Cytokine levels in gingival tissues as an indicator to understand periodontal disease severity

Periodontitis is characterized by gingival regression, alveolar bone resorption and the development of deep periodontal pockets that, if left untreated, can lead to tooth loss. Currently, specific biomarkers are needed for the early, objective diagnosis, monitoring, and management of periodontal patients. In this proteomic study, periodontal pocket tissues from patients with severe periodontitis were analyzed in comparison to periodontally healthy sites with the aim of discovering distinctive protein targets. Gingival tissues were fragmented using a motorized mechanical method and mixture protein was separated via mono-dimensional gel electrophoresis. The examination of protein bands using definite 1D image analysis software allowed for the detection of 22 differentially expressed proteins between pathological and healthy samples that were identified through mass spectrometry. A comparative assessment of these proteins with those previously reported in other studies conducted on periodontal diseases in various types of oral specimens, such as gingival crevicular fluid, dentin, tooth pulp, root canal content, salivary gland secretions, saliva, periodontal ligament cells, and dental stem cells, highlighted a great number of significant common matches. The discovery of a selective cluster of periodontitis-related biomarkers could become particularly important before the clinical manifestation of the disease to promptly stop its progression for a timely preventive diagnosis.

We examined the subgingival bacterial biodiversity in untreated chronic periodontitis patients by sequencing 16S rRNA genes. The primary purpose of the study was to compare the oral microbiome in deep (diseased) and shallow (healthy) sites. A secondary purpose was to evaluate the influences of smoking, race and dental caries on this relationship. A total of 88 subjects from two clinics were recruited. Paired subgingival plaque samples were taken from each subject, one from a probing site depth >5 mm (deep site) and the other from a probing site depth ≤3mm (shallow site). A universal primer set was designed to amplify the V4–V6 region for oral microbial 16S rRNA sequences. Differences in genera and species attributable to deep and shallow sites were determined by statistical analysis using a two-part model and false discovery rate. Fifty-one of 170 genera and 200 of 746 species were found significantly different in abundances between shallow and deep sites. Besides previously identified periodontal disease-associated bacterial species, additional species were found markedly changed in diseased sites. Cluster analysis revealed that the microbiome difference between deep and shallow sites was influenced by patient-level effects such as clinic location, race and smoking. The differences between clinic locations may be influenced by racial distribution, in that all of the African Americans subjects were seen at the same clinic. Our results suggested that there were influences from the microbiome for caries and periodontal disease and these influences are independent.

BackgroundSubgingival microbiome in disease-associated subgingival sites is known to be dysbiotic and significantly altered. In patients with rheumatoid arthritis (RA), the extent of dysbiosis in disease- and health-associated subgingival sites is not clear.Methods8 RA and 10 non-RA subjects were recruited for this pilot study. All subjects received full oral examination and underwent collection of subgingival plaque samples from both shallow (periodontal health-associated, probing depth ≤ 3mm) and deep subgingival sites (periodontal disease-associated, probing depth ≥ 4 mm). RA subjects also had rheumatological evaluation. Plaque community profiles were analyzed using 16 S rRNA sequencing.ResultsThe phylogenetic diversity of microbial communities in both RA and non-RA controls was significantly higher in deep subgingival sites compared to shallow sites (p = 0.022), and the overall subgingival microbiome clustered primarily according to probing depth (i.e. shallow versus deep sites), and not separated by RA status. While a large number of differentially abundant taxa and gene functions was observed between deep and shallow sites as expected in non-RA controls, we found very few differentially abundant taxa and gene functions between deep and shallow sites in RA subjects. In addition, compared to non-RA controls, the UniFrac distances between deep and shallow sites in RA subjects were smaller, suggesting increased similarity between deep and shallow subgingival microbiome in RA. Streptococcus parasanguinis and Actinomyces meyeri were overabundant in RA subjects, while Gemella morbillorum, Kingella denitrificans, Prevotella melaninogenica and Leptotrichia spp. were more abundant in non-RA subjects.ConclusionsThe aggregate subgingival microbiome was not significantly different between individuals with and without rheumatoid arthritis. Although the differences in the overall subgingival microbiome was driven primarily by probing depth, in contrast to the substantial microbiome differences typically seen between deep and shallow sites in non-RA patients, the microbiome of deep and shallow sites in RA patients were more similar to each other. These results suggest that factors associated with RA may modulate the ecology of subgingival microbiome and its relationship to periodontal disease, the basis of which remains unknown but warrants further investigation.

Gingival crevicular fluid (GCF) biomarkers associated with bone resorption may be useful to determine periodontal disease status and response to therapy. The pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP), a bone-specific degradation product, and interleukin 1-beta (IL-1), a potent bone-resorptive cytokine, have both been associated with periodontal disease activity. Minocycline is a tetracycline derivative possessing antimicrobial effects on periodontal pathogens and inhibitory properties on matrix metalloproteinases (MMPs) associated with tissue destruction. The aim of this study was to evaluate the effect of periodontal treatment in the form of scaling and root planing (SRP) and locally administered minocycline microspheres on the GCF levels of ICTP and IL-1. Forty-eight chronic periodontitis patients were randomly assigned to 2 groups (SRP plus subgingival application of vehicle control [SRP + V], or SRP plus subgingival application of minocycline microspheres [SRP + M]) and monitored at 8 sites per subject at baseline and 1, 3, and 6 months. Four shallow (PD < or = 3 mm) and 4 deep (PD > or = 5 mm) sites were evaluated for both marker levels and for probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP). Eight periodontally healthy control subjects with no probing depths >3 mm and no loss of attachment were also monitored at the same time intervals. GCF levels of ICTP and IL-1 were determined using radioimmunoassay and enzyme-linked immunosorbent assay techniques, respectively. Significant differences (P<0.001) in GCF levels of ICTP and IL-1 were found between deep and shallow sites at all time points in both treatment groups. In addition, healthy subjects demonstrated significantly reduced levels of both markers compared to both shallow and deep sites in periodontitis patients (P <0.001). Only the SRP + M treated patients exhibited significant reductions (P <0.05) in both ICTP and IL-1 levels 1 month after treatment. Furthermore, the SRP + M group demonstrated significantly lower IL-1 levels (P <0.02) at 1 month compared to the SRP + V group. Results of this study indicate that GCF levels of ICTP and IL-1 correlate with clinical measures of periodontal disease and may aid in assessing disease status and response to periodontal therapy. Furthermore, local administration of minocycline microspheres led to a potent short-term reduction in GCF IL-1 levels. Additional studies are needed to address whether repeated administration of scaling and root planing along with minocycline microspheres will achieve long-term reductions in GCF ICTP and IL-1 levels.

The relationship between type 1 diabetes mellitus (T1DM) and periodontal disease may exhibit by the alteration of bone metabolism. However, evidence for this relationship is scarce and inconclusive. Thus, the aims of the present study were to investigate salivary receptor activator of nuclear factor kappa-β (RANK), receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG) gene expression and the RANKL:OPG ratio in T1DM and non-T1DM. Secondary objective was to determine the relationships of RANK, RANKL and OPG gene expression to clinical parameters of T1DM and periodontal disease. Twenty patients with T1DM and twenty age-matched non-T1DM were recruited. Clinical periodontal parameters were measured. Total RNA was isolated from non-stimulated saliva, and the relative gene expressions of RANK, RANKL, OPG and RANKL:OPG ratio were determined by quantitative real-time polymerase chain reaction. The T1DM group had significantly higher mean periodontal parameters than the non-T1DM group, while the mean plaque scores of both groups were not significantly different. There was a trend of higher relative gene expression of RANK, RANKL, and the RANKL:OPG ratio and lower expression of OPG in T1DM group but no statistic significant different when compared to non-T1DM. In the T1DM group, RANKL:OPG correlated with the percentage of bleeding sites, whereas RANK, RANKL, and HbA1c levels correlated with pocket depth. Bone metabolisms demonstrating by decreased OPG gene expression and upregulated of RANK, RANKL, RANKL:OPG with higher pocket depth and bleeding in T1DM may play an important role in periodontal destruction in T1DM.

Periodontal disease (PD) is more common and severe in people with diabetes than the general population. The presence of severe PD is correlated with decreased survival, since it can potentially affect glycemic control and severity of complications in people with diabetes. We have reported that Medalists (people with T1DM of 50 years or longer duration) may have endogenous protective factors for the development of diabetic nephropathy (DN) and retinopathy (DR). This study assessed the prevalence of PD in the Medalist cohort and correlated it to the known risk factors of PD and diabetic complications. Severity of PD was defined in a subset (n=170) of Medalists with comparable characteristics of the whole cohort, according to the AAP criteria. The prevalence of severe PD was dramatically decreased in Medalists (13.5%) compared with other studies of people with diabetes of similar age, approximately 33%. Clinical parameters, such as male gender, chronological age, age at diagnosis, and total insulin dose were correlated positively with severity of PD (p=0.04, 0.01, 0.03, and 0.02, respectively), while duration of disease, hemoglobin A1c, BMI, and lipid profiles did not exhibit any correlation. Interestingly, detectible plasma C-peptide levels correlated inversely with severity of PD (p=0.04). Systemic inflammatory markers, such as plasma IL-6, clearly correlated positively with the severity of PD (p=0.01). Serum antibody titer against Porphyromonas gingivalis (Pg), a known pathogen of PD, trended with severity of PD (p=0.08). Amongst the various complications, only the prevalence of CVD correlated positively with severity of PD (p=0.02). These results suggest that Medalists are protected from severe PD even with hyperglycemia. The endogenous protective factors for PD could be similar to those for CVD, possibly related to endogenously produced insulin to neutralize the chronic inflammation caused by residual infection with Pg in the gingival tissues. Disclosure T. Shinjo: None. A. Ishikado: Employee; Self; Sunstar Inc.. Employee; Spouse/Partner; Sunstar Inc.. H. Hasturk: None. L.J. Tinsley: None. D.M. Pober: None. I. Wu: None. T.E. Van Dyke: None. R.J. Genco: None. G.L. King: Research Support; Self; Sanofi-Aventis.

Cell-derived microparticles (MPs) have been described as vital contributors to the inflammatory process. However, its role in the periodontal disease pathogenesis remains unclear. Therefore, we aimed to detect the presence neutrophil (CD66b+) and platelet (CD41b+) derived microparticles in gingival crevicular fluid from individuals having periodontitis aggravated by type 2 diabetes. Twelve patients (56.2 ±7.2 yrs) with severe form of chronic periodontitis aggravated by type 2 diabetes were included. Clinical and metabolic data were gathered. Gingival crevicular fluid was collected using filter strips from deep and shallow sites. MPs were detected by flow cytometry according to their size (< 1 µm) and the expression of surface markers (CD66b for neutrophil-derived MPs and CD41b for platelet-derived MPs). All samples were positive for the antibodies. Median levels of CD66b+ MPs and CD41b+ MPs were, respectively, 3,677.0 (2,553.2 - 9,059.8) MP/µL and 520.7 (432.9 - 766.1) MP/µL in deep sites. In shallow sites, the corresponding values were 2,644.9 (1,451.5 - 3,858.9) MP/µL and 371.2 (287.2 - 692.7) MP/µL. There was no significant difference between deep and shallow sites (p>0.05). In conclusion, this study reported the presence of neutrophil and platelet derived microparticles in gingival crevicular fluid from individuals having severe periodontitis and type 2 diabetes.

PurposeAloe-emodin (AE), a natural anthraquinone abundant in aloe plants and rhubarb (Rheum rhabarbarum), has long been used to treat chronic inflammatory diseases. However, AE’s underlying mechanisms in periodontal inflammation have not been fully elucidated. Acidic mammalian chitinase (AMCase) is a potential biomarker involved in bone remodeling. This study aimed to evaluate AE’s effect on periodontitis in rats and investigate AMCase expression.MethodsEighteen Sprague-Dawley rats were separated into the following groups: healthy (group 1), disease (group 2), vehicle (group 3), AE high-dose (group 4), and AE low-dose (group 5). Porphyromonas gingivalis ligatures were placed in rats (groups 2–5) for 7 days. Groups 4 and 5 were then treated with AE for an additional 14 days. Saliva was collected from all groups, and probing pocket depth was measured in succession. Periodontal pocket tissues were subjected to histomorphometric analysis after the rats were sacrificed. Bone marrow-derived macrophages and murine macrophages were stimulated with receptor activator of nuclear factor-κB ligand (RANKL) and treated with different concentrations of AE. AMCase expression was detected from the analysis of saliva, periodontal pocket tissues, and differentiated osteoclasts.ResultsAmong rats with P. gingivalis-induced periodontitis, the alveolar bone resorption levels and periodontal pocket depth were significantly reduced after treatment with AE. AMCase protein expression was significantly higher in the disease group than in the healthy control (P<0.05). However, AE inhibited periodontal inflammation by downregulating AMCase expression in saliva and periodontal pocket tissue. AE significantly reduced RANKL-stimulated osteoclastogenesis by modulating AMCase (P<0.05).ConclusionsAE decreases alveolar bone loss and periodontal inflammation, suggesting that this natural anthraquinone has potential value as a novel therapeutic agent against periodontal disease.

subjects with periodontitis and chronic pain. Int. J. Odontostomat., 8(2):279-287, 2014. ˚ ABSTRACT: Periodontal disease (PD) is a chronic infection that may have local and systemic rebound. Although a series of inflammatory mediators are involved in PD, the mechanisms involved in chronic craniofacial pain associated with it require elucidation. The aim of this study was to evaluate the immunoreactivity of substance P (SP), neuronal (nNOS) and inducible (iNOS) nitric oxide synthases in gingival samples of patients with severe PD with and without chronic craniofacial pain. Gingival specimens were obtained during routine periodontal surgery while managing 20 patients with both PD and chronic craniofacial pain (CFP Group) and 18 patients with only PD (PD Group). Following surgical removal, the tissue underwent routine histological techniques and was stained by immunohistochemistry with antibodies against SP, nNOS and iNOS. Using an image analysis system, we assessed the SP, nNOS and iNOS content in total gingival tissue as well as in both epithelial and connective gingival area. We observed high expression of nNOS in gingival tissue obtained from CFP patients (p<0.001), particularly in the epithelium area (p<0.001) comparatively to PD patients. In addition, the iNOS expression was also increased in the CFP group in the connective gingival tissue (p=0.003). There was no difference concerning SP expression between the groups. Our results suggest that nitric oxide, particularly derived from nNOS, modulates not only PD but also chronic craniofacial pain, since patients with this association presented an increase in nNOS and iNOS expression in gingival tissue.˚ ˚

Deep and shallow water characteristics of mixed layer response to momentum and buoyancy fluxes off Bombay, west coast of India, during winter

BackgroundThe periodontal disease is caused by a set of inflammatory disorders characterized by periodontal pocket formation that lead to tooth loss if untreated. The proteomic profile and related molecular conditions of pocket tissue in periodontally-affected patients are not reported in literature. To characterize the proteomic profile of periodontally-affected patients, their interproximal periodontal pocket tissue was compared with that of periodontally-healthy patients. Pocket-associated and healthy tissue samples, harvested during surgical therapy, were treated to extract the protein content. Tissues were always collected at sites where no periodontal-pathogenic bacteria were detectable. Proteins were separated using two-dimensional gel electrophoresis and identified by liquid chromatography/mass spectrometry. After identification, four proteins were selected for subsequent Western Blot quantitation both in pathological and healty tissues.ResultsA significant unbalance in protein expression between healthy and pathological sites was recorded. Thirty-two protein spots were overall identified, and four proteins (S100A9, HSPB1, LEG7 and 14-3-3) were selected for Western blot analysis of both periodontally-affected and healthy patients. The four selected proteins resulted over-expressed in periodontal pocket tissue when compared with the corresponding tissue of periodontally-healthy patients. The results of Western blot analysis are congruent with the defensive and the regenerative reaction of injured periodontal tissues.ConclusionsThe proteomic analysis was performed for the first time directly on periodontal pocket tissue. The proteomic network highlighted in this study enhances the understanding of periodontal disease pathogenesis necessary for specific therapeutic strategies setting.

Membrane-bound CD14 (mCD14) is a myeloid differentiation antigen expressed on monocytes/macrophages and neutrophils. It is a key molecule responsible for the innate recognition of bacteria by host cells and functions as an important receptor for bacterial lipopolysaccharide. This study investigated the in vivo expression profile and levels of mCD14 in healthy and diseased gingival tissues. Gingival biopsies were obtained from 24 patients with chronic periodontitis, including 22 periodontal pocket tissues, 13 clinically healthy tissues, and 18 inflamed connective tissues (i.e., granulation tissues). Gingival biopsies from seven periodontally healthy subjects were used as controls. mCD14 was detected by immunohistochemistry. mCD14 was detected in 21 of 22 periodontal pocket tissues and all other categories of tissues. The mCD14-positive cells were mainly confined to the gingival epithelium-connective tissue interface. The expression levels in periodontally healthy subjects were significantly higher than in the patients. Within the patients, clinically healthy tissues showed greater levels of mCD14 than periodontal pocket tissues and granulation tissues. mCD14 was commonly expressed in both healthy and diseased gingival tissues and was predominantly confined to the epithelium-connective tissue interface. The positive relationship observed between mCD14 expression levels and periodontal health may imply that mCD14 is associated with favorable host responses to bacterial challenge and contributes to maintaining periodontal homeostasis.

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Immune responses in women with periodontitis and preterm low birth weight: Levels of CD4+ and CD8+ T cells in gingival granulation tissue

Cytokine expression in gingival and intestinal tissues of patients with periodontitis and inflammatory bowel disease: An exploratory study