Levels of Aggregatibacter actinomycetemcomitans,Porphyromonas gingivalis,inflammatory cytokines and species‐specific immunoglobulin G in generalized aggressive and chronic periodontitis

Casarin RCV, Del Peloso Ribeiro É, Mariano FS, Nociti FH Jr, Casati MZ, Gonçalves RB. Levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, inflammatory cytokines and species‐specific immunoglobulin G in generalized aggressive and chronic periodontitis. J Periodont Res 2010; 45: 635–642. © 2010 John Wiley & Sons A/S Background and Objective: Aggressive periodontitis pathogenesis still is not completely understood in the literature regarding the relationship between microbial and inflammatory aspects. So this study aimed to compare microbial and inflammatory patterns in the gingival crevicular fluid of generalized aggressive and chronic periodontitis patients. Material and Methods:  Forty aggressive and 28 chronic periodontitis patients were selected. Biofilm and gingival crevicular fluid were collected from a deep pocket (periodontal probing depth >7 mm) and a moderate pocket (periodontal probing depth = 5 mm) of each patient, and microbiological and immunoenzymatic assays were performed. Real‐time PCR was used to determine quantities of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Enzyme‐linked immunosorbent assay (ELISA) was employed to determine gingival crevicular fluid levels of interleukin‐1β, interferon‐γ, prostaglandin E₂ and interleukin‐10. In addition, immunoglobulin G (IgG) levels against A. actinomycetemcomitans and P. gingivalis lipopolysaccharide were also determined by ELISA. Analysis of variance/Tukey test, Mann–Whitney U‐test and the Pearson correlation test were used to determine differences and correlations between variables analysed (α = 5%). Results:  Patients suffering from generalized aggressive periodontitis had their mouth colonized by higher amounts of A. actinomycetemcomitans and P. gingivalis than chronic periodontitis patients. Conversely, the gingival crevicular fluid levels of IgG against both pathogens were statistically inferior in aggressive periodontitis patients (p < 0.05). With regard to gingival crevicular fluid levels of cytokines, aggressive periodontitis patients presented reduced levels of interleukin‐10 (p < 0.05). Conclusion:  In comparison to chronic periodontitis, generalized aggressive periodontitis patients have an imbalance in the host response, with reduced levels of interleukin‐10 and IgG, and increased periodontal pathogens.     

Lactoferrin Knockout Mice Demonstrates Greater Susceptibility to Aggregatibacter actinomycetemcomitans–Induced Periodontal Disease

Background : Among the innate defense mechanisms in the oral cavity, lactoferrin (LF) is a vital antimicrobial that can modify the host response against periodontopathogens. Aggregatibacter actinomycetemcomitans is the main periodontopathogen of localized aggressive periodontitis. The aim of this study is to evaluate the role of LF during A. actinomycetemcomitans–induced periodontitis. Methods: Differences in the expression levels of cytokines, chemokines, chemokine receptors, and bone loss markers between wild‐type (WT) and LF knockout mice (LFKO^?/?) were evaluated by real time‐PCR. Serum IgG and LF levels were quantified by ELISA. Alveolar bone loss among the groups was estimated by measuring the distance from cemento‐enamel junction (CEJ) to the alveolar bone crest (ABC) at 20 molar sites. Results: Oral infection with A. actinomycetemcomitans increased LF levels in periodontal tissue (P = 0.01) and saliva (P = 0.0004) of wild‐type infected (WTI) mice compared to wild‐type control mice. Pro‐inflammatory cytokines such as interferon‐γ, tumor necrosis factor‐α, interleukin (IL)‐1β, IL‐6, and IL‐12 were increased in the infected LF knockout (LFKO^?/?I) mice compared to the WTI mice, whereas the anti‐inflammatory cytokines IL‐4 and IL‐10 were decreased. Chemokines and chemokine receptors showed different expression patterns between WTI and LFKO^?/?I mice. The LFKO^?/?I mice developed increased bone loss (P = 0.002), in conjunction with increased expression of receptor activator of nuclear factor‐κB ligand and decrease in osteoprotegerin, compared to WTI mice. Conclusions: These results demonstrate that the infected LFKO^?/? mice were more susceptible to A. actinomycetemcomitans–induced alveolar bone loss, with different patterns of immune responses compared to those of WTI mice.     

Macrophage tolerance response to Aggregatibacter actinomycetemcomitans lipopolysaccharide induces differential regulation of tumor necrosis factor-alpha, interleukin-1 beta and matrix metalloproteinase 9 secretion

Background and Objective: The lipopolysaccharide of Aggregatibacter actinomycetemcomitans, a potent stimulator of the immune system, induces the secretion of inflammatory mediators that modulate periodontal tissue destruction. In this study, we investigated the tolerance response of human macrophages to stimulation with A. actinomycetemcomitans lipopolysaccharide. Material and Methods: U937 monocytes were differentiated into adherent macrophage‐like cells by treatment with phorbol myristic acid. Macrophage‐like cells were then pretreated for 24 h with either 0.01 or 0.1 μg/mL LPS A. actinomycetemcomitans. Culture medium supernatants were removed and cells were restimulated with LPS at 1 μg/mL. Cell‐free supernatants were collected after 24 h of stimulation and analyzed by ELISA for TNF‐α, IL‐1β, IL‐6, IL‐8, PGE₂ and MMP‐9. Results: Phorbol myristic acid‐differentiated U937 macrophages treated with low doses of lipopolysaccharide developed tolerance to subsequent lipopolysaccharide treatments, resulting in significantly reduced secretion of tumor necrosis factor‐α. However, this tolerance response was associated with increased secretion of interleukin‐1β and matrix metalloproteinase 9, whereas the secretion of interleukin‐6, interleukin‐8 and prostaglandin E₂ was unaffected. Phosphatidylinositol‐3′‐kinase inhibitors added during the tolerance‐induction period markedly attenuated the increase in interleukin‐1β secretion but had no effect on tumor necrosis factor‐α. Conclusion: This study showed that A. actinomycetemcomitans lipopolysaccharide can induce a tolerance response in macrophages that alters the secretion of two important inflammatory mediators as well as of the tissue‐degrading enzyme matrix metalloproteinase‐9. This phenomenon may play a role in modulating the host inflammatory response and the progression of periodontitis.     

Characterization of progressive periodontal lesions in chronic periodontitis patients: levels of chemokines, cytokines, matrix metalloproteinase-13, periodontal pathogens and inflammatory cells

Background and aims: Periodontitis is an infection with an episodic nature of tissue support destruction. The aim of this work was to determine the levels of chemokines, cytokines, matrix metalloproteinase‐13, periodontal pathogens and inflammatory cells in periodontal sites characterized by active periodontal connective tissue destruction. Material and Method: Fifty‐six patients with moderate or advanced severity of chronic periodontitis were selected. Periodontitis was characterized by at least six sites with probing depth 5 mm, clinical attachment level 3 mm and radiographic bone loss. Periodontitis progression was determined by the tolerance method. Receptor activator for nuclear factor κ B‐ligand (RANK‐L), monocyte chemoattractant protein‐1 (MCP‐1), tumour necrosis factor‐α (TNF‐α), IL‐1β, MMP‐13, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsithia and inflammatory cells levels were determined. Statistical analysis was performed using the Stata^® 7.0 software. Data were expressed as mean±SD and paired samples t‐test and χ² tests were used. Results: Higher RANK‐L, IL‐1β and MMP‐13 activity levels were observed in active sites (p<0.05). The proportion of P. gingivalis, A. actinomycetemcomitans, T. forsythia and the number of CD4⁺ T were higher in active than in inactive sites (p>0.05). Conclusion: The detection of periodontopathic bacteria, host matrix metalloproteinases and cytokines in periodontitis patients with lesions undergoing episodic attachment loss could partially explain the mechanisms associated with the destruction of the supporting tissues of the tooth.     

Aggregatibacter actinomycetemcomitans,a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis

Aggregatibacter actinomycetemcomitans is a perio‐pathogenic bacteria that has long been associated with localized aggressive periodontitis. The mechanisms of its pathogenicity have been studied in humans and preclinical experimental models. Although different serotypes of A. actinomycetemcomitans have differential virulence factor expression, A. actinomycetemcomitans cytolethal distending toxin (CDT), leukotoxin, and lipopolysaccharide (LPS) have been most extensively studied in the context of modulating the host immune response. Following colonization and attachment in the oral cavity, A. actinomycetemcomitans employs CDT, leukotoxin, and LPS to evade host innate defense mechanisms and drive a pathophysiologic inflammatory response. This supra‐physiologic immune response state perturbs normal periodontal tissue remodeling/turnover and ultimately has catabolic effects on periodontal tissue homeostasis. In this review, we have divided the host response into two systems: non‐hematopoietic and hematopoietic. Non‐hematopoietic barriers include epithelium and fibroblasts that initiate the innate immune host response. The hematopoietic system contains lymphoid and myeloid‐derived cell lineages that are responsible for expanding the immune response and driving the pathophysiologic inflammatory state in the local periodontal microenvironment. Effector systems and signaling transduction pathways activated and utilized in response to A. actinomycetemcomitans will be discussed to further delineate immune cell mechanisms during A. actinomycetemcomitans infection. Finally, we will discuss the osteo‐immunomodulatory effects induced by A. actinomycetemcomitans and dissect the catabolic disruption of balanced osteoclast–osteoblast‐mediated bone remodeling, which subsequently leads to net alveolar bone loss.     

Biology and pathogenesis of cytomegalovirus in periodontal disease

Human periodontitis is associated with a wide range of bacteria and viruses and with complex innate and adaptive immune responses. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Treponema denticola, cytomegalovirus and other herpesviruses are major suspected pathogens of periodontitis, and a combined herpesvirus–bacterial periodontal infection can potentially explain major clinical features of the disease. Cytomegalovirus infects periodontal macrophages and T‐cells and elicits a release of interleukin‐1β and tumor necrosis factor‐α. These proinflammatory cytokines play an important role in the host defense against the virus, but they also have the potential to induce alveolar bone resorption and loss of periodontal ligament. Gingival fibroblasts infected with cytomegalovirus also exhibit diminished collagen production and release of an increased level of matrix metalloproteinases. This article reviews innate and adaptive immunity to cytomegalovirus and suggests that immune responses towards cytomegalovirus can play roles in controlling, as well as in exacerbating, destructive periodontal disease.     

Sustained mitogen-activated protein kinase activation with Aggregatibacter actinomycetemcomitans causes inflammatory bone loss

Aggregatibacter actinomycetemcomitans is a gram‐negative facultative capnophile involved in pathogenesis of aggressive forms of periodontal disease. In the present study, we interrogated the ability of A. actinomycetemcomitans to stimulate innate immune signaling and cytokine production and established that A. actinomycetemcomitans causes bone loss in a novel rat calvarial model. In vitro studies indicated that A. actinomycetemcomitans stimulated considerable production of soluble cytokines, tumor necrosis factor‐α, interleukin‐6 and interleukin‐10 in both primary bone marrow‐derived macrophages and NR8383 macrophages. Immunoblot analysis indicated that A. actinomycetemcomitans exhibits sustained activation of all major mitogen‐activated protein kinase (MAPK) pathways, as well as the negative regulator of MAPK signaling, MAPK phosphatase‐1 (MKP‐1), for at least 8 h. In a rat calvarial model of inflammatory bone loss, high and low doses of formalin‐fixed A. actinomycetemcomitans were microinjected into the supraperiosteal calvarial space for 1–2 weeks. Histological staining and micro‐computed tomography of rat calvariae revealed a significant increase of inflammatory and fibroblast infiltrate and increased bone resorption as measured by total lacunar pit formation. From these data, we provide new evidence that fixed whole cell A. actinomycetemcomitans stimulation elicits a pro‐inflammatory host response through sustained MAPK signaling, leading to enhanced bone resorption within the rat calvarial bone.     

Modulation of Matrix Metalloproteinase and Cytokine Production by Licorice Isolates Licoricidin and Licorisoflavan A: Potential Therapeutic Approach for Periodontitis

Background: Inflammatory cytokines and matrix metalloproteinases (MMPs) produced by resident and inflammatory cells in response to periodontopathogens play a major role in the tissue destruction observed in periodontitis, which is a disease that affects tooth‐supporting structures. In the present study, we investigate the effects of licorice‐derived licoricidin (LC) and licorisoflavan A (LIA) on the secretion of various cytokines and MMPs by human monocyte‐derived macrophages stimulated with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) lipopolysaccharide (LPS). Methods: Macrophages were treated with non‐toxic concentrations of LC or LIA before being stimulated with A. actinomycetemcomitans LPS. The secretion of cytokines and MMPs and the activation of nuclear factor‐kappa B (NF‐κB) p65 and activator protein (AP)‐1 were assessed by enzyme‐linked immunosorbent assays. Results: LC and LIA inhibited the secretion of interleukin (IL)‐6 and chemokine (C‐C motif) ligand 5 in a concentration‐dependent manner but did not affect the secretion of IL‐8 by LPS‐stimulated macrophages. LC and LIA also inhibited the secretion of MMP‐7, ‐8, and ‐9 by macrophages. The suppression of cytokine and MMP secretion by LC and LIA was associated with the reduced activation of NF‐κB p65 but not that of AP‐1. Conclusion: The present study suggests that LC and LIA have potential for the development of novel host‐modulating strategies for the treatment of cytokine and/or MMP‐mediated disorders such as periodontitis.     

Periodontal inflammation and bone loss in aged mice

Liang S, Hosur KB, Domon H, Hajishengallis G. Periodontal inflammation and bone loss in aged mice. J Periodont Res 2010; 45: 574–578. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: Young mice do not develop measurable periodontal bone loss, unless heavily infected with human periodontal pathogens. However, mice with a genetically altered immune system are unable to control their own oral flora and develop periodontitis early in life. Based on the potential of the indigenous oral microbiota to cause periodontitis, we hypothesized that normal mice may ultimately develop inflammatory periodontal bone loss, i.e. as a function of age. If confirmed, this could serve as an aging model of chronic periodontitis. Material and Methods: Periodontal bone levels were measured as the distance from the cementoenamel junction to the alveolar bone crest in young mice (8–10 wk of age), old mice (≥ 18 mo of age) and mice of intermediate ages. Differential expression of inflammatory mediators in the gingivae of young and old mice was determined by quantitative real‐time PCR. Results: In comparison with young mice, old mice displayed significantly (p < 0.05) increased periodontal bone loss, accompanied by elevated expression of proinflammatory cytokines (interleukin‐1β, tumor necrosis factor α and interleukin‐17A) and innate immune receptors involved in the induction or amplification of inflammation (Toll‐like receptor 2, CD14, CD11b, CD18, complement C5a receptor and triggering receptor expressed on myeloid cells 3). Conclusion: Mice develop naturally induced periodontal bone loss as a function of age. This aging model of periodontitis represents a genuinely chronic model to study mechanisms of periodontal tissue destruction.     

Soluble Triggering Receptor Expressed on Myeloid Cells 1 (sTREM‐1) in Gingival Crevicular Fluid: Association With Clinical and Microbiologic Parameters

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. Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Aggregatibacter actinomycetemcomitans levels in subgingival plaque were analyzed by quantitative real‐time 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. gingivalis, 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.     

Systemic Aggregatibacter actinomycetemcomitans Leukotoxin‐Neutralizing Antibodies in Periodontitis

Background: Leukotoxin (Ltx) expressed by Aggregatibacter actinomycetemcomitans is a powerful exotoxin, which can cause imbalance in host response. Immunoreactivity to Ltx is a marker for presence of leukotoxic A. actinomycetemcomitans, a presence that may modify the disease pattern of colonized individuals. The aim of the present study is to examine presence of systemic immunoreactivity to A. actionmycetemcomitans Ltx with respect to clinical and inflammatory findings in individuals with or without periodontitis (N = 88). Methods: Periodontal status was examined in patients with severe periodontitis (n = 49) and healthy controls (n = 39), and patients received periodontal treatment. Systemic biomarkers associated with inflammation and infections as well as clinical parameters were analyzed at baseline and 3 and 6 months after treatment. Results: Presence of immunoreactivity against Ltx was associated with impaired remission of disease after periodontal treatment. This immunoreactivity was also significantly associated with increased systemic levels of A. actinomycetemcomitans‐specific immunoglobulins and increasing age. Conclusion: Presence and levels of systemic immunoreactivity against A. actinomycetemcomitans Ltx are associated with decreased remission after otherwise successful periodontal treatment.     

Periodontal and Systemic Responses in Various Mice Models of Experimental Periodontitis: Respective Roles of Inflammation Duration and Porphyromonas gingivalis Infection

Background: The great variability of periodontal and systemic responses to experimental periodontitis reflects the inherent pathogenic complexity of mice models and could limit the resulting interpretations and their extension to human diseases. This study compared the effect of Porphyromonas gingivalis (Pg) infection and experimental periodontitis duration at local and systemic levels in various models. Methods: Periodontitis was induced in C57BL/6J mice by ligatures previously incubated with Pg (LIGPG group) or not (LIG group) or by oral gavage (GAV) with Pg ATCC 33277. Blood samples were taken, and mice were euthanized at different times. Periodontal tissue destruction, osteoclast number, and inflammation were assessed by histomorphometry, tartrate‐resistant acid phosphatase histoenzymology, and cathepsin B (CATB) and matrix metalloproteinase 9 (MMP9) immunochemistry. Serum levels of interleukin‐6 (IL‐6) and IL‐1β were measured using enzyme‐linked immunosorbent assay bioplex methods. Results: Periodontal tissue destruction and osteoclast numbers were significantly elevated in LIGPG models compared to LIG and GAV models. They increased with time with the exception of osteoclast numbers in the LIG model. CATB and MMP9 expression was related to bone destruction processes and Pg infection. The highest serum levels of IL‐6 and IL‐1β were observed in the LIGPG group. A decrease of IL‐6 and an increase of IL‐1β serum level were observed with time in LIGPG group contrary to LIG group. Conclusions: These data indicate that Pg infection worsened periodontal tissue destruction through specific pathogenic pathways and modified systemic response to periodontal inflammation. Furthermore, the blood cytokine response to ligature models showed their relevance for evaluating the systemic impact of periodontal disease.     

Assessment of the involvement of the macrophage migration inhibitory factor–glucocorticoid regulatory dyad in the expression of matrix metalloproteinase‐2 during periodontitis

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine and counter‐regulator of endogenous glucocorticoids (GCs). It is implicated in acute and chronic inflammatory diseases. This study investigated the role of the MIF–GC regulatory dyad in the expression and release of matrix metalloproteinase‐2 (MMP‐2) during periodontitis, in vivo and in vitro. In a Mif‐knockout (KO) mouse model of ligature‐induced periodontitis, gingival tissues and blood were collected and analysed for levels of interleukin‐6 (IL‐6), MIF, MMP‐2, and corticosterone. In addition, human gingival fibroblasts (HGFs) were tested for production of IL‐6 and MMP‐2 after stimulation with hydrocortisone (HC), MIF, tumour necrosis factor‐alpha (TNF‐α), or Fusobacterium nucleatum, a pathogen known to elicit immune responses during periodontitis. Wild‐type (WT) mice showed a local and systemic increase of MIF levels during inflammation, which was confirmed by increased local IL‐6 concentrations. Systemic GC levels were reduced in WT and Mif‐KO mice during inflammation, with overall lower concentrations in Mif‐KO mice. In vivo and in vitro, MMP‐2 production was not dependent on MIF or inflammatory stimuli, but was inhibited by HC. Therefore, MIF does not appear to stimulate expression of MMP‐2 in the gingival tissues, whereas GC upregulates MIF and downregulates MMP‐2. Our findings further suggest that MIF may regulate systemic GC levels.     

Serum cytokine levels in periodontitis patients in relation to the bacterial load

Background: Periodontitis is a local inflammatory disease that also has some systemic effects. We investigated the levels of interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α, and interleukin (IL)‐2, ‐4, ‐5, and ‐10 in the serum of patients with periodontitis in relation to the bacterial load in the dental plaques. Methods: Serum cytokine levels in patients with generalized periodontitis and healthy control groups were determined using the cytometric bead array kit. Bacterial load in the dental plaque was determined semiquantitatively by real‐time polymerase chain reaction. The proportions of different lymphocyte subsets were determined in the peripheral blood of patients with periodontitis by flow cytometry. Finally, relationships between the bacterial load in the subgingival plaques of patients with periodontitis and levels of cytokines and counts of lymphocyte subsets were established. Results: Serum levels of IFN‐γ, TNF‐α, and IL‐10 were significantly increased, whereas those of IL‐2 were significantly decreased in patients with periodontitis compared to healthy controls. Increased serum levels of IFN‐γ and TNF‐α in patients with periodontitis were associated with the enhanced dental plaque load with Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis, respectively. Finally, as revealed by analysis of lymphocyte populations, the presence of A. actinomycetemcomitans and Trepomena denticola was associated with an increased population of CD3^?/CD16⁺ and CD3⁺/CD8⁺ cells, respectively. Conclusion: Certain periodontal pathogens could be associated with an increased level of proinflammatory cytokines in the peripheral blood and thus increased risk of systemic diseases.     

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

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

Humoral immune response to Aggregatibacter actinomycetemcomitans leukotoxin

Brage M, Holmlund A, Johansson A. Humoral immune response toAggregatibacter actinomycetemcomitans leukotoxin. J Periodont Res 2011; 46: 170–175. © 2010 John Wiley & Sons A/S Background and Objective: Periodontal disease is an inflammatory condition caused by bacterial infections that result in loss of the tooth supporting tissue. The periodontal pathogens produce virulence factors with capacity to affect the host immune response. Aggregatibacter actinomycetemcomitans is a periodontal pathogen that produces a leukotoxin that specifically affects human leukocytes. The aims of the present study were to examine the presence and function of systemic antibodies to the leukotoxin. Material and Methods: One hundred and ninety‐seven middle‐aged (57 ± 5 years) Swedes with well‐documented periodontal status and medical factors related to cardiovascular diseases were studied. These data have been published previously. The serum samples were examined for the presence of leukotoxin antibodies by western blot and the capacity to neutralize leukotoxicity in an activity assay with leukotoxin and cultured leukemic cells. Results: The results showed a high prevalence (57%) of antibodies against A. actinomycetemcomitans leukotoxin in the analyzed population. These antibodies were correlated with leukotoxin neutralizing capacity as well as with the ELISA titers of A. actinomycetemcomitans‐specific IgA and IgG. In addition, high levels of leukotoxin antibodies were correlated with increasing age, but not with periodontal disease parameters or cardiovascular risk factors. Conclusion: Systemic antibodies against A. actionmycetemcomitans leukotoxin were common in this adult Swedish population. These antibodies might contribute to limit the systemic effects of the infection.     

Focal adhesion kinase activation is required for TNF‐α‐induced production of matrix metalloproteinase‐2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF‐α) and its effects on interleukin (IL)‐6 and IL‐8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP‐2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real‐time PCR. Tumor necrosis factor alpha dose‐dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF‐α‐induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL‐6, IL‐8, and MMP‐2 in a dose‐dependent manner. Knockdown of FAK significantly suppressed TNF‐α‐induced expression of IL6 and IL8 mRNA and release of IL‐6 and IL‐8 protein in HPDLFs. Similarly, MMP‐2 down‐regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF‐α‐induced IL‐6, IL‐8, and MMP‐2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.     

Periodontitis-associated up-regulation of systemic inflammatory mediator level may increase the risk of coronary heart disease

Nakajima T, Honda T, Domon H, Okui T, Kajita K, Ito H, Takahashi N, Maekawa T, Tabeta K, Yamazaki K. Periodontitis‐associated up‐regulation of systemic inflammatory mediator level may increase the risk of coronary heart disease. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01209.x. © 2009 John Wiley & Sons A/S Background and Objective: Although an elevation in the concentration of high‐sensitivity C‐reactive protein (hs‐CRP) as a result of periodontal infection may account for an increased risk of developing coronary heart disease (CHD), the effect of periodontal infection on the level of hs‐CRP in an otherwise healthy Japanese population has not yet been reported. The aim of the present study was to confirm, on a larger scale, our previous pilot study findings that both chronic periodontitis and subsequent periodontal treatment alter the serum levels of C‐reactive protein (CRP), interleukin‐6 (IL‐6) and tumor necrosis factor‐α (TNF‐α). Material and Methods: The concentrations of serum hs‐CRP, IL‐6 and TNF‐α were measured in 78 periodontitis patients at baseline and at re‐assessment, and in 40 periodontally healthy subjects at the time of examination. Results: The concentrations of hs‐CRP and IL‐6 in the sera of periodontitis patients were significantly higher than those in control subjects. By contrast, the concentration of TNF‐α was significantly lower in periodontitis patients than in control subjects. Whereas periodontal treatment decreased the levels of serum hs‐CRP and IL‐6, no such effect was observed for TNF‐α. When the patients were subdivided into four groups according to their initial concentration of hs‐CRP, only the CRP and IL‐6 concentrations of the highest quartile group showed a significant reduction following periodontal treatment. No significant difference in the initial clinical parameters was observed in any quartile. Conclusion: Although periodontal infection does affect the concentration of hs‐CRP and IL‐6 in serum, a subgroup of patients exist who are highly susceptible to an increased risk of CHD associated with periodontitis, suggesting that there may be subjects who have an elevated risk of CHD independent of susceptibility to periodontal tissue destruction per se.     

Analysis of matrix metalloproteinase (MMP‐8 and MMP‐2) activity in gingival crevicular fluid from children with Down’s syndrome

Yamazaki‐Kubota T, Miyamoto M, Sano Y, Kusumoto M, Yonezu T, Sugita K, Okuda K, Yakushiji M, Ishihara K. Analysis of matrix metalloproteinase (MMP‐8 and MMP‐2) activity in gingival crevicular fluid from children with Down’s syndrome. J Periodont Res 2010; doi: 10.1111/j.1600‐0765.2009.01214.x. © 2009 John Wiley & Sons A/S Background and Objective: High levels of colonization by periodontopathic bacteria and a high prevalence of chronic inflammatory periodontal disease have been reported in children with Down’s syndrome. Matrix metalloproteinases (MMPs) are mediators of extracellular matrix degradation and remodelling, and are deeply involved in the course of periodontal disease. To clarify the relationship between Down’s syndrome and periodontitis, we investigated levels of MMP‐2 and MMP‐8 in gingival crevicular fluid (GCF) and detection of periodontopathic bacteria from subgingival plaque. Material and Methods: Samples of GCF and plaque were isolated from central incisors. Levels of MMPs were evaluated by enzyme‐linked immunosorbent assay, and periodontopathic bacteria were detected by polymerase chain reaction. Results: Levels of MMP‐2 and MMP‐8 in Down’s syndrome patients were higher than those in healthy control subjects. In the Down’s syndrome group, increases in these MMPs were observed in GCF from patients with an oral hygiene index score of < 2 and in GCF from sites that were negative for bleeding on probing. The detection rate of periodontopathic bacteria in Down’s syndrome patients was higher than that in the control subjects. Matrix metalloproteinase‐2 levels in sites harbouring Porphyromonas gingivalis or Aggregatibacter (Actinobacillus) actinomycetemcomitans were lower than in those without these microorganisms. Conclusion: These results suggest an increase in MMP‐2 and MMP‐8 in Down’s syndrome patients, regardless of whether inflammation of periodontal tissue is present or not.