Host-bacteria crosstalk at the dentogingival junction.

The dentogingival junction is of crucial importance in periodontal host defense both structurally and functionally. Oral bacteria exert a constant challenge to the host cells and tissues at the dentogingival junction. The host response is set up to eliminate the pathogens by the innate and adaptive defense mechanisms. In health, the commensal bacteria and the host defense mechanisms are in a dynamic steady state. During periodontal disease progression, the dental bacterial plaque, junctional epithelium (JE), inflammatory cells, connective tissue, and bone all go through a series of changes. The tissue homeostasis is turned into tissue destruction and progression of periodontitis. The classical study of Slots showed that in the bacterial plaque, the most remarkable change is the shift from gram-positive aerobic and facultatively anaerobic flora to a predominantly gram-negative and anaerobic flora. This has been later confirmed by several other studies. Furthermore, not only the shift of the bacterial flora to a more pathogenic one, but also bacterial growth as a biofilm on the tooth surface, allows the bacteria to communicate with each other and exert their virulence aimed at favoring their growth. This paper focuses on host-bacteria crosstalk at the dentogingival junction and the models studying it in vitro.Peer reviewe

Construction and characterization of a multilayered gingival keratinocyte culture model : the TURK-U model

In construction of epithelial cells as multilayers, the cells are grown submerged to confluence on fibroblast-embedded collagen gels and, then, lifted to air to promote their stratification. We recently demonstrated that gingival epithelial cells form uniform monolayers on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium, which allows the cells to grow at an air-liquid-solid interface from the beginning of the culturing protocol. In this study, the aim was to further develop our previous model to form a multilayered gingival epithelial culture model. Two different epithelial cell lines (HaCaT from skin and HMK from gingiva) were used in all experiments. Both cell lines were grown first as monolayers for 3 days. After that, keratinocytes were trypsinized, counted and seeded on a sterile semi-permeable nitrocellulose membrane placed on the top of a semi-solid growth medium, forming an air-liquid-solid interface for the cells to grow. At days 1, 4, and 7, epithelial cells were fixed, embedded in paraffin, and sectioned for routine Hematoxylin-Eosin staining and immunohistochemistry for cytokeratin (Ck). At day 1, HMK cells grew as monolayers, while HaCaT cells stratified forming an epithelium with two to three layers. At day 4, a stratified epithelium in the HMK model had four to five layers and its proliferation continued up to day 7. HaCaT cells formed a dense and weakly proliferating epithelium with three to four layers of stratification at day 4 but the proliferation disappeared at day 7. At all days, both models were strongly positive for Ck5, Ck7, and Ck 19, and weakly positive for Ck10. Gingival epithelial cells stratify successfully on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium. This technique allows researchers to construct uniform gingival epithelial cell multilayers at an air-liquid-solid interface, without using collagen gels, resulting in a more reproducible method.Peer reviewe

Focussed microarray analysis of apoptosis in periodontitis and its potential pharmacological targeting by carvacrol

AbstractObjectiveThe objective of this study was to perform a landscape analysis of apoptosis-related genes/proteins and to study the differential gene expression by analysing array data from periodontitis patients and, second, to evaluate the anti-apoptotic effects of carvacrol, a monoterpenoid phenol, in vitro.DesignA gene/protein interaction network model ‘APOP’ was developed by using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) version 9.05. Differential gene expression was determined by using the limma package from R and false discovery rate (FDR). With ViaComplex software, gene expression was plotted over the network. The anti-apoptotic effect of carvacrol was tested on sorbitol-treated HaCaT cells, by using a commercial kit for caspase-3 activity.ResultsThe ‘APOP’ model characterised the landscape of interactions between apoptosis-related genes/proteins in silico. Forty-nine out of 70 genes from this model, such as CSF2RB, NFKBIE, ENDOG, CASP10 and CASP3, were differentially expressed (corrected p-value<0.05) in periodontitis samples when compared to those of healthy controls. In addition, carvacrol (0.43%) was able to inhibit the pro-apoptotic effects induced by sorbitol (0.3M), as seen by the reduction in caspase-3 activity on HaCaT cells.ConclusionOur results suggest that caspase-3 can be a target protein to inhibit periodontitis-associated apoptosis of epithelial cells and that carvacrol has therapeutic potential as an anti-apoptotic agent

Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation

Background: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. Methods: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. Results: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and alpha-1-antichymotrypsin, as well as complement C1q. Conclusions: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.Peer reviewe

Long-Term Effect of Heat Shock Protein 60 from Actinobacillus actinomycetemcomitans on Epithelial Cell Viability and Mitogen-Activated Protein Kinases

Our previous studies showed that bacterial heat shock protein 60 (hsp60) induces cultured epithelial cell proliferation within 24 h. Here we investigated the long-term effects of heat shock protein 60 isolated from Actinobacillus actinomycetemcomitans on skin keratinocyte (HaCaT cell line) viability and the cell signaling involved. Prolonged incubation in the presence of hsp60 increased the rate of epithelial cell death. The number of viable cells in hsp60-treated culture was 37% higher than the number in the control at 24 h but 27% lower at 144 h. A kinetics study of the effect of hsp60 on the phosphorylation of mitogen-activated protein kinases (MAPKs) involving Western blotting with phospho-specific antibodies showed that in addition to a transient early increase in p38 levels, a second peak appeared in keratinocytes 24 h after the addition of hsp60. In contrast, prolonged incubation with hsp60 caused a decrease in the level of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) compared with that in the controls, possibly as a result of protein phosphatase activity. We found that hsp60 increased the levels of several phosphatases, including MAP-2, which strongly dephosphorylates ERK1/2. Moreover, hsp60 increased the level of tumor necrosis factor alpha (TNF-α) in culture medium in a dose-dependent manner. TNF-α added to culture showed a cytotoxic effect on epithelial cells, particularly with longer incubation periods. TNF-α also induced the phosphorylation of p38. Finally, our results show that bacterial hsp60 inhibited stress-induced synthesis of cellular hsp60. Therefore, several cell behavior changes caused by long-term exposure to bacterial hsp60 may lead to impaired epithelial cell viability

Human neutrophil peptide-1 affects matrix metalloproteinase-2,-8 and-9 secretions of oral squamous cell carcinoma cell lines in vitro

Objectives: The present study aimed to investigate the effect of HNP-1 on the matrix metalloproteinase (MMP)-2, -8 and -9 secretions of two oral squamous cell carcinoma (OSCC) cell lines (UT-SCC-43A and UT-SCC-43B). Design: In all experiments, the two OSCC cell lines were incubated with graded concentrations (0,1, 5, and 10 mu g/ml) of HNP-1 for 24 and 48 h. Cell viability was measured using a colorimetric proliferation test and cell death was analyzed with a colorimetric cytotoxicity detection kit. Enzyme activity of MMP-2 and MMP-9 was detected by using gelatin zymography, and molecular weight forms of MMP-8 were determined by Western-blot and a densitometric quantitation method. Results: Both cell lines showed a significant increase in LDH toxicity at 24 h (UT-SCC-43A: p = 0.005 & UT-SCC-43B: p = 0.014). Reduced gelatinolytic activities of proMMP-2 were detected in UT-SCC-43B cell line after 24 and 48 h of incubation with HNP-1 (1 mu g/ml: p <0.001, 5 mu g/ml: p <0.001, and 1011g/ml: p = 0.0225). MMP-8 levels of both cell lines decreased at 200-250 kDa after 24 h of incubation, while after 48 h only UT-SCC-43B decreased at 45-50 kDa. Conclusions: Our results indicate that HNP-1 suppresses the secretion of MMP-2,-8, and -9 in OSCC cell lines. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe