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Background: Host inflammatory and immune responses play an important role in aggressive periodontitis (AgP). Thus, this study aims to evaluate levels of the innate immunity-related markers calprotectin, colony-stimulating factor (CSF)-1, macrophage migration inhibitory factor (MIF), monokine induced by interferon-γ (MIG), and matrix metalloproteinase (MMP)-8 in serum and saliva from patients with generalized AgP and those with gingivitis or a healthy periodontium.Methods: This study enrolled 40 individuals (17 males and 23 females; mean age 33.30 ± 9.31 years), 15 with generalized AgP, 15 with gingivitis, and 10 who were periodontally healthy. Full-mouth periodontal examinations were performed, and serum and saliva were collected. Levels of calprotectin, CSF-1, MIF, MIG, and MMP-8 were measured using enzyme-linked immunosorbent assays.Results: In serum, mean levels of calprotectin were 2.06-fold higher in patients with AgP than in healthy patients (P = 0.01). Serum levels of MMP-8 were significantly elevated in patients with AgP compared with both healthy patients and those with gingivitis, by 2.60-fold and 2.77-fold, respectively (P = 0.03 and P = 0.009, respectively). In saliva, levels of MMP-8 were 5.66-fold higher in patients with AgP than in healthy patients (P = 0.02). CSF-1, MIF, and MIG levels in both serum and saliva did not differ significantly among the groups.Conclusions: Serum levels of calprotectin and MMP-8 are elevated in patients with AgP. MMP-8 levels are also increased in saliva from patients with AgP. These results support involvement of innate immune response in the pathogenesis of AgP. [ABSTRACT FROM AUTHOR]

Background: The single nucleotide polymorphism (SNP) context of a previously identified periodontitis-associated locus is investigated, and its association with microbial, biologic, and periodontal disease clinical parameters is examined.Methods: A 200-kb spanning region of 1q12 previously highlighted in a genome-wide association scan among 4,766 European American individuals (SNP rs1633266) was annotated. Two haplotype blocks were selected. Association of these polymorphisms with data on microbial plaque composition, gingival crevicular fluid (GCF)-interleukin (IL)-1β levels, and clinical parameters of periodontal disease were examined. Descriptive analysis of IFI16 and AIM2 protein expression in gingival tissues from healthy individuals (n = 2) and individuals with chronic periodontitis (n = 2) was done via immunohistochemistry.Results: The highlighted locus is a 100-kb region containing the interferon γ-inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) genes. Two haplotype blocks, rs6940 and rs1057028, were significantly associated with increased extent bleeding on probing and levels of microorganisms Porphyromonas gingivalis, Tannerella forsythia, and Campylobacter rectus (P ≤0.05). Haplotype block rs1057028 was also significantly associated with pathogens Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, increased GCF-IL-1β levels, and extent of probing depth ≥4 mm (P ≤0.05). Prevalence of severe periodontitis (biofilm-gingival interface P3 classification) was positively associated with haplotype block rs1057028. Similar trends were observed for haplotype block rs1057028. IFI16 and AIM2 protein expression was observed in multiple cell types of gingival tissues, including inflammatory cells.Conclusion: This study found IFI16 and AIM2 SNPs associated with higher levels of periodontal microorganisms and an increased percentage of periodontal disease clinical parameters, suggesting the need for functional studies and additional fine-mapping of variants in the 1q12-locus. [ABSTRACT FROM AUTHOR]

Background: Rosuvastatin exhibits anti-inflammatory effects and reduces periodontal diseases and atherosclerosis; however, its role in regulating periodontopathogen-induced endothelial proinflammatory responses remains unclear. The purpose of this study is to determine whether rosuvastatin can reduce the proinflammatory response induced by Aggregatibacter actinomycetemcomitans (Aa) in human coronary artery endothelial cells (HCAECs). Methods: HCAECs were stimulated with purified Aa serotype b lipopolysaccharide (LPS) (Aa-LPS), heat-killed (HK) bacteria (Aa-HK), or live bacteria. Expression of Toll-like receptors and cellular adhesion molecules were evaluated by fluorometric enzyme-linked immunosorbent assay. Endothelial cell activation was evaluated by quantifying nuclear factor (NF)-kappa B-p65 and cytokine expression levels by quantitative polymerase chain reaction and flow cytometry. Effect of rosuvastatin in expression of the atheroprotective factor Krüppel-like factor 2 (KLF2) and cytokines were also studied using similar approaches. Results: HCAECs showed increased interleukin (IL)-6, IL-8, intercellular adhesion molecule 1, and platelet endothelial cell adhesion molecule 1 expression when stimulated with Aa-LPS or Aa-HK. NF-κB-p65 activation was induced by all antigens. Aa-induced IL-6 and IL-8 production was inhibited by rosuvastatin, particularly at higher doses. Interestingly, reduced IL-6 and IL-8 levels were observed in HCAECs stimulated with Aa in the presence of higher concentrations of rosuvastatin. This anti-inflammatory effect correlated with a significant increase of rosuvastatin-induced KLF2. Conclusions: These results suggest Aa-induced proinflammatory endothelial responses are regulated by rosuvastatin in a mechanism that appears to involve KLF2 activation. Use of rosuvastatin to prevent cardiovascular disease may reduce risk of endothelial activation by bacterial antigens. [ABSTRACT FROM AUTHOR]

Background: Obesity is associated with infiltration of macrophages into adipose tissue. However, effects of obesity on macrophage infiltration and activation in periodontal tissues with periodontitis are still to be elucidated.Methods: A diet-induced obesity 16-week mouse model was constructed, and periodontitis was induced by periodontal ligation for 10 days. The model consisted of periodontitis (P) and control (C) groups, with high fat (HF) and normal (N) diet conditions. Bone loss (BL) was analyzed by microcomputed tomography. In periodontal tissues, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) detected expressions of: 1) nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) pathway; 2) macrophage-specific marker (F4/80); and 3) macrophage chemotactic protein 1 (MCP1). Bone marrow-derived macrophages (BMDMs) from the mouse model were stimulated by Porphyromonas gingivalis lipopolysaccharide (LPS) in vitro (NC/NC + LPS: BMDMs from NC group without/with LPS stimulation; HFC/HFC + LPS: BMDMs from HFC group without/with LPS stimulation). Expressions of NLRP3 pathway in BMDMs were detected by immunocytochemical staining and qPCR.Results: BL increased significantly with periodontitis (NC versus NP; HFC versus HFP) and obesity (NP versus HFP). Expressions of NLRP3 pathway were significantly elevated in gingival tissues with periodontitis (NC versus NP; HFC versus HFP), but not with obesity (NC versus HFC; NP versus HFP). F4/80 and MCP1 expressions were significantly upregulated in gingival tissues with periodontitis (NC versus NP; HFC versus HFP) but significantly downregulated in the context of obesity (NP versus HFP). In vitro, NLRP3 pathway expressions were significantly upregulated in BMDMs after LPS stimulation (NC + LPS versus NC; HFC + LPS versus HFC), but significantly downregulated in HFC groups (HFC versus NC; HFC + LPS versus NC + LPS).Conclusion: Obesity may paralyze innate immune response of periodontium via attenuating infiltration and activation of macrophages and further aggravate periodontal disease. [ABSTRACT FROM AUTHOR]

Background: Innate and adaptive immunosurveillance mechanisms in response to the normal commensal bacteria can affect periodontal innate defense status. However, it is still unclear how commensal bacteria contribute to the inflammatory responses of junctional epithelium (JE) and periodontal connective tissue (PCT). The aim of the present study is to investigate the contribution of commensal bacteria on inflammatory responses in JE and PCT in mice. Methods: The periodontal tissue of germ-free (GF) and specific-pathogen-free (SPF) mice were compared at age 11 to 12 weeks (n = 6 per group). In this study, the number of neutrophils and expression of intercellular adhesion molecule (ICAM)-l, fibroblast growth factor receptor (FGFR)-l, matrix metalloproteinase (MMP)-l, and MMP-8 within the JE and the PCT are evaluated. The collagen density was also determined in PCT stained with picrosirius red (PSR). PSR staining combined with or without polarized light microscopy has been used to assess the organization and maturation of collagen matrix. Results: In the present findings, the area of JE in SPF mice was significantly greater than that in GF mice (P<0.05). In addition, the JE and PCT in SPF mice showed greater migration of neutrophils and higher expression of ICAM-1, FGFR-1, MMP-1, and MMP-8 than those in GF mice (P<0.05). Furthermore, the density of collagen in PCT in SPF mice was lower compared to GF mice (P<0.05). Conclusion: These results indicate that commensal bacteria induced a low-grade inflammatory state in JE and that such conditions may contribute to degradation of collagen in PCT in mice. [ABSTRACT FROM AUTHOR]

Background: The NLRP3 inflammasome is essentially a family of intracellular innate immune sensors that can respond to bacterial challenge and initiate early host immunity responses. However, the involvement and possible molecular mechanism of the NLRP3 pathway in the context of chronic periodontitis (CP) and diabetes mellitus have yet to be fully elucidated. Methods: Gingival tissues were collected from patients with CP and/or type 2 diabetes mellitus (T2DM), and the expression of NLRP3 and interleukin (IL)-1β was analyzed by immunohistochemistry. To explore the possible molecular mechanism, human gingival epithelial cells (HGECs) were established in vitro and challenged with lipopolysaccharide (LPS) and/or high glucose. High extracellular K+ was applied as an inhibitor of NLRP3. The NLRP3 pathway was analyzed by immunocytochemistry and quantitative polymerase chain reaction. Results: Compared with control individuals, NLRP3 and IL-1β were significantly upregulated in oral gingival epithelium of patients with CP and/or T2DM (P<0.05). The expression of NLRP3 was significantly upregulated in HGECs when stimulated in vitro by LPS or high glucose (P = 0.00). The simultaneous stimulation of LPS and high glucose contributed to significant upregulation of NLRP3 expression versus LPS or high glucose alone (P= 0.00). Although expression of caspase 1 and IL-1β protein were increased in HGECs when stimulated by LPS, they were partially inhibited after the NLRP3 was successfully blocked. Conclusion: For patients with T2DM and CP, hyperglycemic status may exacerbate the inflammation state of gingival tissue by activating the NLRP3 pathway, and this abnormal host inflammatory response may contribute to further tissue breakdown. [ABSTRACT FROM AUTHOR]

Background: Aggregatibacter actinomycetemcomitans resists killing by neutrophils and is inhibited by azithromycin (AZM) and amoxicillin (AMX). AZM actively concentrates inside host cells, whereas AMX enters by diffusion. The present study is conducted to determine whether AZM is more effective than AMX at enhancing phagocytic killing of A. actinomycetemcomitans by neutrophils. Methods: Killing assays were conducted in the presence of either 2 (µg/rnL AZM or 16 (µg/mL AMX (equipotent against A. actinomycetemcomitans). Neutrophils were loaded by incubation with the appropriate antibiotic. Opsonized A. actinomycetemcomitans strain Y4 was incubated with the indicated antibiotic alone, with loaded neutrophils and antibiotic, or with control neutrophils (without antibiotic) at multiplicities of infection (MOIs) of 30 and 90 bacteria per neutrophil. Results: Meutrophil incubation with 2 µg/mL AZM yielded an intracellular concentration of 10 (xg/mL. At an MOI of 30, neutrophils loaded with AZM failed to kill significantly more bacteria than control neutrophils during the 60- and 90-minute assay periods. At an MOI of 90, neutrophils loaded with AZM killed significantly more bacteria than either AZM alone or control neutrophils during 60- and 90-minute incubations (P <0.05), and killed significantly more bacteria after 90 minutes than the sum of the killing produced by AZM alone or neutrophils alone. Neutrophils incubated with AMX under identical conditions also killed significantly more bacteria than either AMX alone or control neutrophils, but there was no evidence of synergism between AMX and neutrophils. Conclusions: Neutrophils possess a concentrative transport system for AZM that may enhance killing of A. actinomycetemcomitans. Its effects are most pronounced when neutrophils are greatly outnumbered by bacteria. [ABSTRACT FROM AUTHOR]

Background: A multitude of studies suggest an association between periodontal disease and adverse birth outcomes, but the findings have been equivocal. Although the evidence is controversial, it is biologically plausible, and the key link may be inflammation. Because periodontitis is at times either active or inactive, trying to correlate the presence of pockets, for example, to adverse outcome of pregnancy might be preordained to failure or at least confusion. Alternatively, if inflammatory activity associated with periodontitis could be measured, it might be possible to correlate oral inflammatory load (OIL) to adverse pregnancy outcomes more precisely, but given the low incidence of adverse pregnancy outcomes, large populations must be studied. This underscores the need to use a means for assessment of OIL that is reliable, reproducible, and so simple to perform that it does not require dental expertise and can be used for large numbers of patients attending obstetrics units. The objective of this study was to demonstrate that OIL can be measured in a cohort of pregnanffemales presenting for obstetric care and secondarily to ensure that there was a realistic correlation to the presence of periodontitis. Methods: Sixty-three pregnant females were recruited, and 15-second saline rinses were collected to measure OIL as represented by counts of oral neutrophil levels. Periodontal examinations were performed to determine the extent of the correlation between the presence of clinical markers of periodontitis, such as pockets and clinical attachment loss to the OIL. Results: using this small cohort of patients, a test for oral inflammatory disease could be administered successfully in a non-dental setting. In addition, there was a statistically significant increase (two-fold, P<0.05) in oral neutrophil counts found in patients with periodontitis compared with those without periodontitis. Conclusion: The rinse assay can be used as a screening tool for oral inflammation, which was also related to the presence of periodontitis, in pregnant females attending a medical clinic. [ABSTRACT FROM AUTHOR]

Background: Protease activated receptor-1 (PAR1) activation by thrombin may play a role in repair and homeostasis of periodontal tissues. The main objective of this study is to investigate PAR1 expression in patients with periodontitis, before and after non-surgical periodontal treatment, and to associate its expression with the presence of inflammatory biomarkers and PAR2 expression. Methods: Gingival crevicular fluid (GCF) samples and clinical parameters, including probing depth, clinical attachment level, bleeding on probing, and gingival and plaque indices, were collected from periodontally healthy individuals and patients with moderate chronic periodontitis (CP) before and 6 weeks after periodontal non-surgical treatment. PAR1 and PAR2 messenger RNA (mRNA) at the GCF were evaluated by quantitative polymerase chain reaction (qPCR). Flow cytometry analysis identified the GCF PAR1-expressing cells. GCF inflammatory biomarkers were also determined. Results: Clinical parameters were significantly improved after therapy (P<0.01). The qPCR analysis showed that, before therapy, PAR1 mRNA levels in CP were similar to controls. Periodontal treatment led to increased PAR1 expression in CP (P <0.05). PAR1 expression was inversely correlated to PAR2 expression and with interleukins 6 and 8, tumor necrosis factor-a, interferon-7, and matrix metalloproteinase -2 levels. Conclusions: Periodontal treatment results in PAR1 overexpression in the GCF, and PAR1 expression is associated with decreased expression of inflammatory biomarkers and inversely correlated to PAR2 expression in the GCF. Therefore, the data suggest the importance of PAR1 mediating the known anabolic actions of thrombin in the periodontium. [ABSTRACT FROM AUTHOR]

Background: Soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) belongs to the immunoglobulin superfamily and is involved in amplification of the inflammatory response to bacterial infection. This cross-sectional study aims to investigate the levels of sTREM-1 in gingival crevicular fluid (GCF) of individuals without periodontitis and with chronic periodontitis (CP) or generalized aggressive periodontitis (GAgP) and their association with the levels of key periodontal pathogens in subgingival plaque. Methods: GCF and subgingival plaque samples were obtained from healthy sites of participants without periodontitis (n = 20) and periodontitis sites of patients with CP (n = 22) and GAgP (n = 20). sTREM-1 levels in GCF were measured by enzyme-linked immunosorbent assay. Porphyr-omonas gingiualis, Treponema denticola, Tannerella for-sythia, and Aggregatibacter actinomycetemcomitans levels in subgingival plaque were analyzed by quantitative realtime polymerase chain reaction. Results: sTREM-1 levels in GCF were higher in CP and GAgP than healthy sites by 3.6- and 4.4-fold, respectively, with no significant differences between the two forms of periodontitis. Moreover, sTREM-1 levels in GCF were positively correlated with site-specific clinical periodontal parameters and levels of P. gingiualis, T. denticola, and T. forsythia, but not A. actinomycetemcomitans, in subgingival plaque. Conclusion: Increased GCF levels of sTREM-1 at diseased sites and their positive correlation with clinical and microbiologic parameters strengthen the association of this inflammatory marker with periodontitis. [ABSTRACT FROM AUTHOR]

Background: Periodontitis, activated by oral bacteria and orchestrated by innate immune response, is regulated by primary nociceptive neurons, which are generally considered to have small- to medium-sized perikaryons. Bacterial byproducts (e.g., lipopolysaccharides) activate primary nociceptive neurons directly through Toll-like receptors (TLRs). Therefore, this study aims to morphometrically characterize rat trigeminal neurons, which express TLR4, and to investigate the changes in the TLR4 expression in neurons during peri-odontal inflammation. Methods: Trigeminal neurons innervating gingivomucosa were identified by application of the retrograde tracer hydroxystilbamidine into the gingival sulcus of the maxillary molar in 14 rats. Periodontitis was induced by ligature around the same molar in seven rats. TLR4 expression was investigated by immunohistochemistry on paraffin sections of the trigeminal ganglia (TG). Semiquantitative method was used to identify the intensity of TLR4 expression. Results: In the control group without the ligatures, TLR4 was detected in 19% of the neurons in the maxillary region of TG and in 29% of neurons innervating gingivomucosa. Expression of TLR4 was more frequent and intensive in small-to medium-sized neurons than in large-sized neurons. One week after ligature-induced periodontitis, the percentage of TLR4-positive neurons in the maxillary region and among the neurons innervating inflamed gingivomucosa significantly increased statistically to 32% and 41%, respectively. Conclusions: TLR4 is predominantly, but not exclusively, expressed in smaller trigeminal nociceptive neurons in the rat. Experimental periodontitis upregulates TLR4 expression in the trigeminal neurons. The hypothesis that bacterial byproducts regulate the pathogenesis of periodontitis by activation of trigeminal nociceptors through TLR4 should be explored. [ABSTRACT FROM AUTHOR]

Background: Mast cells appear to be associated with human periodontal disease. However, to the best of our knowledge, no studies have evaluated the presence of mast cells in individuals with human immunodeficiency virus (HIV) undergoing highly active antiretroviral therapy (HAART). Methods: Gingival samples were obtained from 50 individuals with and without HIV who presented with chronic gingivitis and periodontitis, immunohistochemical staining was performed to identify c-kit and tryptase mast cells. Inflammatory infiltrate was evaluated and quantified in the specimens of gingival tissue. In the inflammatory infiltrate subjacent to the pocket epithelium, the densities of the mast cells were calculated. These results were compared between the groups with and without HIV. Correlations could be drawn between the densities of c-kit and tryptase mast cells and the density of inflammatory infiltrate, cluster of differentiation 4 (CD4)-positive and CD8+ T-lymphocyte levels, and viral loads. Results: Although the individuals with HIV had higher densities of c-kit or tryptase mast cells than those without HIV, both groups presented with chronic gingivitis or periodontitis, and no statistically significant differences could be observed. Both strong and negative correlations could be observed among the inflammatory infiltrate, c-kit, and tryptase in individuals both with and without HIV. Concerning the chronic gingivitis of the individuals with HIV, both strong and positive correlations could be observed between the density of c-kit and CD8+ T-lymphocyte levels. Conclusions: Individuals with HIV undergoing HAART, compared with individuals without HIV, had no statistically significant differences in mast cell densities in gingival tissue. [ABSTRACT FROM AUTHOR]