Refractory neutrophil activation in type 2 diabetics with chronic periodontitis.

BACKGROUND: Inflammation increases diabetes mellitus type 2 (T2DM) progression and severity. T2DM patients are at high risk of the rapid development of chronic periodontitis (CP). Topical presence, high numbers, and bactericidal effects of immune cells are challenged by augmented antigen-induced inflammation, which promotes both diseases. OBJECTIVES: To investigate gingival cellular inflammatory responses in individuals with previously undiagnosed T2DM with CP or CP alone and in systemically and periodontally healthy controls (H) in vivo and to establish an ex vivo technique permitting quantitative and qualitative assessments of gingival crevicular immune cells. MATERIALS AND METHODS: T2DM + CP, CP, and H individuals (n = 10, each) received a 2-week oral hygiene regimen (OHR). Afterwards, a noninvasive sampling technique was performed to evaluate gingival inflammation induced under standardized conditions in vivo, that is, in the absence of severe periodontal destruction and inflammation at clinically healthy sites. Stimuli (casein/test or phosphate-buffered saline w/o. Ca2+ or Mg2+ , PBS(-/-) /control) were randomly applied contralaterally in the gingival sulci of participants' upper dentes canini. One day after completion of the OHR, gingival crevicular fluid (GCF) was kinetically assayed between the time of the baseline (BL) measurement and 55 minutes. Polymorphonuclear leukocyte (PMN) content (PMNGCF ) was quantitated at an optimum time of 35 minutes. PMNGCF counts reflect local inflammation. Ex vivo samples were fluorimetrically labeled, gated according to the donor's peripheral blood polymorphonuclear neutrophils (PMNPB ), and then counted, employing flow cytometry. RESULTS: PMNGCF counts in unstimulated gingival crevices (at BL) in the T2DM + CP group were higher than those in the CP and H groups. PMNGCF counts were elevated in casein vs PBS(-/-) -stimulated gingival crevices in all groups. Patients with T2DM + CP showed increased PMNGCF counts compared to those with CP (P = .035) according to scatter plots. CD45+ counts in the stimulated sites in T2DM + CP patients were higher than those in CP and H patients (P = .041). Under stimulation conditions, the CD45+ counts differed from those under placebo conditions (P = .019), indicating augmented, inducible inflammatory leukocyte infiltrate in T2DM + CP patients. CONCLUSIONS: This noninvasive technique permits quantitative assessment of (experimental) gingival inflammation in vivo, revealing an influence of T2DM + CP on the number of primary immune cells in the gingival crevice. Patients who are challenged with (local) leukocytosis are likely at risk of collateral damage to the gingival crevice neighboring tissues, favoring the severity and progression of CP and consequently T2DM (www.clinicaltrials.gov NCT01848379).

Cellular populations of periimplant tissues: cytological analysis with sulcular microbrushing.

PURPOSE: Cellular populations of gingival crevicular fluid cytological analysis of integrated implants sites have been investigated by using sulcular cytological brushing, as a means of providing an objective and reproducible technique for monitoring periimplant tissue health. MATERIALS AND METHODS: A total of 60 patients with osteointegrated implants bearing at least for 2 years were divided in 2 groups, A and B. Group A consisted of 30 subjects who presented scarce oral hygiene. In Group B, 30 subjects with a good oral hygiene were included. RESULTS: Comparative analysis of the data obtained by sulcular microbrushing of the 2 groups put into evidence significative differences in the expression of the microbiological and the cytological parameters. CONCLUSION: Clinical monitoring of parodontal and periimplant tissues makes use of several diagnostic tests ranging from clinical and radiological tests to biological assays. However, none of these techniques allows to evaluate periimplant tissue cytological status. This preliminary study suggested sulcular microbrushing might be a useful tool in the early diagnosis and in the micrological monitoring of peri-implantitis.

Periodontal treatment downregulates protease-activated receptor 2 in human gingival crevicular fluid cells.

Protease-activated receptor 2 (PAR2) is implicated in the pathogenesis of chronic inflammatory diseases, including periodontitis; it can be activated by gingipain and produced by Porphyromonas gingivalis and by neutrophil protease 3 (P3). PAR2 activation plays a relevant role in inflammatory processes by inducing the release of important inflammatory mediators associated with periodontal breakdown. The effects of periodontal treatment on PAR2 expression and its association with levels of proinflammatory mediators and activating proteases were investigated in chronic periodontitis patients. Positive staining for PAR2 was observed in gingival crevicular fluid cells and was reflective of tissue destruction. Overexpression of PAR2 was positively associated with inflammatory clinical parameters and with the levels of interleukin-6 (IL-6), IL-8, tumor necrosis factor alpha, matrix metalloprotease 2 (MMP-2), MMP-8, hepatocyte growth factor, and vascular endothelial growth factor. Elevated levels of gingipain and P3 and decreased levels of dentilisin and the protease inhibitors secretory leukocyte protease inhibitor and elafin were also associated with PAR2 overexpression. Healthy periodontal sites from individuals with chronic periodontitis showed diminished expression of PAR2 mRNA and the PAR2 protein (P < 0.05). Furthermore, periodontal treatment resulted in decreased PAR2 expression and correlated with decreased expression of inflammatory mediators and activating proteases. We concluded that periodontal treatment resulted in decreased levels of proteases and that proinflammatory mediators are associated with decreased PAR2 expression, suggesting that PAR2 expression is influenced by the presence of periodontal infection and is not a constitutive characteristic favoring periodontal inflammation.

[Crystallographic picture of the gingival fluid in healthy teeth and by inflammatory periodontal disease].

The research revealed crystallographic structure of the gingival fluid in healthy teeth and by inflammatory periodontal disease. Comparing the results with clinical data both crystallographic patterns in healthy teeth gingival fluid and markers of periodontal tissue pathology were established.

The neutrophil subset defined by CD177 expression is preferentially recruited to gingival crevicular fluid in periodontitis.

In recent years, the concept of distinct subpopulations of human neutrophils has attracted much attention. One bona fide subset marker, exclusively expressed by a proportion of circulating neutrophils in a given individual, and therefore dividing neutrophils in two distinct subpopulations, is the glycoprotein CD177. CD177 is expressed on the plasma and granule membranes of 0-100% of circulating neutrophils depending on the donor. Several in vitro studies have linked CD177 to neutrophil transmigration, yet very few have looked at the role of CD177 for tissue recruitment in vivo. We investigate whether the CD177+ and CD177- neutrophil subsets differ in their propensity to migrate to both aseptic- and microbe-triggered inflamed human tissues. Microbe-triggered neutrophil migration was evaluated in samples of gingival crevicular fluid (GCF) from patients with periodontitis, whereas neutrophil migration to aseptic inflammation was evaluated in synovial fluid from patients with inflammatory arthritis, as well as in exudate from experimental skin chambers applied on healthy donors. We found that the proportion of CD177+ neutrophils was significantly higher in GCF from patients with periodontitis, as compared to blood from the same individuals. Such accumulation of CD177+ neutrophils was not seen in the two models of aseptic inflammation. Moreover, the proportion of CD177+ neutrophils in circulation was significantly higher in the periodontitis patient group, as compared to healthy donors. Our data indicate that the CD177+ neutrophil subset is preferentially recruited to the gingival crevice of periodontitis patients, and may imply that this subtype is of particular importance for situations of microbe-driven inflammation.

Neutrophil fate in gingival crevicular fluid.

The fate of the neutrophils within the inflammatory exudate in the periodontal crevice and their possible participation in the formation of neutrophil extracellular traps (NETs) are of clinical interest. However, the cytological analysis of clinical samples of inflammatory exudate is restricted by the obtainable quantities, which do not enable employing the routine approaches. Clinical examinations, ACLAR strip sampling, scanning electron microscopy, and confocal laser scanning microscopy were employed to analyze purulent crevicular exudate and gingival crevicular fluid in periodontitis. Bacteria, neutrophil activation, NETosis stages, and NETs were identified by molecular probe, expression of citrullinated histone H3, enzymatic digestion, and ultrastructurally. Crevicular neutrophils, all in diverse NETosis stages marked by the histone citrullination, and an abundance of NETs were found in both purulent crevicular exudate and gingival crevicular fluid. Largely varying quantities of dispersed crevicular bacteria were entrapped by NETs, but no phagocytized bacteria were evident in gingival crevicular fluid. The offered method enables for the first time the demonstration NETs in gingival crevicular fluid. The histone citrullination of all the floating crevicular neutrophils indicates that they all undergo NETosis.

Fibrin mimics neutrophil extracellular traps in SEM.

Neutrophil extracellular traps (NETs) are extracellular web-like structures produced by activated polymorphonuclear neutrophils. NETs kill bacteria extracellularly, but their role in human pathology remains largely unclear. One possible way of studying NETs is through the SEM approach. However, web-like structures observed with SEM in sites of inflammation have been interpreted either as NETs or as fibrin. Thus, the question arises whether a reliable SEM discrimination between NETs and fibrin is at all possible. NET samples were collected as purulent crevicular exudate from periodontal pockets. DNase-digested controls for SEM were employed to demonstrate the DNA backbone and immuno-staining for confocal laser scanning microscopy was used to show the citrullinated histones of NETs. Blood clot samples were treated in the same way as the exudate samples to demonstrate that fibrin and fibrinolysis can mimic NETs and DNA digestion, respectively. No discrimination between fibrin and NETs based on morphological criteria in SEM was possible. Furthermore, only a vague distinction between DNA digestion and fibrinolysis could be made. These findings unambiguously indicate that the discrimination between NETs and fibrin by means of SEM is untrustworthy for samples of inflammatory exudate.

Oral extracellular vesicles in early pregnancy can identify patients at risk of developing gestational diabetes mellitus.

AIM: To isolate and characterize oral extracellular vesicles from gingival crevicular fluid at 11-14 weeks and evaluate their capacity to identify patients at risk of developing gestational diabetes mellitus. METHODS: A case-control study was conducted, including patients who developed gestational diabetes mellitus (n = 11) and healthy pregnant controls (n = 23). Obstetric and periodontal histories were recorded at 11-14 weeks of gestation, and samples of gingival crevicular fluid obtained. Extracellular vesicles were isolated from gingival crevicular fluid by ExoQuick. Nanoparticle tracking analysis, ELISA and transmission electron microscopy were used to characterize extracellular vesicles. RESULTS: Total extracellular vesicles isolated from gingival crevicular fluid were significantly higher in patients who developed gestational diabetes mellitus later in pregnancy compared to normoglycemic pregnant women (6.3x109 vs 1.7 x1010, p value = 0.0026), and the concentration of the extracellular vesicles delivered an area under the ROC curve of 0.81. The distribution size of extracellular vesicles obtained using ExoQuick was around 148 ± 57 nm. There were no significant differences in the periodontal status between cases and controls. The exosome transmembrane protein CD63 was also detected in the extracellular vesicles of gingival crevicular fluid. CONCLUSION: We were able to isolate extracellular vesicles from gingival crevicular fluid using a method that is suitable to be applied in a clinical setting. Our results provide an insight into the potential capacity of first trimester oral extracellular vesicles as early biomarkers for the prediction of gestational diabetes mellitus in pre-symptomatic women.

Influence of amine fluoride/stannous fluoride mouthwashes with and without chlorhexidine on secretion of proinflammatory molecules by peri-implant crevicular fluid cells.

AIM: Patients with dental implants need optimal plaque control. Peri-implantitis is an inflammation of soft and hard tissues around implants characterized by bone loss mediated by proinflammatory molecules such as IL-1beta, PGE(2), vascular endothelial growth factor (VEGF). The aim of this study was to evaluate the influence of amine fluoride/stannous fluoride (AmF-SnF(2)) vs chlorhexidine 0.12% (CHX) combined with Am-SnF(2) on IL-1beta, PGE(2) and EGF secretion by cells of crevicular peri-implant fluid. METHODS: Thirty patients with dental implants were included in this study. The test group used AmF-SnF(2) rinsing for 14 days, the control group used CHX rinsing during the first 7 days and AmF-SnF(2) during the following 7 days. Crevicular samples were collected using filter paper strips and assayed for level of IL-1beta, PGE(2) and VEGF with ELISA test. Data were analyzed with paired and unpaired t test. RESULTS: IL-1beta, VEGF and PGE(2) levels were significantly lower in test compared to control group. Comparing first with second week of treatment, a greater decrease of IL-1beta and VEGF was evident in sample group during the second week. There was a lower decrease of IL-1beta and VEGF during the entire treatment in control group. Differences of PGE(2) levels after 7 days in both the groups were not significant while there was a significant difference during the second week. CONCLUSION: The following data suggest that the use of AmF-SnF(2) could decrease the production of IL-1beta, PGE(2) and VEGF by inflammatory cells.AmF-SnF(2) could be an alternative to CHX mouth rinses in plaque control of patients with implants.

Influence of Dental Restorations on Oxidative Stress in Gingival Crevicular Fluid.

Biocompatibility of dental materials (DM) can be evaluated by gingival crevicular fluid (GCF) oxidative stress (OS) status. The goal of the study was to ascertain influence of dental caries degree, teeth position, and type and amount of applied DM on GCF OS profile. For this purpose, we tested six DMs that were sealed in one session: amalgam (Amg), composites: Tetric EvoCeram and Beautifil (BF), phosphate cement-zinc phosphate and polycarboxylate cements-zinc polycarboxylate cements, and glass ionomer cement (GIC). The study included 88 dental outpatients. Follow-up was scheduled at 7th and 30th day. Oxidative stress parameters (malondialdehyde (MDA) and glutathione (GSH) levels and total superoxide dismutase (tSOD) activity) were measured before (0th day) and after the treatment (7th and 30th day) in GCF. Control teeth were mirror-positioned healthy teeth. The DM accomplished the following effects (listed in descending order): increase of GSH in GCF was realized by ZPoC > BF > GIC > Amg; tSOD activity increase by ZPoC > BF > Amg; and MDA decrease by ZPoC > ZPhC > Amg > TEC. Dental caries provokes insignificant rise of OS in GCF. ZPoC and ZPhC showed the highest antioxidant effect, contrary to GIC. Restorations with antioxidant properties may reduce gum diseases initiated by caries lesion, what is of great clinical relevance in dentistry.

Identification of Gingival Crevicular Fluid Sampling, Analytical Methods, and Oral Biomarkers for the Diagnosis and Monitoring of Periodontal Diseases: A Systematic Review.

Background. Several studies in the last decades have focused on finding a precise method for the diagnosis of periodontal disease in its early stages. Aim. To evaluate from current scientific literature the most common and precise method for gingival crevicular fluid (GCF) sample collection, biomarker analytical methods, and the variability of biomarker quantification, even when using the same analytical technique. Methodology. An electronic search was conducted on in vivo studies that presented clinical data on techniques used for GCF collection and biomarker analysis. Results. The results showed that 71.1%, 24.7%, and 4.1% of the studies used absorption, microcapillary, and washing techniques, respectively, in their gingival crevicular fluid collection. 73.1% of the researchers analyzed their samples by using enzyme-linked immunosorbent assay (ELISA). 22.6%, 19.5%, and 18.5% of the researchers included interleukin-1 beta (IL-1ß), matrix metalloproteinase-8 (MMP-8), and tumor necrosis factor-alpha (TNF-α), respectively, in their studies as biomarkers for periodontal disease. Conclusion. IL-1ß can be considered among the most common biomarkers that give precise results and can be used as an indicator of periodontal disease progression. Furthermore, paper strips are the most convenient and accurate method for gingival crevicular fluid collection, while enzyme-linked immunosorbent assay can be considered the most conventional method for the diagnosis of biofluids.

Aberrant neutrophil reactions in periodontitis.

BACKGROUND: The aim of this study was to compare the activity of neutrophilic granulocytes in patients with severe periodontitis and patients with gingivitis alone. METHODS: The study population comprised 22 patients with gingivitis and 44 with periodontitis. Samples of gingival crevicular fluid (GCF) were collected from untreated patients with gingivitis and from shallow and deep pockets in untreated patients with periodontitis. GCF samples were analyzed for lactoferrin, elastase, matrix metalloproteinase-8 and -9, and collagenolytic activity. RESULTS: The free elastase activity and the neutrophil activity, estimated as the ratio between elastase and lactoferrin, were significantly higher in the samples from the periodontitis patients. These differences were also observed in shallow pockets in periodontitis patients compared to similar pockets in patients with gingivitis. CONCLUSION: This study shows higher levels of free elastase in untreated patients with periodontitis, relative to inflammation-matched controls, which may explain the tissue destruction seen in periodontitis.

Phagocytic and killing activity of human blood, gingival crevicular, and salivary polymorphonuclear leukocytes for oral streptococci.

The phagocytosis and killing of oral streptococci by blood, crevicular, and salivary polymorphonuclear leukocytes (PMNL) were examined using a visual assay based on differential staining of viable and non-viable microorganisms by acridine orange. Crevicular PMNL were 83% viable, 19% contained bacteria on collection, and phagocytosis occurred in vitro in 66% of glass-adherent leukocytes. Salivary PMNL were 56% viable, 11% contained bacteria on collection, and 44% phagocytosed streptococci in vitro. Crevicular and salivary PMNL were capable of phagocytosis and killing of oral streptococci, but both were impaired. Crevicular fluid was not significantly leukotoxic; mixed saliva caused a significant reduction in PMNL viability and in phagocytic and killing activity for oral streptococci. Crevicular PMNL may be actively functional phagocytes, but salivary PMNL are unlikely to be significant in oral defenses.

Amplified crevicular leukocyte activity in aggressive periodontal disease.

Exaggerated neutrophil responses are a critical component in the pathogenesis of periodontal disease. We investigated whether leukocyte activity in aggressive periodontitis (AP) is increased compared with that in chronic periodontitis (CP) by gingival crevicular fluid (GCF) analysis of myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH), cathepsin D (CD), and elastase-alpha-1-proteinase inhibitor complex (alpha-1-EPI) before and 6 months after therapy. Initial AP neutrophil responses were significantly amplified compared with those in CP (MPO, 3.2-fold; beta-NAH, 37.5-fold; CD, 2.2-fold; alpha-1-EPI, 1.4-fold; p < 0.05). Surgical therapy resulted in a significant reduction of GCF markers compared with non-surgical treatment. However, the changes in clinical parameters were not different between AP and CP (P > 0.05). Analysis of the results suggests that the local inflammatory response in AP is characterized by increased release of inflammatory mediators of neutrophil origin into the GCF. Analysis of the data further suggests that surgical therapy is a more predictable method for removal of the pro-inflammatory etiology.

Gingival crevice neutrophil function in periodontosis.

This study examined gingival crevicular polymorphonuclear leucocyte function in periodontosis patients. Cells were examined for viability, function and ultrastructure. Eighty percent or more of the cells in each sample were viable as assessed by the fluorescein diacetate technique, but the test organism, Candida guillermondiae, was not phagocytosed. Gingival crevicular fluid contained many lysing neutrophils and nonphagocytosed organisms. Recognizable polymorphs contained Gram-negative and Gram-positive organisms. On the basis of this and previous studies it is concluded that gingival crevice neutrophils from periodontosis sites show reduced phagocytic function compared with cells from normal or periodontitis-affected gingival crevices. It is possible that the behavior of neutrophils from gingival crevices may be irrelevant. Original changes by that stage may have obscured their capabilities.

Short-term effects of phase I therapy on crevicular cell populations.

The purpose of the present study was to use a novel intracrevicular lavage technique to evaluate short-term effects of phase I therapy on crevicular cell populations. Nineteen patients with untreated advanced adult periodontitis were selected for phase I therapy. One side of the dentition was treated with ultrasonic curets (U), the other side with manual curets (M). Nine months before (-9), immediately prior to (0), and 1 month after treatment (+1) gingival index (GI), plaque index (PI), bleeding index (BI), bleeding on probing (BOP), probing depth (PD), and probing attachment levels (PAL) were measured at all sites in the dentition. Crevicular lavages were obtained from 3 to 4 selected sites per patient at the same time points. Crevicular leukocytes were vital stained with ethydium bromide-fluorescein-diacetate (EB-FDA). The total number of cells and the percentage of vital cells (%) were calculated for each sample. Clinical and lavage parameters obtained from the selected sites were compared between U and M sites, and between pre- and post-treatment values. The results showed that without treatment PAL remained at the same level at both pretreatment time points -9 and -0 (control). At 1 month after treatment there were statistically significant reductions in GI, PI, BOP, and PD (P < 0.001 for each comparison), and a statistically significant gain in PAL from 4.9 to 4.1 mm (P = 0.014). The total number of leukocytes per sample was similar at both pretreatment time points, but numerically reduced at 1 month after treatment. The percent of vital leukocytes was above 74% at both pretreatment time points (control). After treatment these values were below 70%. This reduction was statistically significant (P < 0.002). These results suggest that periodontal phase I therapy leads to shifts in crevicular cell populations.

In vitro phagocytosis by crevicular phagocytes in various forms of periodontitis.

Phagocytes from the gingival crevice fluid (CF-cells) of 11 patients with localized juvenile and post-juvenile periodontitis (LJP/PJP), 14 with rapidly progressive periodontitis (RPP), 11 with adult periodontitis (AP), and 14 controls without periodontal disease were examined. Phagocytic activity in vitro was assessed. Crevicular washings were obtained from healthy sites of controls and diseased sites of patients after completion of the oral hygiene phase (professional and home care). The cells were carefully processed to avoid mechanical damage. The in vitro phagocytosis by uptake of opsonized C. albicans was performed in a moist chamber (30 minutes, 37 degrees C) and examined by light microscopy. CF-cells were differentiated on the basis of their morphological appearance. The majority of cells in crevicular washings were PMNs, some macrophages, and few lymphocytes. Phagocytic activity in patients with LJP/PJP and RPP was significantly decreased in comparison with that from AP and the control group. The decreased percentage of cells phagocytosing opsonized C. albicans was associated with the enhanced adherence of opsonized C. albicans. Moreover cell viability of CF-cells from LJP/PJP sites was significantly reduced. The data from the present study suggest that the in vitro phagocytosis of crevicular phagocytes in juvenile and rapidly progressive periodontitis lesions is diminished.

An electron microscopic study of human neutrophils obtained by crevicular washing.

The Application of electron microscopic methods to the study of neutrophils obtained from single sites by crevicular washing is described. A subsequent preliminary study of neutrophils and neutrophil-bacterial interactions at advanced lesions in patients with juvenile periodontitis and juvenile diabetes indicated that the neutrophils are phagocytically active at these sites. It is suggested that in vitro assays of crevicular neutrophil function may exaggerate the extent of impairment of phagocytosis because of cellular activity which has taken place prior to the time of sampling.

A rapid chairside test for the severity of periodontal disease using gingival fluid.

A study was done in an attempt to develop a simple test for the severity of periodontal disease. Gingival fluid collected on filter paper was tested for protein content, and the resulting color was compared to standard color filters. Tissue was excised and prepared for histologic examination. The inflammatory cell infiltrate on each slide was graded on a scale of zero to three. Zero was least and three was the highest number of inflammatory cells. The results indicate that the white cell infiltrate graded 0 or 1 on the histologic inflammatory index has a color index of B 1/8 TO B1 whereas the tissue graded 2 or 3 HII has a range of B2 to B6 on the color scale. This test can give the dental practitioner a general idea of the severity of the inflammation.

Effect of toothbrushing with a fluoride-free and fluoride-containing dentifrice on oral hygiene and number of leukocytes in the gingival fluid.

The number of leukocytes in the gingival exudate and the oral hygiene index during toothbrushing with a fluoride-free and fluoride-containing dentifrice was investigated in humans during a 3-month period. Significant correlations existed between the average number of leukocytes in the gingival exudate and the oral hygiene indices. The number of leukocytes increased significantly during the period of toothbrushing with the Fluoride dentifrice, whereas in the OHI no change was evident.