A single application of hydrogen sulphide induces a transient osteoclast differentiation with RANKL expression in the rat model

Objective

Oral malodor is mainly attributed to volatile sulphur compounds (VSCs) such as hydrogen sulphide (H₂S), methyl mercaptan and dimethyl sulphide. VSC accelerate periodontal soft tissue destruction. However, there is little information about the potential role of H₂S in alveolar bone loss. The purpose of this animal study was to examine the effects of sodium hydrogen sulphide (NaHS), H₂S donor drug, on osteoclast differentiation in rat periodontal tissue.

Design

Twenty-four male Wistar rats (8 weeks old) were divided into four groups: a control group and three experimental groups, which were examined at 3 h, 1 day, and 3 days after topical application of 3 μl NaHS (l M in physiological saline) into the gingival sulcus of rat first molar. Expression of tumour necrosis factor (TNF)-α, RANKL, NF-κB and tartrate-resistant acid phosphatase (TRAP) was evaluated in the periodontal tissue.

Results

Three hours after NaHS application, TNF-α expression increased in the periodontal ligament. The numbers of RANKL-positive osteoblasts and TRAP-positive osteoclasts significantly increased progressively with time and reached a maximum level after 1 day. Significant up-regulation of RANKL and NF-κB mRNA was observed at 3 h after NaHS application.

Conclusions

H₂S application caused a transient increase of osteoclast differentiation with up-regulation of RANKL expression in osteoblasts. H₂S, which is primarily responsible for halitosis, may also contribute to alveolar bone resorption through RANKL expression.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Toll‐like receptor 5 activation promotes migration and invasion of salivary gland adenocarcinoma

J Oral Pathol Med (2011) 40 : 187–193 Background: Toll‐like receptor (TLR) signaling has been found to be closely associated with tumor development. The aim of this study was to examine whether activation of TLRs promote migration and invasion of salivary gland adenocarcinoma. Materials and Methods: TLR expression in SGT and HSG cells was examined by RT‐PCR. Wound scratch and chemotaxis cell migration assay were performed. Invasiveness was determined by Matrigel invasion assay. Results: All the tested TLRs including TLR1, TLR2, TLR4, and TLR5 and myeloid differentiation factor‐2 (MD‐2) were expressed on SGT and HSG cells. Treatment of flagellin, but not Pam₃CSK₄ and LPS, led to the production of IL‐6 and IL‐8, suggesting TLR5 is functional in both cells. Stimulation by flagellin also accelerated wound closure of SGT and HSG cells in a dose‐dependent manner. In addition, flagellin promoted migration and invasion ability of SGT cells. Blocking of TLR5 using antibody restored the promoting effect of flagellin on migration and invasion of SGT cells. Conclusion: These findings suggest that TLR5 activation by flagellin can promote migration and invasion of salivary gland adenocarcinoma.     

Involvement of prostaglandin E2 and interleukin-1α in the differentiation and survival of osteoclasts induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans Y4

Lipopolysaccharide (LPS) is a bacterial cell component that plays multifunctional roles in inflammatory reactions. LPS from various periodontal pathogens is supposed to be a major virulence factor of periodontal diseases. In the present study, we demonstrated that LPS from periodontopathic bacterium Actinobacillus actinomycetemcomitans Y4 (Y4 LPS) stimulated osteoclast formation in mouse bone marrow culture systems. Addition of anti-interleukin-lα (IL-lα) antibody or indomethacin in the marrow cultures resulted in the suppression of osteoclast differentiation. Quantitative analyses revealed that Y4 LPS stimulated the production of IL-lα and prostaglandin E₂ (PGE₂) by bone marrow cells. Furthermore, an immunoblot analysis showed that Y4 LPS stimulated bone marrow cells to upregulate the expression of cyclooxygenase-2, a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids. These findings suggest that both IL-lα and PGE₂ are involved in the LPS-mediated osteoclast differentiation. In addition, we found that Y4 LPS supported the survival of osteoclasts. Addition of anti-IL-lα antibody in the osteoclast culture resulted in a reduction of osteoclast survival. Indomethacin, however, showed no effect on osteoclast survival. These findings suggest that the increased PGE₂ and IL-lα synthesis by bone marrow cells may play an important role in the differentiation and survival of osteoclasts induced by A. actinomycetemcomitans LPS.     

TLR4 and TLR9 are induced in oral lichen planus

J Oral Pathol Med (2012) 41 : 741–747 Background: The role of Toll‐like receptors (TLRs) has been elucidated in many human infectious, autoimmune and neoplastic diseases. Previously, TLR2 and TLR4 expression in oral lichen planus (OLP) was described. The aim of our study was to examine expression patterns of TLR4 and TLR9 in normal oral mucosa and OLP and describe the effect of topical tacrolimus treatment on the expression of TLR4 and TLR9 in OLP. Methods: Toll‐like receptor 4 and TLR9 expression was analysed by immunohistochemistry in five samples of normal oral mucosa and 50 samples of OLP (31 representing clinically white and 19 clinically erythematous/erosive lesions). We evaluated also the effect of topical tacrolimus on TLR4 and TLR9 expression in a patient with OLP. Results: Toll‐like receptor 4 and TLR9 expression was increased in OLP epithelium compared with normal epithelium (P < 0.001); no significant difference between the two clinical types of OLP was observed. TLR9 expression was strongest in the superficial layer of the epithelium (P < 0.001), while the expression of TLR4 was strongest in the basal layer (P < 0.001). Treatment of OLP lesions with topical tacrolimus resulted in clinical improvement but had no effect on TLR expression levels. Conclusions: Toll‐like receptor 4 and TLR9 are induced in OLP; our finding confirms the results of a previous study. TLR4 and TLR9 may play a part in the pathogenesis of OLP. Further studies are needed to dissect the definitive role of TLRs in OLP pathogenesis and progression and to determine the effect of tacrolimus on the function of TLRs.     

Contrasting responses of human gingival and periodontal ligament fibroblasts to bacterial cell‐surface components through the CD14/Toll‐like receptor system

We compared human periodontal ligament fibroblasts with human gingival fibroblasts isolated from the same donor to examine interleukin‐8 (IL‐8) responses of the cells to Salmonella lipopolysaccharide, a water‐soluble peptidoglycan from Staphylococcus epidermidis and the synthetic muramyldipeptide, with special reference to the possible involvement of the CD14/Toll‐like receptor (TLR) system of the cells in the responses. Human gingival fibroblasts expressed CD14 on their surfaces and strongly expressed CD14 mRNA, while human periodontal ligament fibroblasts showed considerably lower levels of expression in both respects. Both cells expressed mRNA of TLR‐related molecules, i.e. TLR2, TLR4, MD‐2 and MyD88, although human periodontal ligament fibroblasts expressed TLR2 more strongly than human gingival fibroblasts. Human gingival fibroblasts exhibited a stronger IL‐8 response than human periodontal ligament fibroblasts to lipopolysaccharide, while human periodontal ligament fibroblasts exhibited a response comparable to, or slightly stronger than, that of human gingival fibroblasts to S. epidermidis peptidoglycan and muramyldipeptide. The IL‐8 responses of both cells to lipopolysaccharide and S. epidermidis peptidoglycan were completely inhibited by antihuman CD14 monoclonal antibody (MAb). The responses of both cells to lipopolysaccaride were significantly inhibited by antihuman TLR4 MAb, while those to S. epidermidis peptidoglycan were inhibited by antihuman TLR2 MAb. In contrast, muramyldipeptide activated both types of cells in a TLR2‐ and TLR4‐independent manner, although the activities of muramyldipeptide on human gingival fibroblasts, but not human periodontal ligament fibroblasts, were significantly inhibited by anti‐CD14 MAb.     

Toll‐like receptors 2 and 5 in human gingival epithelial cells co‐operate with T‐cell cytokine interleukin‐17

Background/aim: Periodontitis begins as the result of perturbation of the gingival epithelial cells caused by subgingival bacteria interacting with the epithelial cells via pattern recognition receptors. Toll‐like receptors (TLRs) have been shown to play an important role in the recognition of periodontal pathogens so we have studied the interaction of TLR ligands with TLR2 and TLR5 for cytokine production in the cultures of gingival epithelial cells. Methods: Immunohistochemistry was used for the localization of TLR2 and TLR5 in tissue specimens. Enzyme‐linked immunosorbent assays were performed to detect the levels of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α), released from gingival epithelial cell cultures following stimulation with TLR ligand alone or in combination with IL‐17. Results: Both TLR2 and TLR5 were increased in periodontitis (2128 ± 159 vs. 449 ± 59 and 2456 ± 297 vs. 679 ± 103, respectively, P < 0.001) including gingival epithelial cells that stained strongly. Cultured gingival epithelial cells stimulated with their respective ligands (HKLM, a TLR2 ligand that is also found in Porphyromonas gingivalis, and flagellin, a TLR5 ligand that is also found in Treponema denticola) produced both IL‐1β and TNF‐α. To mimic T‐cell help, IL‐17 was added. This further greatly enhanced TLR ligand‐induced IL‐1β (P < 0.001) and TNF‐α (P < 0.01) production. Conclusions: These findings show how pathogen‐associated molecular patterns, shared by many different periodontopathogenic bacteria, stimulate the resident gingival epithelial cells to inflammatory responses in a TLR‐dependent manner. This stimulation may be particularly strong in periodontitis and when T helper type 17 cells provide T‐cell help in intercellular cooperation.     

Salivary and plasma levels of Toll-like receptor 2 and Toll-like receptor 4 in chronic periodontitis

Background: This cross‐sectional study was planned to investigate whether patients with chronic periodontitis exhibit different salivary or plasma concentrations of Toll‐like receptor (TLR) 2 and TLR4 compared to subjects who are clinically healthy. Methods: Whole saliva and plasma samples were obtained and full‐mouth clinical periodontal measurements were recorded from 22 otherwise healthy patients with chronic periodontitis and 21 systemically and periodontally healthy control subjects. Salivary and plasma TLR2 and TLR4 levels were determined by enzyme‐linked immunoassays. Data were tested statistically using Mann‐Whitney U test. Results: The healthy group exhibited significantly lower values in all clinical measurements (P <0.001). The salivary TLR2 levels were similar in the two study groups (P >0.05). The patients with chronic periodontitis exhibited significantly higher salivary TLR4 (P <0.01) and plasma TLR2 and TLR4 levels (P <0.05). Conclusion: The present findings support a hypothesis that inflammation increases expression of TLRs which leads to an increased detection of TLRs in saliva and plasma, which could be useful as a diagnostic test for periodontal diseases.     

Global TLR2 and 4 deficiency in mice impacts bone resorption,inflammatory markers and atherosclerosis to polymicrobial infection

Toll‐like‐receptors (TLRs) play a significant role in the generation of a specific innate immune response against invading pathogens. TLR2 and TLR4 signaling contributes to infection‐induced inflammation in periodontal disease (PD) and atherosclerosis. Observational studies point towards a relationship between PD and atherosclerosis, but the role of TLR2 and TLR4 in the recognition of multiple oral pathogens and their modulation of host response leading to atherosclerosis are not clear. We evaluated the role of TLR2 and TLR4 signaling in the induction of both PD and atherosclerosis in TLR2^?/? and TLR4^?/? mice to polymicrobial infection with periodontal pathogens Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Fusobacterium nucleatum. Polybacterial infections have established gingival colonization in TLR2^?/? and TLR4^?/? mice and induction of a pathogen‐specific immunoglobulin G immune response. But TLR deficiency dampened accelerated alveolar bone resorption and intrabony defects, indicating a central role in infection‐induced PD. Periodontal bacteria disseminated from gingival tissue to the heart and aorta through intravascular dissemination; however, there was no increase in atherosclerosis progression in the aortic arch. Polybacterial infection does not alter levels of serum risk factors such as oxidized low‐density lipoprotein, nitric oxide, and lipid fractions in both mice. Polymicrobial‐infected TLR2^?/? mice demonstrated significant levels (P < 0.05 to P < 0.01) of T helper type 2 [transforming growth factor‐β₁, macrophage inflammatory protein‐3α, interleukin‐13 (IL‐13)] and T helper type 17 (IL‐17, IL‐21, IL‐22, IL‐23) splenic T‐cell cytokine responses. Increased heat‐shock protein expression, hspa1a for Hsp 70, was observed for both TLR2^?/? and TLR4^?/? mice. This study supports a role for TLR2 and TLR4 in PD and atherosclerosis, corroborating an intricate association between two inflammatory diseases.     

Prediabetes Enhances Periodontal Inflammation Consistent With Activation of Toll‐Like Receptor–Mediated Nuclear Factor‐κB Pathway in Rats

Background: Clinical studies have showed that prediabetes (preDM) is a predisposing factor for periodontitis. However, the pathogenic mechanism involved is unclear. Because it is known that the activation of Toll‐like receptor (TLR)‐mediated nuclear factor‐kappa B (NF‐κB) signaling pathway plays a crucial role in periodontitis, it is hypothesized that preDM enhances periodontal inflammation by activation of the TLR‐mediated NF‐κB pathway. Methods: In this study, a preDM rat model is established by feeding a high‐fat diet (HFD). HFD‐induced rats with preDM (n = 7) and normal chow–fed rats (n = 7) were studied. The animal model was characterized in terms of body weight and the glycemic and insulinemic profiles. The following parameters were assessed to evaluate possible early periodontal alterations and underlying mechanisms: 1) histology analysis of periodontal tissue; and 2) serum and mRNA levels and/or the tissue protein expression of TLRs, myeloid differentiation factor 88 (MyD88), tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6), NF‐κB, cytokines, advanced glucose ends (AGEs), and free fatty acids (FFAs). Results: Rats with preDM presented higher expression of TLR2 and TLR4 in periodontal tissue in the HFD group compared with the control group. The TLR2 and TLR4 was mostly expressed in gingiva, and TLR4 was expressed in periodontal ligament in rats. Furthermore, the MyD88 and TRAF6 protein levels were significantly increased in gingiva in rats with preDM compared with normal rats. The activity of NF‐κB signals was higher in rats with preDM than in normal rats. Regarding cytokines expression, the TNF‐α protein levels and interleukin‐1β mRNA levels were significantly increased in the HFD group compared with the control group. In the serum, AGEs levels were significantly increased in the rats with preDM. Mean FFAs concentrations were increased in rats with preDM compared with normal rats, but it did not reach statistical significance. Conclusion: In rats with preDM, TLR2 and TLR4 gene and protein levels were higher in periodontal tissue, and the activation of NF‐κB may, through TLRs/MyD88, cause more cytokine secretion, which is associated with the onset or development of periodontal disease.     

Cimetidine Reduces Interleukin‐6, Matrix Metalloproteinases‐1 and ‐9 Immunoexpression in the Gingival Mucosa of Rat Molars With Induced Periodontal Disease

Background: Histamine seems to act, via H₂ receptor, on inflammatory processes by stimulating interleukin (IL)‐6 and matrix metalloproteinase (MMP) release. As cimetidine is an H₂ receptor antagonist, the authors hypothesize that this antiulcer drug reduces IL‐6, MMP‐1, and MMP‐9 immunoexpression in gingiva with induced periodontal disease (PD). To confirm a possible modulatory role of IL‐6 on MMPs, the relationship between IL‐6/MMP‐1 and IL‐6/MMP‐9 immunoexpression was evaluated. Methods: Forty‐six male rats were distributed into the cimetidine group (CimG: received daily intraperitoneal injections of 100 mg/kg of body weight of cimetidine) or the saline group (SG). PD was induced by cotton ligature around the maxillary left first molars (PDSG and PDCimG). The right molars were used as controls (SG and CimG). After 7, 15, 30, and 50 days, maxillary fragments were processed for paraffin embedding or for transmission electron microscopy. Tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in the alveolar process surface and number of IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells in the gingival mucosa were quantified. Statistical analyses were performed (P ≤0.05). Results: In PDSG and PDCimG, gingival mucosa exhibited few collagen fibers among numerous inflammatory cells. In PDCimG, the number of TRAP‐positive osteoclasts and IL‐6, MMP‐1, and MMP‐9‐immunolabeled cells was significantly lower than in PDSG at all periods. A positive correlation between IL‐6/MMP‐1 and IL‐6/MMP‐9 was detected in PDSG and PDCimG. Conclusion: Cimetidine decreases bone loss through reduction of osteoclast number and induces reduction of IL‐6, MMP‐1, and MMP‐9 immunoexpression, reinforcing the idea that the beneficial effect of cimetidine in PD may be due to reduction of IL‐6 immunolabeling in the inflamed gingival mucosa.     

In vivo expression of Toll‐like receptor 2, Toll‐like receptor 4, CSF2 and LY64 in Chinese chronic periodontitis patients

Oral Diseases (2010) 16 , 343–350 Objective: Toll‐like receptors (TLRs) are the essential components in the innate and adaptive immune systems. Colony stimulating factor 2 (CSF2) is a cytokine that may prevent endotoxin tolerance, and LY64 has the ability to interfere with the recognition of bacteria via TLR4. The aim of this study was to explore the in vivo expressions of TLR2, TLR4, CSF2 and LY64 in Chinese chronic periodontitis patients. Methods: Gingival biopsies were collected from 24 chronic periodontitis patients and 19 healthy controls. The gene expression profiles of TLR2, TLR4, CSF2 and LY64 were investigated by real‐time polymerase chain reaction, and the protein expressions of TLR2 and TLR4 were detected by immunohistochemistry. In addition, the levels of CSF2 in gingival crevicular fluid (GCF) were determined by ELISA. Results: The higher mRNA expressions of TLR2, TLR4 and CSF2, and the lower mRNA expression of LY64 were detected in chronic periodontitis patients. And the increased protein expressions of TLR2 and TLR4 were confirmed by immunohistochemistry. In addition, the increase of total amount of CSF2 in GCF was observed in chronic periodontitis patients. Conclusions: Our results suggest that TLR2 and TLR4 may play a role in periodontal pathogenesis. In addition, CSF2 and LY64 may contribute to the regulation of inflammatory response and maintaining periodontal homeostasis.     

Invasive Streptococcus mutans induces inflammatory cytokine production in human aortic endothelial cells via regulation of intracellular toll‐like receptor 2 and nucleotide‐binding oligomerization domain 2

Streptococcus mutans, the primary etiologic agent of dental caries, can gain access to the bloodstream and has been associated with cardiovascular disease. However, the roles of S. mutans in inflammation in cardiovascular disease remain unclear. The aim of this study was to examine cytokine production induced by S. mutans in human aortic endothelial cells (HAECs) and to evaluate the participation of toll‐like receptors (TLRs) and cytoplasmic nucleotide‐binding oligomerization domain (NOD) ‐like receptors in HAECs. Cytokine production by HAECs was determined using enzyme‐linked immunosorbent assays, and the expression of TLRs and NOD‐like receptors was evaluated by real‐time polymerase chain reaction, flow cytometry and immunocytochemistry. The involvement of TLR2 and NOD2 in cytokine production by invaded HAECs was examined using RNA interference. The invasion efficiencies of S. mutans strains were evaluated by means of antibiotic protection assays. Five of six strains of S. mutans of various serotypes induced interleukin‐6, interleukin‐8 and monocyte chemoattractant protein‐1 production by HAECs. All S. mutans strains upregulated TLR2 and NOD2 mRNA levels in HAECs. Streptococcus mutans Xc upregulated the intracellular TLR2 and NOD2 protein levels in HAECs. Silencing of the TLR2 and NOD2 genes in HAECs invaded by S. mutans Xc led to a reduction in interleukin‐6, interleukin‐8 and monocyte chemoattractant protein‐1 production. Cytokine production induced by invasive S. mutans via intracellular TLR2 and NOD2 in HAECs may be associated with inflammation in cardiovascular disease.     

Analysis of Subgingival Plaque Ability to Stimulate Toll‐Like Receptor 2 and 4

Background: It has been shown that toll‐like receptor (TLR) 2‐ and TLR4‐stimulating abilities of supragingival plaque (SPP) are associated with periodontal conditions. It is hypothesized that SPP might affect the periodontium through its influence on subgingival plaque (SBP). This study investigates relationships between TLR2‐ and TLR4‐stimulating abilities of SBP and periodontal conditions. Methods: One hundred thirteen SBP samples were collected from the deepest pockets in patients with chronic periodontitis. TLR2‐ and TLR4‐stimulating abilities were measured using genetically engineered nuclear factor‐kappa B reporter cells. Numbers of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in each plaque sample were determined by real‐time polymerase chain reaction. Peripheral blood mononuclear cells (PBMCs) were stimulated with SBP samples in presence or absence of TLR4 or TLR2 inhibitor. Production of tumor necrosis factor (TNF)‐α and interleukin (IL)‐8 was analyzed by enzyme‐linked immunosorbent assay. Results: TLR4‐stimulating ability of SBP was associated with plaque index (PI), but not with other clinical parameters at sampling sites. TLR2‐stimulating ability of SBP was associated with none of the parameters. Number of P. gingivalis and A. actinomycetemcomitans in each plaque sample was not associated with TLR2‐ or TLR4‐stimulating ability of SBP. PBMCs stimulated with SBP samples produced TNF‐α and IL‐8, which was inhibited by TLR4 but not by TLR2 inhibitor. Conclusion: TLR4‐ but not TLR2‐stimulating ability of SBP is associated with PI. Enhanced TLR4‐stimulating ability at sites with accumulated plaque may mediate gingival inflammation.     

Innate Immune Receptor Expression in Peri‐Implant Tissues of Patients With Different Susceptibility to Periodontal Diseases

Background: Although inflammation mediates the pathogenesis of periodontal diseases, the effects of innate immune responses on implant therapies have not been evaluated. Innate immune receptors, including toll‐like‐receptors (TLRs) and the receptor for advanced glycated end‐products (RAGE), are upregulated within inflamed gingiva and are responsible for initiation of detrimental host responses. The aim of this study is to compare the expression of TLR2, TLR4, and RAGE in gingival tissues from participants susceptible to periodontitis and participants not susceptible to periodontitis before and after implant therapy. Methods: Periodontally healthy participants received implant therapy for non‐periodontal edentulism. Participants susceptible to periodontitis were diagnosed with chronic periodontitis prior to implant therapy. Gingival biopsies were collected from edentulous ridges before implant installation and from peri‐implant mucosa 2 months after treatment. Histology, real‐time PCR, and Western blot were used to evaluate levels of inflammatory infiltrate, TLR2, TLR4, and RAGE expression. Results: Before implant therapy, elevated levels of RAGE were detected in gingival tissues from participants susceptible to periodontitis when compared to those from participants with healthy periodontiums, whereas no differences in the expression of TLR2 or TLR4 were detected. After implant therapy, there was an upregulation of RAGE and TLR4 levels that coincided with a downregulation of TLR2 levels in biopsies from participants susceptible to periodontitis. Levels of RAGE and TLR4 remained unchanged in biopsies from participants with healthy periodontiums, whereas TLR2 levels were significantly upregulated. Histologically, post‐implant biopsies from participants susceptible to periodontitis displayed higher levels of inflammatory infiltrate. Conclusion: Elevated levels of inflammatory potential were found after implant therapy in participants susceptible to periodontitis.     

Expression of transient receptor potential vanilloid receptor 1 and toll-like receptor 4 in aggressive periodontitis and in chronic periodontitis

Öztürk A, Y?ld?z L. Expression of transient receptor potential vanilloid receptor 1 and toll‐like receptor‐4 in aggressive periodontitis and in chronic periodontitis. J Periodont Res 2011; 46: 475–482. © 2011 John Wiley & Sons A/S Background and Objective: The objective of the present study was to evaluate the expression and the distribution of the transient receptor potential vanilloid receptor 1 (TRPV1) and of toll‐like receptor 4 (TLR4) in tissue samples from patients with periodontal disease (aggressive periodontitis and chronic periodontitis) and from healthy controls. Material and Methods: Ten tissue samples from each disease group (aggressive periodontitis and chronic periodontitis) and from healthy subjects were obtained during routine oral surgical procedures. Subgingival specimens were collected from sites with advanced loss of support (probing depth > 5 mm) and specimens from the corresponding healthy controls were obtained during tooth extraction for orthodontic reasons or following surgical extraction of an impacted third molar. The distribution of TRPV1 and TLR4 receptors in human gingival tissue was studied by immunohistochemistry. Results: Both TLR4 and TRPV1 were detected in gingival tissues from healthy subjects, and from patients with chronic periodontitis and aggressive periodontitis, particularly in gingival keratinocytes, fibroblasts, inflammatory cells and the endothelial lining of capillaries in connective tissues. Histologic examination of the samples from healthy controls disclosed that clinically healthy gingiva does not correspond to histologically healthy gingiva. Subsequently, these samples were redesignated as gingivitis samples. TRPV1 was down‐regulated in all cell types in samples obtained from patients with chronic periodontitis compared to samples obtained from patients with gingivitis, whereas TLR4 was down‐regulated only in the epithelium and in gingival fibroblasts. In contrast, the levels of these markers in patients with aggressive periodontitis were similar to those in healthy patients. Conclusion: Local expression of TRPV1 and TLR4 in gingival tissues may contribute to both physiological and pathological processes in the periodontium. Our data suggest that TRPV1 and TLR4 may play a role specifically in the pathophysiology of chronic periodontitis.     

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis

Scheres N, Laine ML, Sipos PM, Bosch‐Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis. J Periodont Res 2011; 46: 407–416. © 2011 John Wiley & Sons A/S Background and Objective: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. Material and Methods: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n = 14) and healthy control subjects (n = 8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll‐like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor‐κB1 and its putative inhibitor NF‐κB inhibitor‐like protein 1, and of interleukin‐1β, interleukin‐6, interleukin‐8, tumour necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cel expressed, and secreted, were assessed by real‐time PCR. Results: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture‐positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis‐negative persons. Conclusion: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis‐positive donors are more responsive to an in vitro P. gingivalis challenge.