiltrate include B and T lymphocytes and monocyte/macrophages. Active sites contained a higher number of B lymphocytes and macrophages. IL-8 and IL-1 beta and RANTES in GCF were detected in the majority of sites from periodontal patients (100%, 94% and 87%, respectively); IL-10 was found in only 43%. IL-8 was the only cytokine detected in the GCF (75%) of the control group. Moreover, IL-1 beta levels were significantly higher in active sites versus inactive sites (P <0.05). IL-8 and IL-10 and RANTES were increased in active sites; however, differences were not significant (P>0.05). A positive correlation between the IL-8 and RANTES (r = 0.677, P<0.05) was observed in periodontitis patients. Periodontal therapy reduced the total amount of IL-1 beta, IL-8, and IL-10 and RANTES. Data showed a weak correlation between the clinical parameters and the total amount of cytokines in periodontitis. CONCLUSIONS: These data suggest that the amount of crevicular IL-1 beta, IL-8, and IL-10 and RANTES is associated with periodontal status. Removal of the bacterial plaque reduces the antigenic stimuli and consequently could modulate the chemokines present in GCF. We propose that the dynamic interactions between cytokines, their production rates, and their quantity could represent factors controlling the induction, perpetuation, and collapse of the cytokine network present in the periodontal disease.     

Identification of the osteoprotegerin/receptor activator of nuclear factor-kappa B ligand system in gingival crevicular fluid and tissue of patients with chronic periodontitis

BACKGROUND AND OBJECTIVE: Recent findings have suggested that osteoclastogenesis is directly regulated by receptor activator of nuclear factor-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG). However, no studies have described interactions of OPG/RANKL and the gp130 cytokine family in periodontal disease. This study aimed to identify and quantify OPG/RANKL in the gingival crevicular fluid (GCF) and connective tissue of patients with periodontitis, and to clarify possible correlations with disease severity and interleukin-6 (IL-6) cytokines. MATERIAL AND METHODS: Ninety-five sites in 20 patients with generalized chronic periodontitis were divided into four groups by site based on probing depth (PD) and bleeding on probing (BOP). In periodontitis patients, GCF was obtained using sterile paper strips from clinically healthy sites (PD 6 mm with BOP, n = 27). Fourteen clinically healthy sites from four periodontally healthy individuals were used as the control group. The levels of OPG, RANKL and two gp130 cytokines - IL-6 and oncostatin M (OSM) - in the GCF were determined by an enzyme-linked immunosorbent assay (ELISA) and are expressed as total amounts (pg/site). Immunohistochemical localization of OPG- and RANKL-positive cells was also performed on gingival connective tissues harvested from patients with periodontitis (inflammatory group, n = 8 biopsies) and from non-diseased individuals (healthy group, n = 8 biopsies). RESULTS: GCF RANKL, but not OPG, was elevated in diseased sites of patients with periodontitis. However, the expressions of OPG and RANKL showed no correlation with disease severity (r = 0.174 and 0.056, respectively), but the content of RANKL in the GCF was significantly positively correlated with those of IL-6 (r = 0.207) and OSM (r = 0.231) (p < 0.01). Immunohistochemical staining showed that RANKL-positive cells were significantly distributed in the inflammatory connective tissue zone of diseased gingiva, compared with those of samples from non-diseased persons (p < 0.01). However, few OPG-positive cells were found in connective tissue zones of either the diseased gingiva or healthy biopsies. CONCLUSION: These findings imply that in this cross-sectional study of GCF, RANKL, IL-6 and OSM were all prominent in periodontitis sites, whereas OPG was inconsistently found in a few samples of diseased sites but was undetectable in any of the control sites. The results also imply that the expression of RANKL was positively correlated with IL-6 and OSM in the GCF.     

Crevicular interleukin-1β in moderate and severe periodontitis patients and the effect of phase I periodontal treatment

Abstract Interleukin-1β (IL-1β), a potent stimulator of bone resorption, has been implicated in the pathogenesis of periodontal destruction. However, the relationship between cytokines and periodontal disease has not been studied sufficiently to allow definitive conclusions. The aims of this study are to investigate crevicular IL-1β and the clinical status of patients with periodontitis and the effect of phase I periodontal therapy on levels of IL-1β. For this study, 130 gingival crevicular fluid (GCF) samples were harvested from non-inflamed (15) and diseased sites (115) in 11 patients with periodontitis. The gingival index (GI) and probing depth (PD) of each site was recorded initially and one month after treatment. The amount of IL-1β in the GCF was measured by enzyme-linked immunosorbent assay (ELISA) using an antibody specific for this cytokine. Before treatment, IL-1β was found in 12 of 15 non-inflamed gingival crevices and in 112 of 115 diseased pockets. The amount of IL-1β varied from 4.03 to 511.12 pg/site. The average amount of IL-1/7 from diseased sites was 3-fold greater than that from non-inflamed sites. Both total amount of IL-1β and the GCF volume, but not IL-1β concentration, were found to be correlated, positively, with GI score and PD. After therapy, 63 sites from 7 patients were re-examined, and the amount of 1L-1β in 49 of 63 sites was found to have declined. These data suggest that the amount of Crevicular IL-1β is closely associated with periodontal status. This relationship may be valuable in monitoring periodontal disease activity.     

Relationship between IL-1A polymorphisms and gingival overgrowth in renal transplant recipients receiving Cyclosporin A

AIM: Levels of interleukin-1alpha (IL-1alpha) are elevated in periodontal inflammation. IL-1A gene polymorphisms are associated with inflammatory diseases. This study aimed to investigate IL-1A gene polymorphism in Cyclosporin A (CsA)-treated renal transplant patients and investigate the association between this polymorphism and gingival crevicular fluid (GCF) levels of several cytokines. MATERIALS AND METHODS: Fifty-one renal transplant patients on CsA treatment (25 with and 26 without gingival overgrowth) and 29 healthy controls were recruited for the study. Demographic, pharmacological and periodontal parameters were recorded and gingival overgrowth was assessed. RESULTS: Multiple regression analysis showed that genotype was significantly associated with gingival overgrowth (p=0.02). Carriage of the IL-1A (-889) T allele was strongly protective [95% confidence interval (CI): 0.046-0.77], although not significantly associated with IL-1alpha protein levels in GCF. IL-1alpha, IL-1beta and IL-8, but not IL-6, were detected in GCF of CsA-treated patients, but none of them was significantly associated with gingival overgrowth. CONCLUSIONS: This study is the first to associate a gene polymorphism as a risk factor for CsA-induced gingival overgrowth in renal transplant patients, demonstrating that IL-1A polymorphism might alter individual susceptibility to CsA. However, there was no association between GCF cytokine levels and the presence of gingival overgrowth or patient IL-1A genotype.     

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

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

龈沟液中的炎症调节因子——白细胞介素8

为研究白细胞介素８（ｉｎｔｅｒｌｅｕｋｉｎ－８，ＩＬ－８）在牙周炎中的作用，作者应用ＥＬＩＳＡ法检测了５４例（１０５个牙）成人牙周炎和２４名（５４个牙）健康对照者龈沟液中ＩＬ－８的水平。结果显示：龈沟液中ＩＬ－８的浓度低于３０μｇ／Ｌ时，ＩＬ－８与龈沟出血指数呈正相关（Ｐ＜０．０１）。龈沟液中ＩＬ－８的浓度高于３０μｇ／Ｌ时，ＩＬ－８与出血指数和探诊深度呈负相关（Ｐ值均小于０．０１）。９１％的牙周炎患牙龈沟液中ＩＬ－８低于３０μｇ／Ｌ。结果提示：ＩＬ－８可能具有抗炎和致炎的双重调节作用；其抗炎和致炎作用的分界值约为３０μｇ／Ｌ。高浓度的ＩＬ－８具有抗炎作用，低浓度的ＩＬ－８具有致炎作用。牙周炎时，龈沟液中的ＩＬ－８主要起致炎作用。     

Gingival crevicular fluid and plasma acute-phase cytokine levels in different periodontal diseases

Background: The aim of the present study is to investigate gingival crevicular fluid (GCF) and plasma acute-phase cytokines, interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-11 (IL-11), oncostatin M (OSM), and leukemia inhibitory factor (LIF) levels in patients with different periodontal diseases. Methods: Eighty individuals were included in this study; 20 with chronic periodontitis (CP), 20 with generalized aggressive periodontitis (GAgP), 20 with gingivitis, and 20 classified as healthy (H). Probing depth, clinical attachment level, plaque index, and papilla bleeding index were recorded. Plasma and GCF IL-1β, IL-6, IL-11, OSM, and LIF levels were analyzed by enzyme-linked immunosorbent assay. Results: CP and GAgP groups had significantly higher GCF IL-1β, IL-6, and IL-11 levels when compared with the H group (P <0.05). Conversely, GCF LIF levels of the CP and GAgP groups were lower than those of the H group (P <0.05). GCF OSM levels did not differ significantly among study groups. Plasma levels of all the cytokines studied were not significantly different among the study groups. Conclusions: Based on the present data, elevated IL-1β, IL-6, and IL-11 GCF levels, but not plasma levels, are suggested as reliable inflammatory biomarkers in periodontal diseases. Decreased LIF levels in diseased groups might reflect the possible beneficial effects of LIF in the modulation of inflammatory response in gingiva.     

慢性牙周炎病人基础治疗前后Th17型细胞因子及RORC的表达变化

目的:研究慢性牙周炎(CP)病人牙周基础治疗前后龈沟液(GCF)中的Th17型细胞因子及其外周血特异性转录因子RORC的表达变化规律。方法:选择CP病人30例,使用ELISA检测牙周基础治疗前后GCF中Th17型细胞因子(IL-17、IL-21)的水平变化,并利用荧光定量PCR检测牙周基础治疗前后外周血CD4+T细胞中特异性转录因子RORC的表达水平。结果:牙周基础治疗后龈沟液中的Th17型细胞因子IL-17、IL-21水平以及外周血CD4+T细胞中特异性转录因子RORC表达水平均较治疗前显著下降,差异均有统计学意义(P<0.05)。结论:Th17型细胞因子在慢性牙周炎发生发展中发挥重要的致炎作用。     

The evaluation of cystatin C, IL-1beta, and TNF-alpha levels in total saliva and gingival crevicular fluid from 11- to 16-year-old children

BACKGROUND: The aim of this study was to evaluate the levels of cystatin C, interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) in the total saliva and gingival crevicular fluid (GCF) of periodontally healthy children (PHC) and children with gingivitis (CG) who were between 11 and 16 years old. METHODS: The study was carried out with 10 PHC and 25 CG. Unstimulated total saliva and GCF samples were obtained. Clinical parameters, including probing depth (PD), clinical attachment loss (CAL), plaque index (PI), gingival index (GI), and gingival bleeding index (GBI), were assessed. GCF samples were collected from four maxillary upper incisors. After sampling, biochemical analyses were performed using latex particle-enhanced turbidimetric immunoassay for cystatin C and enzyme-linked immunosorbent assay for IL-1beta and TNF-alpha. The multivariate analysis of variance test was used for statistical evaluation. RESULTS: In total saliva, cystatin C and TNF-alpha levels were higher in PHC, and IL-1beta levels were higher in CG, but the differences were not statistically significant. In GCF, cystatin C levels were higher in PHC (P >0.05), whereas TNF-alpha and IL-1beta levels were higher in CG (P >0.05). In the CG group, there were positive correlations between the GCF cystatin C level and the PI of the sampled site (r = 0.488; P <0.05); also, GCF IL-1beta (r = 0.603; P <0.05) and TNF-alpha (r = 0.456; P <0.05) levels were positively correlated with PD and CAL. For the whole mouth and the sampled sites, PI, GI, GBI, PD, and CAL values were higher in CG (P <0.05), but no significant differences were detected between GCF volumes of the two groups. CONCLUSIONS: To the best of our knowledge, this study represents the first evaluation of cystatin C in the gingival disease mechanism in children. Our results showed that total saliva and GCF cystatin C levels were higher in PHC (P >0.05), but there was no correlation between cystatin C levels and IL-1beta or TNF-alpha levels in total saliva or GCF.     

母体唾液及龈沟液细胞因子水平与早产低体重新生儿关系的病例对照研究

目的 验证牙周炎是否为早产低体重新生儿(preterm low birth weight,PLBW)母亲体内低度炎症的来源之一,初步探讨PLBW与炎症因子之间的关系.方法 以83例PLBW的母亲(PLBW组)及44名健康新生儿(normal birth weight,NBW)的母亲(NBW组)为研究对象,留取受试对象的静息全唾液、龈沟液,应用酶联免疫吸附试验检测唾液和龈沟液中白细胞介素(IL)-1β和IL-6水平,比较两组间唾液和龈沟液中细胞因子水平的差异,并分析细胞因子与分娩孕周、新生儿体重之间的相关性.结果 唾液IL-1β水平在PLBW组[(78.32±11.81)ng/L]显著高于NBW组[(39.66±11.89)ng/L],差异有统计学意义(P<0.05);分娩孕周与唾液IL-6之间呈显著的负相关关系(r=-0.274,P<0.01);新生儿体重与龈沟液IL-1β(r=-0.231,P<0.01)、唾液IL-6(r=-0.424,P<0.01)之间呈显著的负相关关系.结论 唾液和龈沟液中炎性因子水平越高,分娩孕周越短、新生儿体重越轻,母体牙周炎程度与PLBW之间可能存在一定程度的相关关系.     

龈沟液中的IL—8抑制因子及其生物活性

In order to confirm the existence of Interleukin-8 (IL-8) inhibitor and its biological activity in gingival crevicular fluid (GCF), this study examined the GCF taken from 7 adult periodontitis (AP) patients. In neutralization test of IL-8, the results showed that the mean level of IL-8 was less than 1 ng/ml, which had been added into the GCF before ELESAs was performed to measure the amount of IL-8 in GCF. The mean level of IL-8 in the GCF of AP group was significantly lower than that of healthy group (P < 0.001). In biological activity test of IL-8 inhibitor, using pooled GCF taken from 8 AP patients (23 teeth), the results showed that the GCF (without recombinant human IL-8, rhIL-8) caused more white blood cell (WBC) migration than blank control group (physiological saline) did. When the amount of rhIL-8 increased in GCF from 0.1 microgram to 1 microgram, the WBC count increased by 18.6% which was less than the increase rate (49.1%) in control group with same dose of IL-8. In the saline group containing rhIL-8, the WBC chemotactic response appeared as an inverted "V"-shape curve. All these data suggested that 1. Certain kinds of IL-8 inhibitor exist in GCF which can "cleave" IL-8. 2. The level of IL-8 inhibitor(s) increases significantly in the GCF from periodontitis sites. 3. The GCF of adult periodontitis patient has strong chemotactic effects on WBC. IL-8 inhibitor(s) in GCF can slightly suppress the chemotactic effect induced by IL-8. When assessing the role of IL-8 in pathophysiology, the high and low dose of IL-8 might have different sense.     

The influence of diabetes on gingival crevicular fluid beta-glucuronidase and interleukin-8

OBJECTIVES: Polymorphonuclear neutrophil (PMN) dysfunction is associated with diabetes. We examined the gingival crevicular fluid (GCF) beta-glucuronidase (BG) and interleukin-8 (IL-8) levels of periodontitis patients with and without type 2 diabetes mellitus (DM). MATERIAL AND METHODS: Forty five adults with type 2 DM and 32 adults without DM, both with chronic periodontitis were enrolled. GCF was collected from eight posterior sites in each quadrant, and periodontal parameters were recorded. GCF was assayed for IL-8 by ELISA and BG by a fluorometric assay. RESULTS: GCF IL-8 was positively correlated with probing depth (PD), and GCF BG but not clinical attachment level (CAL), bleeding on probing (BOP), or plaque index (PI). In contrast, GCF BG was strongly correlated with each of the clinical measures of periodontal disease. Subjects with DM significantly lower levels of both BG (73.0+/-44.8 versus 121.9+/-84.6 pg/sample; p=0.002) and IL-8 (32.1+/-33.1 versus 90.8+/-83.2 pg/sample; p<0.0001) even after adjustments for age, gender, PD, CAL, BOP, and PI. Neither BG nor IL-8 was correlated with HbA1c levels in subjects with DM. CONCLUSION: These data suggest that an inadequate local response by PMN, partially explained by an altered chemokine gradient, may contribute to periodontal disease in patients with type 2 DM.     

Optimization of buffer solutions to analyze inflammatory cytokines in gingival crevicular fluid by multiplex flow cytometry

Objective: the aim of this study was to test two buffer solutions in order to attain a reliable and reproducible analysis of inflammatory cytokines (IL-1β, IL-6, TNF-α, OPG, OPN and OC), in gingival crevicular fluid (GCF) by flow cytometry. Material and Methods: GCF samples from healthy volunteers were collected with perio-paper strips and diluted either in phosphate buffered saline (PBS) or Tris-HCl buffer, with and without protease inhibitors (PI). Cytokine immunoassays were carried out by flow cytometry (Luminex Xmap 200) generating standard curves. Results: standards curves generated with the use of phosphate-buffered saline (PBS) demonstrated best adjustment for cytokines IL-1ß, IL-6 and TNF- α levels, when using Tris-HCl (p<0.05). Conclusions: The use of PBS buffer with the addition of PI provided reliable measurements of inflammatory biomarkers in GCF samples of healthy volunteers. Key words:Curve fitting, flow cytometer, immunoassay buffer, crevicular fluid, cytokines.