Insulin‐like growth factor‐binding protein‐2 and ‐3 in gingival crevicular fluid

Takenouchi Y, Ohshima M, Yamaguchi Y, Nishida T, Senda N, Idesawa M, Otsuka K, Ito K. Insulin‐like growth factor‐binding protein‐2 and ‐3 in gingival crevicular fluid. J Periodont Res 2010; 45: 803–808. © 2010 John Wiley & Sons A/S Background and Objective: Insulin‐like growth factor‐binding proteins (IGFBPs) are crucial regulators of insulin‐like growth factor (IGF). They enhance or inhibit IGF functions, but also exhibit IGF‐independent effects. In a previous study, we detected, qualitatively, IGFBP‐2 and ‐3 in gingival crevicular fluid using a cytokine antibody array. Here we extended these results using an ELISA to determine the concentrations of IGFBP‐2 and ‐3 in gingival crevicular fluid. In addition, we explored whether the expression of IGFBP‐2 and IGFBP‐3 correlates with periodontal disease severity. Material and Methods: Gingival crevicular fluid samples from 92 sites of 12 patients affected with periodontal disease and from 100 sites of 19 healthy volunteers, were collected, divided into two groups and analyzed by ELISA for IGFBP‐2 and ‐3 expression. The potential correlation among probing depth, gingival index and the concentrations of IGFBP‐2 and ‐3 was analyzed. Results: Positive correlations were observed between the concentration of IGFBP‐2 and probing depth and gingival index, but not for IGFBP‐3. The IGFBP‐2 concentrations at bleeding on probing‐positive sites and at sites with a probing depth of ≥ 4 mm were higher than at bleeding on probing‐negative sites and at sites with a probing depth of ≤ 3 mm. Conclusion: These results indicate that IGFBP‐2 is a potential novel marker for periodontal disease progression. As IGFBP‐2 modulates bone metabolism and cell migration, IGFBP‐2 in the gingival crevicular fluid may reflect periodontal disease activity.     

Attenuation of radiation‐ and chemoradiation‐induced mucositis using gamma‐d‐glutamyl‐l‐tryptophan (SCV‐07)

Oral Diseases (2010) 16 , 655–660 Objective: To evaluate the efficacy of a novel immunomodulating peptide (SCV‐07) in attenuating the course of radiation‐induced mucositis in an established animal model of oral mucositis (OM). Material and Methods: In three separate experiments, golden Syrian hamsters received either an acute radiation challenge to the buccal mucosa of eight fractionated doses of 7.5 Gy of radiation over a 2‐week‐period, or a combination of acute radiation and cisplatin. In each experiment, animals were treated with varying doses or schedules of SCV‐07 or placebo. OM was scored in a blinded fashion using digital images obtained during the experimental period. Results: We found that SCV‐07 reduced the severity and duration of both acute and fractionated radiation‐induced OM. Similarly, when radiation and chemotherapy were used to induce OM, treatment with SCV‐07 significantly reduced the duration of ulcerative OM. The therapeutic benefit was dependent on both dose and schedule of administration. Conclusion: Taken together, we found SCV‐07 was able to modify the duration and severity of oral mucositis and was dependent on schedule and dose.     

The expressions of IGF‐1, BMP‐2 and TGF‐β1 in cartilage of condylar hyperplasia

Summary Condylar hyperplasia is a complex post‐natal growth abnormality of the mandible and condyle, which leads to facial asymmetry. We investigated the distributions of insulin‐like growth factors (IGF‐1), bone morphogenetic protein‐2 (BMP‐2) and transforming growth factor‐β1 (TGF‐β1) in cartilage of condylar hyperplasia and revealed relationships between age and the cartilaginous thickness. Twenty patients with condylar hyperplasia were divided into four histopathological types. The cartilaginous thickness and age in different histological types were analysed, and the localizations of IGF‐1, BMP‐2 and TGF‐β1 were detected by immunohistochemistry analysis. The cartilaginous thickness of condylar hyperplasia significantly increased. The cartilaginous thickness of type III was significantly thicker than type I and type II, Bivariate correlation revealed a significant correlations between age and the cartilaginous thickness (r = 0·68, P = 0·01). However, the expressions of IGF‐1, BMP‐2 and TGF‐β1 were the strongest in type I. In almost all types of condylar hyperplasia, the presence of IGF‐1 and BMP‐2 was found mainly in the proliferative chondrocyte layer and the hypertrophic chondrocyte layer, and only a few in the calcified chondrocyte layer. The presence of TGF‐β1 widely distributed from the fibrous articular surface to the calcified cartilage. These findings suggest that the proliferative activity of cartilage in condylar hyperplasia is strongly associated with age and cartilaginous thickness. Therefore, the four pathological types of condylar hyperplasia seem more likely to be four discontinuous stages.     

Involvement of MT1‐MMP and TIMP‐2 in human periodontal disease

Oral Diseases (2010) 16 , 388–395 Objectives: Periodontal disease is characterized by an increased collagen metabolism. Although membrane type‐1 matrix metalloproteinase (MT1‐MMP) plays a critical role in collagen degradation, its involvement in human periodontitis remains to be determined. Methods: MT1‐MMP and TIMP‐2 expression and distribution were evaluated in gingival tissue samples derived from 10 healthy and 12 periodontitis‐affected human subjects. MT1‐MMP and TIMP‐2 expression were assessed through Western‐blot of tissue homogenates. The main cell types involved in MT1‐MMP and TIMP‐2 production were evaluated by means of immunohistochemistry. Results: Both MT1‐MMP and TIMP‐2 were significantly increased in periodontitis‐affected gingival tissues when compared to healthy gingiva. Moreover, the balance between MT1‐MMP and its inhibitor TIMP‐2 was altered in periodontitis‐affected tissues, suggesting an imbalance in this proteolytic axis. Immunohistochemistry demonstrated the expression of MT1‐MMP in fibroblasts and macrophages in gingival tissues. MT1‐MMP was detected in cells in close association with the gingival collagen matrix. TIMP‐2 expression was identified in fibroblasts, macrophages and epithelial cells. Conclusions: Our observations show an increased expression of MT1‐MMP and TIMP‐2 in periodontitis‐affected gingival tissues. The altered balance between these two molecular mediators of collagen remodeling suggests their involvement in human periodontal disease.     

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.     

Protease‐activated receptor 2 mediates interleukin‐8 and intercellular adhesion molecule‐1 expression in response to Aggregatibacter actinomycetemcomitans

Introduction:  We investigated the mechanisms by which extracts of Aggregatibacter actinomycetemcomitans affect the inflammatory response in gingival epithelial cells. Methods:  Human gingival cells (Ca9‐22) were cultured in bacterial extracts prepared from A. actinomycetemcomitans ATCC 29522. The cells were pretreated with protease inhibitors or transfected with small interfering RNA (siRNA) specific for protease‐activated receptor 2 (PAR‐2). Results:  The pretreatment of cells with serine protease inhibitors significantly inhibited A. actinomycetemcomitans extract‐induced expression of interleukin‐8 (IL‐8) and intercellular adhesion molecule‐1 (ICAM‐1) at both the messenger RNA and protein levels. In addition, A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression was significantly decreased in PAR‐2/siRNA‐transfected cells. Conclusions:  A. actinomycetemcomitans extract‐induced IL‐8 and ICAM‐1 expression in gingival epithelial cells is mediated by PAR‐2.     

Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E2 synthesis in human gingival fibroblasts

Chotjumlong P, Khongkhunthian S, Ongchai S, Reutrakul V, Krisanaprakornkit S. Human β‐defensin‐3 up‐regulates cyclooxygenase‐2 expression and prostaglandin E ₂ synthesis in human gingival fibroblasts. J Periodont Res 2010; 45: 464–470. © 2010 John Wiley & Sons A/S Background and Objective: Oral epithelial cells express three antimicrobial peptide human β‐defensins (hBDs) that have previously been demonstrated to exert proinflammatory effects on various immune cells. We wanted to examine whether hBDs could induce cyclooxygenase‐2 (COX‐2) expression and prostaglandin E₂ (PGE₂) synthesis in non‐immune cells, such as human gingival fibroblasts. Material and Methods: Cultured fibroblasts were treated with different concentrations of hBD‐1, ‐2, ‐3 or interleukin‐1β, as a positive control, for various times, in the presence or absence of NS‐398, a specific COX‐2 inhibitor. The levels of COX‐1 and COX‐2 mRNA expression were analyzed using RT‐PCR and real‐time PCR. Whole cell lysates were analyzed for COX‐1 and COX‐2 protein expression by western blotting. Cell‐free culture supernatants were assayed for PGE₂ levels by ELISA. The lactate dehydrogenase assay was performed to determine the cytotoxicity of hBDs. Results: Ten and 40 μg/mL of hBD‐3 up‐regulated COX‐2 mRNA and protein expression, consistent with COX‐2 up‐regulation by interleukin‐1β, whereas hBD‐1 and hBD‐2 did not. However, COX‐1 mRNA and protein were constitutively expressed. The time‐course study revealed that hBD‐3 up‐regulated COX‐2 mRNA and protein expression at 6 and 12 h, respectively. Consistent with COX‐2 up‐regulation, 10 and 40 μg/mL of hBD‐3 significantly increased PGE₂ levels in cell‐free culture supernatants (p < 0.05), and this was inhibited by NS‐398 in a dose‐dependent manner. Neither of the hBD concentrations tested in this study was toxic to the cells. Conclusion: These findings indicate that epithelial human β‐defensin‐3 functions as a proinflammatory mediator in controlling arachidonic acid metabolism in underlying fibroblasts.