The role of programmed death receptor (PD-)1/PD-ligand (L)1 in periodontitis and cancer.

The programmed-death-ligand-1 (PD-L1) is an immune-modulating molecule that is constitutively expressed on various immune cells, different epithelial cells and a multitude of cancer cells. It is a costimulatory molecule that may impair T-cell mediated immune response. Ligation to the programmed-death-receptor (PD)-1, on activated T-cells and further triggering of the related signaling pathways can induce T-cells apoptosis or anergy. The upregulation of PD-L1 in various cancer types, including oral squamous cell carcinomas, was demonstrated and has been linked to immune escape of tumors and poor prognosis. A bidirectional relationship exists between the increased PD-L1 expression and periodontitis as well as the epithelial-mesenchymal transition (EMT), a process of interconversion of epithelial cells to mesenchymal cells that may induce immune escape of tumors. Interaction between exosomal PD-L1 and PD-1 on T-cells may cause immunosuppression by blocking the activation and proliferation of T-cells. The efficacy and importance of treatment with PD-1/PD-L1 checkpoint inhibitors and their prognostic influence on human cancers was demonstrated. Regarding PD-1/PD-L1 checkpoint inhibitors, resistances exist or may develop, basing on various factors. Further investigations of the underlying mechanisms will help to overcome the therapeutic limitations that result from resistances and to develop new strategies for the treatment of cancer.

Orthodontic Compression Enhances Macrophage M2 Polarization via Histone H3 Hyperacetylation.

Orthodontic tooth movement is a complex periodontal remodeling process triggered by compression that involves sterile inflammation and immune responses. Macrophages are mechanically sensitive immune cells, but their role in orthodontic tooth movement is unclear. Here, we hypothesize that orthodontic force can activate macrophages, and their activation may be associated with orthodontic root resorption. After force-loading and/or adiponectin application, the migration function of macrophages was tested via scratch assay, and Nos2, Il1b, Arg1, Il10, ApoE, and Saa3 expression levels were detected using qRT-PCR. Furthermore, H3 histone acetylation was measured using an acetylation detection kit. The specific inhibitor of H3 histone, I-BET762, was deployed to observe its effect on macrophages. In addition, cementoblasts were treated with macrophage-conditioned medium or compression force, and OPG production and cellular migration were measured. We further detected Piezo1 expression in cementoblasts via qRT-PCR and Western-blot, and its effect on the force-induced impairment of cementoblastic functions was also analyzed. Compressive force significantly inhibited macrophage migration. Nos2 was up-regulated 6 h after force-loading. Il1b, Arg1, Il10, Saa3, and ApoE increased after 24 h. Meanwhile, higher H3 histone acetylation was detected in the macrophages subjected to compression, and I-BET762 dampened the expression of M2 polarization markers (Arg1 and Il10). Lastly, even though the activated macrophage-conditioned medium showed no effect on cementoblasts, compressive force directly impaired cementoblastic function by enhancing mechanoreceptor Piezo1. Compressive force activates macrophages; specifically, it causes M2 polarization via H3 histone acetylation in the late stage. Compression-induced orthodontic root resorption is macrophage-independent, but it involves the activation of mechanoreceptor Piezo1.

Influence of leptin and compression in GAS-6 mediated homeostasis of periodontal ligament cell.

Growth arrest-specific protein 6 (GAS-6) regulates immunomodulatory and inflammatory mechanisms in periodontium and may participate in obesity predisposition. This study aimed to determine whether GAS-6 is associated with the homeostasis of periodontal ligament (SV-PDL) cells in the presence of adipokines or compressive forces. The SV-PDL cell line was used. Western blots were employed for TAM receptors detection. Cells were stimulated using different concentrations of GAS-6. The migration, viability, and proliferation were measured by a standard scratch test, MTS assay, and immunofluorescent staining. The mRNA expression was analyzed by RT-PCR. Release of TGF-ß1, GAS-6, and Axl were verified by ELISA. Western blot shows that TAM receptors are expressed in SV-PDL cells. GAS-6 has a promoting effect on cell migration and proliferation. RT-PCR analysis showed that GAS-6 induces Collagen-1, Collagen-3, Periostin, and TGF-ß1 mRNA expression whereas it reduces Caspase-3, Caspase-8, Caspase-9, and IL-6 mRNA expression. Further, secreted GAS-6 in SV-PDL is reduced in response to both compressive forces and leptin and upregulated by IL-6. Additionally, ADAM-10 inhibition reduces GAS-6 and Axl release on SV-PDL cells. TAM receptors especially Axl are identified as the receptors of GAS-6. GAS-6/TAM interactions contribute to periodontal ligament cells homeostasis. Leptin inhibits the GAS-6 release independently of ADAM-10 metalloprotease.

Ciliary Neurotrophic Factor (CNTF) and Its Receptors Signal Regulate Cementoblasts Apoptosis through a Mechanism of ERK1/2 and Caspases Signaling.

Ciliary neurotrophic factor (CNTF) was identified as a survival factor in various types of peripheral and central neurons, glia and non-neural cells. At present, there is no available data on the expression and localization of CNTF-receptors in cementoblasts as well as on the role of exogenous CNTF on this cell line. The purpose of this study was to determine if cementoblasts express CNTF-receptors and analyze the mechanism of its apoptotic regulation effects on cementoblasts. OCCM-30 cementoblasts were cultivated and stimulated kinetically using CNTF protein (NBP2-35168, Novus Biologicals). Quantified transcriptional (RT-qPCR) and translational (WB) products of CNTFRα, IL-6Rα (CD126), LIFR, p-GP130, GP130, p-ERK1/2, ERK1/2, Caspase-8, -9, -3 and cleaved-caspase-3 were evaluated. Immunofluorescence (IF) staining was applied to visualize the localization of the CNTF-receptors within cells. The apoptosis ratio was measured with an Annexin-V FITC/PI kit. The ERK1/2 antagonist (FR180204, Calbiochem) was added for further investigation by flow cytometry analysis. The CNTF-receptor complex (CNTFRα, LIFR, GP130) was functionally up-regulated in cementoblasts while cultivated with exogenous CNTF. CNTF significantly attenuated cell viability and proliferation for long-term stimulation. Flow cytometry analysis shows that CNTF enhanced the apoptosis after prolonged duration. However, after only a short-term period, CNTF halts the apoptosis of cementoblasts. Further studies revealed that CNTF activated phosphorylated GP130 and the anti-apoptotic molecule ERK1/2 signaling to participate in the regulation of the apoptosis ratio of cementoblasts. In conclusion, CNTF elicited the cellular functions through a notable induction of its receptor complex in cementoblasts. CNTF has an inhibitory effect on the cementoblast homeostasis. These data also elucidate a cellular mechanism for an exogenous CNTF-triggered apoptosis regulation in a mechanism of ERK1/2 and caspase signaling and provides insight into the complex cellular responses induced by CNTF in cementoblasts.

PD-L1 Up-Regulation in Prostate Cancer Cells by Porphyromonas gingivalis.

Chronic inflammation is known to contribute to various human cancers. Porphyromonas gingivalis (P. gingivalis), is a gram-negative oral keystone pathogen that may cause severe periodontitis and expresses several virulence factors to affect the host immune system. Periodontitis is a chronic infectious disease that while progression, may cause loss of attachment and destruction of the tooth supporting tissues. Prostate cancer is one of the most common malignancies in men. Increasing evidence links periodontitis with prostate cancer, however the mechanisms explaining this relationship remain unclear. The aim of this study was to investigate the expression and signaling pathway of programmed death ligand 1 (PD-L1) in a prostate cancer cell line after infection with P. gingivalis and stimulation with P. gingivalis components to reveal the mechanism of tumor-induced immune evasion associated with bacterial infection in the tumor environment. Prostate cancer cells were infected with different concentrations of viable P. gingivalis and treated with different concentrations of heat-killed P. gingivalis and P. gingivalis cell components, including the total membrane fraction, inner membrane fraction, outer membrane fraction, cytosolic fraction and peptidoglycan (PGN). Chemical inhibitors were used to block different important molecules of signaling pathways to assess the participating signal transduction mechanisms. PD-L1 expression was detected by Western blot after 24 h of infection. PD-L1 was demonstrated to be upregulated in prostate cancer cells after infection with viable and with heat-killed P. gingivalis membrane fractions. Also isolated PGN induced PD-L1 up-regulation. The upregulation was mediated by the NOD1/NOD2 signaling pathway. No upregulation could be detected after treatment of the cells with P. gingivalis lipopolysaccharide (LPS). These results indicate, that chronic inflammatory disease can contribute to tumor immune evasion by modifying the tumor microenvironment. Thus, chronic infection possibly plays an essential role in the immune response and may promote the development and progression of prostate cancer.

Recombinant Porphyromonas gingivalis W83 FimA alters immune response and metabolic gene expression in oral squamous carcinoma cells.

OBJECTIVES: The Gram-negative anaerobic rod Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in periodontitis and expresses a multitude of virulence factors iincluding fimbriae that are enabling adherence to and invasion in cells and tissues. The progression of periodontitis is a consequence of the interaction between the host immune response and periodontal pathogens. The aim of this study was to investigate the genome-wide impact of recombinant fimbrial protein FimA from P. gingivalis W83 on the gene expression of oral squamous carcinoma cells by transcriptome analysis. MATERIALS AND METHODS: Human squamous cell carcinoma cells (SCC-25) were stimulated for 4 and 24 h with recombinant FimA. RNA sequencing was performed and differential gene expression and enrichment were analyzed using gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and REACTOME. The results of transcriptome analysis were validated using quantitative real-time polymerase chain reaction (PCR) with selected genes. RESULTS: Differential gene expression after 4 and 24 h revealed upregulation of 464 (4 h) and 179 genes (24 h) and downregulation of 69 (4 h) and 312 (24 h) genes. GO, KEGG, and REACTONE enrichment analysis identified a strong immunologic transcriptomic response signature after 4 h. After 24 h, mainly those genes were regulated, which belonged to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response and signaling demonstrated strong upregulation of CCL20, TNFAIP6, CXCL8, TNFAIP3, and NFkBIA after both stimulation times. CONCLUSIONS: These data shed light on the RNA transcriptome of human oral squamous carcinoma epithelial cells following stimulation with P. gingivalis FimA and identify a strong immunological gene expression response to this virulence factor. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate basic mechanisms that may provide new prospects for periodontitis therapy and give new insights into the development and possible treatments of cancer.

Porphyromonas gingivalis W83 Membrane Components Induce Distinct Profiles of Metabolic Genes in Oral Squamous Carcinoma Cells.

Periodontitis, a chronic inflammatory disease is caused by a bacterial biofilm, affecting all periodontal tissues and structures. This chronic disease seems to be associated with cancer since, in general, inflammation intensifies the risk for carcinoma development and progression. Interactions between periodontal pathogens and the host immune response induce the onset of periodontitis and are responsible for its progression, among them Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic rod, capable of expressing a variety of virulence factors that is considered a keystone pathogen in periodontal biofilms. The aim of this study was to investigate the genome-wide impact of P. gingivalis W83 membranes on RNA expression of oral squamous carcinoma cells by transcriptome analysis. Human squamous cell carcinoma cells (SCC-25) were infected for 4 and 24 h with extracts from P. gingivalis W83 membrane, harvested, and RNA was extracted. RNA sequencing was performed, and differential gene expression and enrichment were analyzed using GO, KEGG, and REACTOME. The results of transcriptome analysis were validated using quantitative real-time PCR with selected genes. Differential gene expression analysis resulted in the upregulation of 15 genes and downregulation of 1 gene after 4 h. After 24 h, 61 genes were upregulated and 278 downregulated. GO, KEGG, and REACTONE enrichment analysis revealed a strong metabolic transcriptomic response signature, demonstrating altered gene expressions after 4 h and 24 h that mainly belong to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response, signaling, and metabolism revealed upregulated expression of CCL20, CXCL8, NFkBIA, TNFAIP3, TRAF5, CYP1A1, and NOD2. This work sheds light on the RNA transcriptome of human oral squamous carcinoma cells following stimulation with P. gingivalis membranes and identifies a strong metabolic gene expression response to this periodontal pathogen. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate the basic mechanisms of periodontitis and the development of cancer.

Pathogenic Mechanisms of Fusobacterium nucleatum on Oral Epithelial Cells.

Periodontitis is an oral chronic inflammatory disease and may cause tooth loss in adults. Oral epithelial cells provide a barrier for bacteria and participate in the immune response. Fusobacterium nucleatum (F. nucleatum) is one of the common inhabitants of the oral cavity and has been identified as a potential etiologic bacterial agent of oral diseases, such as periodontitis and oral carcinomas. F. nucleatum has been shown to be of importance in the development of diverse human cancers. In the dental biofilm, it exhibits a structural role as a bridging organism, connecting primary colonizers to the largely anaerobic secondary colonizers. It expresses adhesins and is able to induce host cell responses, including the upregulation of defensins and the release of chemokines and interleukins. Like other microorganisms, its detection is achieved through germline-encoded pattern-recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs). By identification of the pathogenic mechanisms of F. nucleatum it will be possible to develop effective methods for the diagnosis, prevention, and treatment of diseases in which a F. nucleatum infection is involved. This review summarizes the recent progress in research targeting F. nucleatum and its impact on oral epithelial cells.

Selection and validation of reference gene for RT-qPCR studies in co-culture system of mouse cementoblasts and periodontal ligament cells.

OBJECTIVE: RT-qPCR is a reliable method for gene expression analysis, but the accuracy of the quantitative data depends on the appropriate selection of reference genes. A Co-culture system consisting of periodontal ligament cells (SV-PDL) and cementoblasts (OCCM-30) to investigate the crosstalk between these two cell lines under orthodontic condition is essential for experimental orthodontic setups in-vitro. Therefore, we aimed to identify a set of reliable reference genes suitable for RT-qPCR studies for prospective co-culture systems of OCCM-30 and SV-PDL cells. RESULTS: The results demonstrated that PPIB, GUSB and RPLP0 turned out to be the three most stable reference genes for OCCM-30 in the co-culture system, while PPIB, POLR2A and RPLP0 have the three highest rankings for SV-PDL cells in the co-culture system. The most stable gene combination were PPIB and POLR2A in the co-culture system. In conclusion, PPIB is overall the most stably expressed reference gene for OCCM-30 or SV-PDL cell line in the system. The combination of PPIB and POLR2A as reference genes are indicated to be the potential and mandatory to obtain accurate quantification results for normalizing RT-qPCR data in genes of interest expression in these two cell lines co-culture systems.

MAPK and ß-Catenin signaling: implication and interplay in orthodontic tooth movement.

Orthodontic tooth movement (OTM) requires the orthodontic forces (compressive and tensile strain) to subject to the periodontal ligament and mechanosensory cells in the periodontium and to achieve mechanotransduction by mechanoreceptors. In the context of OTM, a diverse array of signaling pathways are activated in mechanosensory cells that modulate bone resorption and formation in in vitro and in vivo models. The underlying molecular signal transduction, such as MAPK and ß-Catenin signaling, that is involved in OTM, has been partially identified. It includes, but is not limited to genes and proteins which are related to osteogenesis, osteoclastogenesis, cementogenesis and inflammation. However, the interactive relation of ß-Catenin and MAPK signaling remains ambiguous and diverse cross-talks are acting with each other. In this comprehensive text, we review the biology of OTM and reported experimental results on the activation/inhibition of these two signaling pathways during OTM. Here, we also focus on the implications and interplays between the MAPK and ß-Catenin signaling in mechanosensory cells in response to orthodontic forces. Finally, the potential of further investigation strategies aimed at supporting orthodontic interventions are discussed. This review provides a conceptual framework for more comprehensive knowledge about signaling interaction during OTM.

Effect of Low-Level Er: YAG (2940 nm) laser irradiation on the photobiomodulation of mitogen-activated protein kinase cellular signaling pathway of rodent cementoblasts.

BACKGROUNDS: Dental avulsion due to trauma, especially in young patients, is a worldwide problem, requiring tooth replacement. Delayed replantation could cause tooth loss when the cementum is severely damaged. A small number of studies has reported that photobiomodulation (PBM) therapy using Er: YAG laser irradiation activates cellular signaling responses in different cell types, resulting in a variety of favorable biological effects. The aim of this in vitro study was to evaluate the potential biostimulatory effect of low-level Er: YAG laser irradiation on the biological responses of cultured mouse cementoblasts (OCCM-30), including the mitogen-activated protein kinases (MAPKs). METHODS: OCCM-30 cells were exposed to 2940 nm Er: YAG laser irradiation for 15 s at 0.34 W (pulse duration of 100 or 1000 µs, 17 mJ/pulse) at energy densities of 1 or 2 J/cm2. Irradiated and non-irradiated OCCM-30 cells were tested for migration (Scratch assay), proliferation (MTS assay) and functional differentiation (Alizarin Red S assay). Lumican (Lum) and Fibromodulin (Fmod) gene expression, and activation of MAPKs, were assessed by RT-PCR and Western blotting, respectively. RESULTS: Low-level Er: YAG laser irradiation at 2 J/cm2 and pulse duration of 1000 µs resulted in the highest migration rate and proliferation. Moreover, the pulse duration irradiation of 100 µs increased Lum expression. Fmod expression was increased after 1000 µs pulse duration laser stimulation. Low-level Er: YAG laser irradiation increased the mineralization of OCCM-30 cells after 7 days and activated ERK1/2, P38 and JNK signaling. CONCLUSIONS: Low-level Er: YAG laser irradiation induces OCCM-30 cell migration, proliferation and differentiation, and activates the MAPK signaling pathway.

Hypoxia-inducible factor 1-alpha acts as a bridge factor for crosstalk between ERK1/2 and caspases in hypoxia-induced apoptosis of cementoblasts.

Hypoxia-induced apoptosis of cementoblasts (OCCM-30) may be harmful to orthodontic treatment. Hypoxia-inducible factor 1-alpha (HIF-1α) mediates the biological effects during hypoxia. Little is known about the survival mechanism capable to counteract cementoblast apoptosis. We aimed to investigate the potential roles of HIF-1α, as well as the protein-protein interactions with ERK1/2, using an in-vitro model of chemical-mimicked hypoxia and adipokines. Here, OCCM-30 were co-stimulated with resistin, visfatin or ghrelin under CoCl2 -mimicked hypoxia. In-vitro investigations revealed that CoCl2 -induced hypoxia triggered activation of caspases, resulting in apoptosis dysfunction in cementoblasts. Resistin, visfatin and ghrelin promoted the phosphorylated ERK1/2 expression in OCCM-30 cells. Furthermore, these adipokines inhibited hypoxia-induced apoptosis at different degrees. These effects were reversed by pre-treatment with ERK inhibitor (FR180204). In cells treated with FR180204, HIF-1α expression was inhibited despite the presence of three adipokines. Using dominant-negative mutants of HIF-1α, we found that siHIF-1α negatively regulated the caspase-8, caspase-9 and caspase-3 gene expression. We concluded that HIF-1α acts as a bridge factor in lengthy hypoxia-induced apoptosis in an ERK1/2-dependent pathway. Gene expressions of the caspases-3, caspase-8 and caspase-9 were shown to be differentially regulated by adipokines (resistin, visfatin and ghrelin). Our study, therefore, provides evidence for the role of ERK1/2 and HIF-1α in the apoptotic response of OCCM-30 cells exposed to CoCl2 -mimicked hypoxia, providing potential new possibilities for molecular intervention in obese patients undergoing orthodontic treatment.

Porphyromonas gingivalis induced up-regulation of PD-L1 in colon carcinoma cells.

Programmed death-ligand-1 (PD-L1) is a ligand for programmed death receptor (PD-1) that plays a major role in cell-mediated immune response; it regulates T-cell activation and regulates survival and functions of activated T cells. Expression of PD-L1 can induce chronic inflammation and activate mechanisms of immune evasion. PD-L1 is expressed in most of human carcinomas. Porphyromonas gingivalis (P. gingivalis) is a major keystone pathogen in periodontitis that invade host cells and disposes a variety of virulence factors. The aim of the present study was to clarify the signaling pathway of P. gingivalis molecules that induce PD-L1 up-regulation in colon carcinoma cells. Additionally, it was investigated which components of P. gingivalis are responsible for PD-L1 induction. Colon cancer cells (CL-11) were stimulated with total membrane (TM) fractions, peptidoglycans (PDGs) and viable P. gingivalis bacteria. Seven signaling molecule inhibitors were used: receptor-interacting serine/threonine-protein kinase 2 (RIP2) tyrosine kinase inhibitor, nucleotide-binding oligomerization domain (NOD)-like receptor 1&2 inhibitor, NOD-like receptor, nuclear factor kappa B inhibitor, c-Jun N-terminal kinases inhibitor, mitogen-activated protein/extracellular signal-regulated kinase inhibitor, mitogen activated kinase (MAPK) inhibitor. PD-L1 protein expression was examined by western blot analysis and quantitative real time PCR. It was demonstrated that the TM fraction and PDG induced up-regulation of PD-L1 expression in colon cancer cells. In conclusion, the results of this study suggest that PDG of P. gingivalis plays a major role in PD-L1 up-regulation in colon cancer cells. In addition, the mechanism of PD-L1 up-regulation depends on NOD 1 and NOD 2 and involves activation of RIP2 and MAPK signaling pathways.

Reactivity of Titanium Dental Implant Surfaces in Simulated Body Fluid.

Osseointegration of titanium (Ti) implants in bone is crucial for dental implant treatment. Bacterial challenge possibly leading to peri-implantitis threatens long-term success. For the improvement of osseointegration, an understanding of materials and tissue intervention is needed. This in vitro study analyzed changes of different implant surfaces exposed to simulated body fluid (SBF). Implants were analyzed by scanning electron microscopy/X-ray photoelectron spectroscopy. Supernatants (SNs) were assessed using inductively coupled plasma-mass spectrometry (ICP-MS). Additional calcium (Ca) and phosphate (P) crystals developed (Hank's buffered salt solution (HBSS)) on implants with layered surfaces. ICP of SN demonstrated a decreased Ca/P ratio. After incubation with human serum (HS), layers appeared flattened containing <1% Ca/P. The etched/machined implants showed the formation of a surface transformation layer or coating consisting of crystalline Ca/P precipitations and a decrease in the Ca/P ratio in the supernatant. Incubation in HS induced noncrystalline coverage, and increased Ti/Ca/P was detected in supernatants. HBSS induced crystals on surfaces of modified implants and crystalline covers on nonmodified implants containing Ca/P. The serum did not show the development of HA-like structures but showed dissolving effects. Titanium surfaces show completely altered behavior when incubated in serum-containing SBF.

Refractory neutrophil activation in type 2 diabetics with chronic periodontitis.

BACKGROUND: Inflammation increases diabetes mellitus type 2 (T2DM) progression and severity. T2DM patients are at high risk of the rapid development of chronic periodontitis (CP). Topical presence, high numbers, and bactericidal effects of immune cells are challenged by augmented antigen-induced inflammation, which promotes both diseases. OBJECTIVES: To investigate gingival cellular inflammatory responses in individuals with previously undiagnosed T2DM with CP or CP alone and in systemically and periodontally healthy controls (H) in vivo and to establish an ex vivo technique permitting quantitative and qualitative assessments of gingival crevicular immune cells. MATERIALS AND METHODS: T2DM + CP, CP, and H individuals (n = 10, each) received a 2-week oral hygiene regimen (OHR). Afterwards, a noninvasive sampling technique was performed to evaluate gingival inflammation induced under standardized conditions in vivo, that is, in the absence of severe periodontal destruction and inflammation at clinically healthy sites. Stimuli (casein/test or phosphate-buffered saline w/o. Ca2+ or Mg2+ , PBS(-/-) /control) were randomly applied contralaterally in the gingival sulci of participants' upper dentes canini. One day after completion of the OHR, gingival crevicular fluid (GCF) was kinetically assayed between the time of the baseline (BL) measurement and 55 minutes. Polymorphonuclear leukocyte (PMN) content (PMNGCF ) was quantitated at an optimum time of 35 minutes. PMNGCF counts reflect local inflammation. Ex vivo samples were fluorimetrically labeled, gated according to the donor's peripheral blood polymorphonuclear neutrophils (PMNPB ), and then counted, employing flow cytometry. RESULTS: PMNGCF counts in unstimulated gingival crevices (at BL) in the T2DM + CP group were higher than those in the CP and H groups. PMNGCF counts were elevated in casein vs PBS(-/-) -stimulated gingival crevices in all groups. Patients with T2DM + CP showed increased PMNGCF counts compared to those with CP (P = .035) according to scatter plots. CD45+ counts in the stimulated sites in T2DM + CP patients were higher than those in CP and H patients (P = .041). Under stimulation conditions, the CD45+ counts differed from those under placebo conditions (P = .019), indicating augmented, inducible inflammatory leukocyte infiltrate in T2DM + CP patients. CONCLUSIONS: This noninvasive technique permits quantitative assessment of (experimental) gingival inflammation in vivo, revealing an influence of T2DM + CP on the number of primary immune cells in the gingival crevice. Patients who are challenged with (local) leukocytosis are likely at risk of collateral damage to the gingival crevice neighboring tissues, favoring the severity and progression of CP and consequently T2DM (www.clinicaltrials.gov NCT01848379).

Oral Mucosal Epithelial Cells.

Cellular Phenotype and Apoptosis: The function of epithelial tissues is the protection of the organism from chemical, microbial, and physical challenges which is indispensable for viability. To fulfill this task, oral epithelial cells follow a strongly regulated scheme of differentiation that results in the formation of structural proteins that manage the integrity of epithelial tissues and operate as a barrier. Oral epithelial cells are connected by various transmembrane proteins with specialized structures and functions. Keratin filaments adhere to the plasma membrane by desmosomes building a three-dimensional matrix. Cell-Cell Contacts and Bacterial Influence: It is known that pathogenic oral bacteria are able to affect the expression and configuration of cell-cell junctions. Human keratinocytes up-regulate immune-modulatory receptors upon stimulation with bacterial components. Periodontal pathogens including P. gingivalis are able to inhibit oral epithelial innate immune responses through various mechanisms and to escape from host immune reaction, which supports the persistence of periodontitis and furthermore is able to affect the epithelial barrier function by altering expression and distribution of cell-cell interactions including tight junctions (TJs) and adherens junctions (AJs). In the pathogenesis of periodontitis a highly organized biofilm community shifts from symbiosis to dysbiosis which results in destructive local inflammatory reactions. Cellular Receptors: Cell-surface located toll like receptors (TLRs) and cytoplasmatic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) belong to the pattern recognition receptors (PRRs). PRRs recognize microbial parts that represent pathogen-associated molecular patterns (PAMPs). A multimeric complex of proteins known as inflammasome, which is a subset of NLRs, assembles after activation and proceeds to pro-inflammatory cytokine release. Cytokine Production and Release: Cytokines and bacterial products may lead to host cell mediated tissue destruction. Keratinocytes are able to produce diverse pro-inflammatory cytokines and chemokines, including interleukin (IL)-1, IL-6, IL-8 and tumor necrosis factor (TNF)-α. Infection by pathogenic bacteria such as Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) can induce a differentiated production of these cytokines. Immuno-modulation, Bacterial Infection, and Cancer Cells: There is a known association between bacterial infection and cancer. Bacterial components are able to up-regulate immune-modulatory receptors on cancer cells. Interactions of bacteria with tumor cells could support malignant transformation an environment with deficient immune regulation. The aim of this review is to present a set of molecular mechanisms of oral epithelial cells and their reactions to a number of toxic influences.

Green tea polyphenols enhance gingival keratinocyte integrity and protect against invasion by Porphyromonas gingivalis.

The gingival epithelium, a stratified squamous tissue that acts as an interface between the external environment and the underlying connective tissue, plays an active role in maintaining periodontal health. The aim of the present study was to investigate the ability of green tea catechins to enhance gingival epithelial barrier function and protect against the disruption of epithelial integrity induced by Porphyromonas gingivalis. Both the green tea extract and epigallocatechin-3-gallate (EGCG) dose- and time-dependently increased the transepithelial electrical resistance (TER) of a gingival keratinocyte model and decreased the permeability of the cell monolayer to fluorescein isothyocyanate-conjugated 4.4-kDa dextran. This was associated with the increased expression of zonula occludens-1 (ZO-1) and occludin, two tight junction proteins. Treating the gingival keratinocyte monolayer with P. gingivalis caused a reduction in TER and affected the distribution of ZO-1 and occludin, allowing P. gingivalis to translocate through the cell monolayer. These deleterious effects mediated by P. gingivalis were abolished by the green tea extract and EGCG. This protection may be in part related to the ability of tea catechins to inhibit the protease activities of P. gingivalis. Given the above properties, green tea catechins may represent promising preventive and therapeutic molecules against periodontal disease.

The innate host response in caries and periodontitis.

INTRODUCTION: Innate immunity rapidly defends the host against infectious insults. These reactions are of limited specificity and exhaust without providing long-term protection. Functional fluids and effector molecules contribute to the defence against infectious agents, drive the immune response, and direct the cellular players. AIM: To review the literature and present a summary of current knowledge about the function of tissues, cellular players and soluble mediators of innate immunity relevant to caries and periodontitis. METHODS: Historical and recent literature was critically reviewed based on publications in peer-reviewed scientific journals. RESULTS: The innate immune response is vital to resistance against caries and periodontitis and rapidly attempts to protect against infectious agents in the dental hard and soft tissues. Soluble mediators include specialized proteins and lipids. They function to signal to immune and inflammatory cells, provide antimicrobial resistance, and also induce mechanisms for potential repair of damaged tissues. CONCLUSIONS: Far less investigated than adaptive immunity, innate immune responses are an emerging scientific and therapeutic frontier. Soluble mediators of the innate response provide a network of signals to organize the near immediate molecular and cellular response to infection, including direct and immediate antimicrobial activity. Further studies in human disease and animal models are generally needed.

Porphyromonas gingivalis activates NFκB and MAPK pathways in human oral epithelial cells.

BACKGROUND: The bacterial biofilm at the gingival margin induces a host immune reaction. In this local inflammation epithelial cells defend the host against bacterial challenge. Porphyromonas gingivalis (P. gingivalis), a keystone pathogen, infects epithelial cells. The aim of this study was to investigate the activation of signaling cascades in primary epithelial cells and oral cancer cell lines by a profiler PCR array. RESULTS: After infection with P. gingivalis membranes the RNA of 16 to 33 of 84 key genes involved in the antibacterial immune response was up-regulated, amongst them were IKBKB (NF-κB signaling pathway), IRF5 (TLR signaling) and JUN, MAP2K4, MAPK14 and MAPK8 (MAPK pathway) in SCC-25 cells and IKBKB, IRF5, JUN, MAP2K4, MAPK14 and MAPK8 in PHGK. Statistically significant up-regulation of IKBKB (4.7 ×), MAP2K4 (4.6 ×), MAPK14 (4.2 ×) and IRF5 (9.8 ×) (p < 0.01) was demonstrated in SCC-25 cells and IKBKB (3.1 ×), MAP2K4 (4.0 ×) MAPK 14 (3.0 ×) (p < 0.05), IRF5 (3.0 ×) and JUN (7.7 ×) (p < 0.01) were up-regulated in PHGK. CONCLUSIONS: P. gingivalis membrane up-regulates the expression of genes involved in downstream TLR, NFκB and MAPK signaling pathways involved in the pro-inflammatory immune response in primary and malignant oral epithelial cells.

Oral Squamous Carcinoma Cells Express B7-H1 and B7-DC Receptors in Vivo.

B7-H1 and B7-DC ligands are members of the B7 family with important regulatory functions in cell-mediated immune response. Both receptors are ligands of the programmed death receptor PD-1. B7-H1 expression has been detected in the majority of human carcinomas in vivo. B7-H1 mediated signals are able to negatively regulate activated T cell functions and survival, and enable tumor cells to overcome host response. The aim of this study was to investigate the expression of B7-H1 and B7-DC proteins in oral squamous cell carcinomas (OSCC) in vivo. Tissues from 15 samples were cryo-sected and following histological routine staining (HE), incubated with antibodies against human B7-H1 and B7-DC. Immuno-staining of pan-cytokeratin was performed to ascertain the epithelial origin of the tissue and CK 19 to demonstrate the proliferating stage. Confocal laser scanning microscopy confirmed the presence of both B7-H1 and B7-DC in all 15 OSCC. The B7-H1 and B7-DC staining was located in areas of the tissue that were identified as cancerous lesions in the previously stained HE sections before. Staining with Pan-CK and CK19 provided evidence for the epithelial origin and the proliferating stage of the tissue. The in vivo expression of the B7-H1 and B7-DC receptors in oral squamous cell carcinomas suggest that general mechanisms for immune evasion of tumors are also found in OSCC.