Sabine Groeger

B7-H1 and B7-DC receptors of oral squamous carcinoma cells are upregulated by Porphyromonas gingivalis.

The up-regulation of the B7-H1 receptors in host cells might influence the chronicity of inflammatory disorders that frequently precede the development of human cancers. B7-H1 expression has been detected in the majority of human cancers, leading to anergy and apoptosis of activated T cells, and enabling tumor cells to overcome host response. Porphyromonas gingivalis (P. gingivalis), a putative periodontal pathogen, is an etiologic agent of periodontitis and expresses a variety of virulence factors. In this study, the expression of B7-H1 and B7-DC receptors on squamous cell carcinoma cells SCC-25 and BHY and primary human gingival keratinocytes (PHGK) was analyzed after infection with two virulent P. gingivalis strains in vitro. After 48h, the cells were stained with antibodies for human B7-H1 and B7-DC and further analyzed by flow cytometry. RNA was extracted and gene expression of B7-H1 or B7-DC was quantified by real time PCR. After infection with P. gingivalis, both B7-H1 and B7-DC receptors were up-regulated. The mean fluorescence intensity (MFI) increased from 4.5 to 9.9 (B7-H1) and from 6.9 to 15.0 (B7-DC) (p<0.05, respectively) in SCC-25 cells. PHGK showed an increase from 4.8 to 12.4 (B7-H1) and from 5.5 to 15.6 (B7-DC) (p<0.05, respectively). Streptococcus salivarius K12, a commensal bacterium, caused no up-regulation. After 24h, the expression of B7H1 and B7-DC mRNA in infected cells, normalized to GAPDH and in relation to non-infected cells, was 6.4 fold (B7-H1) and 8.6 fold (B7-DC) higher. In PHGK B7-H1/DC mRNA expression increased 8.2 fold (B7-H1) and 5.9 fold (B7DC) (p<0.05) respectively. The results of the study demonstrate that in contrast to S. salivarius K12 virulent P. gingivalis strains are able to induce the expression of the B7-H1 and B7-DC receptors in squamous carcinoma cells and human gingival keratinocytes, which might facilitate immune evasion by oral cancers.

Human monocytes represent a competitive source of interferon-α in peripheral blood

Interferon-alpha (IFN-alpha) has a critical role in antiviral immunity and plasmacytoid dendritic cells (pDCs) have been demonstrated as the principal IFN-alpha source after Toll-like receptor (TLR) 7 and 9 stimulation. Little is known about the contribution of pDC-independent IFN-alpha sources to total IFN-alpha production capacity of human peripheral blood. Using an array of pathogen associated molecular patterns (PAMPs), Poly(I:C)/Dotap represented the second strongest IFN-alpha stimulus in total PBMC. Poly(I:C)/Dotap induced three times more IFN-alpha, when compared to TLR7-stimulation (R848) and four times less, when compared to TLR9-stimulation. Dotap (mediator of cellular uptake) dramatically increased Poly(I:C)-induced IFN-alpha production. Sorting experiments and ELISpot assays revealed that monocytes and not myeloid DCs are the main IFN-alpha source after Poly(I:C)/Dotap stimulation. ELISpot analyses demonstrated the highest IFN-alpha spot numbers after Poly(I:C)/Dotap stimulation. Although pDCs produced highest IFN-alpha levels per cell, monocytes represent a competing IFN-alpha source in total PBMC due to their high frequency.

Epithelial barrier and oral bacterial infection

The oral epithelial barrier separates the host from the environment and provides the first line of defense against pathogens, exogenous substances and mechanical stress. It consists of underlying connective tissue and a stratified keratinized epithelium with a basement membrane, whose cells undergo terminal differentiation resulting in the formation of a mechanically resistant surface. Gingival keratinocytes are connected by various transmembrane proteins, such as tight junctions, adherens junctions and gap junctions, each of which has a specialized structure and specific functions. Periodontal pathogens are able to induce inflammatory responses that lead to attachment loss and periodontal destruction. A number of studies have demonstrated that the characteristics of pathogenic oral bacteria influence the expression and structural integrity of different cell-cell junctions. Tissue destruction can be mediated by host cells following stimulation with cytokines and bacterial products. Keratinocytes, the main cell type in gingival epithelial tissues, express a variety of proinflammatory cytokines and chemokines, including interleukin-1alpha, interleukin-1beta, interleukin-6, interleukin-8 and tumor necrosis factor-alpha. Furthermore, the inflammatory mediators that may be secreted by oral keratinocytes are vascular endothelial growth factor, prostaglandin E2 , interleukin-1 receptor antagonist and chemokine (C-C motif) ligand 2. The protein family of matrix metalloproteinases is able to degrade all types of extracellular matrix protein, and can process a number of bioactive molecules. Matrix metalloproteinase activities under inflammatory conditions are mostly deregulated and often increased, and those mainly relevant in periodontal disease are matrix metalloproteinases 1, 2, 3, 8, 9, 13 and 24. Viral infection may also influence the epithelial barrier. Studies show that the expression of HIV proteins in the mucosal epithelium is correlated with the disruption of epithelial tight junctions, suggesting a possible enhancement of human papilloma virus infection by HIV-associated disruption of tight junctions. Altered expression of matrix metalloproteinases was demonstrated in keratinocytes transformed with human papilloma virus-16 or papilloma virus-18,. To summarize, the oral epithelium is able to react to a variety of exogenous, possibly noxious influences.

Effects of Porphyromonas gingivalis infection on human gingival epithelial barrier function in vitro.

The gingival epithelium plays an important role in the protection of oral tissues from microbial challenge. Oral keratinocytes form various cellular contacts, including tight junctions, and thus are able to create an epithelial barrier. A measurable indicator of barrier function in vitro is the transepithelial electrical resistance (TER). Porphyromonas gingivalis is recognized as a major aetiologic agent of periodontal disease and exhibits a variety of virulence factors. The aim of the study was to investigate the effect, in vitro, of infection with P. gingivalis on gingival barriers composed of primary and immortalized human keratinocytes. Primary and immortalized human gingival keratinocytes were infected with different strains of P. gingivalis. The impact of the bacterial challenge on the barrier was analysed by measuring the TER. The destructive effects of gingipains were blocked by specific enzyme inhibitors. After an initial increase of about 20-30% in infected wells, the TER decreased to zero. Gingipain inhibitors delayed the destruction of the barrier by 12 ± 4 h. In all cases, the loss of TER was accelerated if the system was infected from the basolateral side. A distinct effect of P. gingivalis on the epithelial barrier function of three-dimensional cultured epithelial cell models was demonstrated, which can partly be attributed to the activity of gingipains.

Localization and functional importance of a conserved zona pellucida 2 protein domain in the human and bovine ovary using monoclonal anti-ZP2 peptide antibodies

In mammals, gamete recognition and sperm binding to the oocyte are mediated by the zona pellucida (ZP), an acellular coat surrounding the plasma membrane of the oocyte that consists of particular ZP proteins. The ZP2 protein mediates secondary sperm binding to the ZP. Its primary structures are highly conserved as revealed by cDNA cloning. In the present study, we investigated the localization of ZP2 in human and bovine ovaries and oocytes and the influence of monoclonal anti-ZP2 peptide antibodies upon bovine sperm-egg interactions. We generated a monoclonal anti-ZP2 synthetic peptide antibody, mAb ZP2-20, against a sequence that is strongly conserved in the mammalian ZP2 amino acid sequence. Specificity of mAb ZP2-20 was determined by ELISA and immunoblotting, respectively. Our results show that mAb ZP2-20 specifically detected the peptide used as an antigen and reacted with its corresponding protein antigen in human and bovine ovaries. In order to elucidate effects of mAb ZP2-20 upon bovine sperm-ZP binding, we used the competitive hemizona assay (cHZA) and found that the antibodies clearly inhibit sperm binding to the ZP. We conclude that (i). monoclonal antibodies against ZP2 peptides react with ZP proteins present in bovine and human ovaries and can be used as a specific marker for ZP2; and that (ii). mAb ZP2-20 detects a ZP2 epitope that is of functional relevance for sperm-ZP interactions.

Influence of retinoic acid on human gingival epithelial barriers.

BACKGROUND AND OBJECTIVES The gingival epithelium plays an important role in the protection of oral tissues from microbial challenge. Oral keratinocytes form a barrier and show various cellular contacts, including tight junctions (TJ). To analyse the barrier function in vitro the transepithelial electrical resistance (TER) is commonly used. Retinoic acid (RA) is an important signalling molecule in most tissues, including epithelial differentiation. RA signalling is mediated through three RA receptors. The aim of the study was to investigate the influence of RA on human gingival barriers in vitro. MATERIAL AND METHODS Immortalized human gingival keratinocytes were seeded on culture plate inserts. The effect of RA with and without infection with Porphyromonas gingivalis W83 on the barrier was analysed by TER measurements. The expression of TJ proteins was investigated by western blot. RESULTS During differentiation, mean TER increased from 16 (1 h), 43 (4 h) to 62 (6 h) Ohm × cm2 . Addition of 15 μm RA increased TER by +19 after 1 h, +25 after 4 h and +16 Ohm × cm2 after 6 h. The pan-RA receptor inhibitor BMS 493 resulted in TER values comparable to the control. The mean established TER of the control was approximately 110 Ohm × cm2 . Addition of 15 μm RA elevated TER to 127 Ohm × cm2 after 1 h, 150 Ohm × cm2 after 4 h and 189 Ohm × cm2 after 6 h (p ≤ 0.01). RA plus infection with P. gingivalis W83 further increased the TER increasing effect but could not prevent the destruction of TER induced by bacterial infection. The protein expression of the TJ proteins claudin 4 and occludin was enhanced while ZO-1 was downregulated after 1 h of RA incubation. CONCLUSION RA provides barrier-positive elements to the gingival epithelial cell model that is accompanied by altered expression of TJ proteins.

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

BackgroundThe 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.ResultsAfter 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.ConclusionsP. 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.

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.

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

Abstract 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.

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.