Differential regulation of NPY and SP receptor expression in STRO-1+ve PDLSCs by inflammatory cytokines.

OBJECTIVES: The aims of this study were to investigate neuropeptide receptor expression regulation on STRO-1 +ve periodontal ligament stem cells (PDLSCs) in response to inflammatory cytokines and to investigate a potential osteogenic effect of neuropeptides. BACKGROUND: Nerve fibres innervating the periodontal tissues in humans contain several neuropeptides including neuropeptide Y and substance P. The role of neuropeptide receptors on PDLSCs, including their response to the local inflammatory environment of periodontitis, is currently unknown. METHODS: A homogenous population of STRO-1 +ve PDLSCs was prepared by immunomagnetic separation of cells obtained by the tissue out-growth method from healthy premolar teeth from a single donor. Regulation of gene expression of the neuropeptide Y Y1 receptor and substance P receptor tachykinin receptor 1 was investigated. A potential osteogenic effect of neuropeptide Y and substance P was also investigated by measuring alkaline phosphatase (ALP) activity, Alizarin red staining and quantifying osteogenic gene expression. RESULTS: Treatment of STRO-1 +ve PDLSCs with tumour necrosis factor-alpha or interleukin 1-beta up-regulated the expression of the neuropeptide Y's Y1 receptor, but down-regulated substance P's receptor. Significantly increased ALP activity was observed in STRO-1 +ve PDLSCs treated with neuropeptide Y but not substance P. Further studies showed that neuropeptide Y had a modest osteogenic effect on cells at both a functional level and a gene level. CONCLUSIONS: Expression of the neuropeptide Y Y1 receptor gene on STRO-1 +ve PDLSCs was sensitive to local inflammatory cytokines. Treatment of cells with neuropeptide Y was found to produce a modest enhanced osteogenic effect.

Altered Toll-like receptor 2-mediated endotoxin tolerance is related to diminished interferon beta production.

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-α and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-α after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-α but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-κB activation, whereas TNF-α expression is blocked at the gene transcription level. Interferon ß plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon ß. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon ß and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Natural Antimicrobials in the Dental Pulp.

INTRODUCTION: Like many tissues, the dental pulp is equipped with innate and adaptive immune responses, designed to defend against infection and limit its spread. The pulp's innate immune response includes the synthesis and release of antimicrobial peptides by several dental pulp cell types. These naturally-occurring antimicrobial peptides have broad spectrum activity against bacteria, fungi and viruses. There is a resurgence of interest in the bioactivities of naturally-occurring antimicrobial peptides, largely driven by the need to develop alternatives to antibiotics. METHODS: This narrative review focused on the general properties of antimicrobial peptides, providing an overview of their sources and actions within the dental pulp. RESULTS: We summarized the relevance of antimicrobial peptides in defending the dental pulp, highlighting the potential for many of these antimicrobials to be modified or mimicked for prospective therapeutic use. CONCLUSION: Antimicrobial peptides and novel peptide-based therapeutics are particularly attractive as emerging treatments for polymicrobial infections, such as endodontic infections, because of their broad activity against a range of pathogens.

Sialylation of Porphyromonas gingivalis LPS and its effect on bacterial-host interactions.

Porphyromonas gingivalis produces different LPS isoforms with significant structural variations of their lipid A and O-antigen moieties that can affect its pro-inflammatory and bone-resorbing potential. We show here, for the first time, that P. gingivalis LPS isolated from W83 strain is highly sialylated and possesses significantly reduced inflammatory potential compared with less sialylated ATCC 33277 strain LPS. Nevertheless, the reduction in the endotoxin activity is not mediated by the presence of sialic acid LPS moieties as the sialic acid-free LPS produced by the mutant W83 strain exhibits a similar inflammatory potential to the wild type strain. Furthermore, our findings suggest that the interaction between the sialic acid LPS moieties and the inhibitory CD33 receptor is prevented by endogenously expressed sialic acid on the surface of THP-1 cells that cannot be out-competed by sialic acid containing P. gingivalis LPS. The present study also highlights the importance of endogenous sialic acid as a 'self-associated molecular pattern' and CD33 receptors in modulation of innate immune response as human gingival fibroblasts, which do not express CD33 receptors, and desialylated THP-1 cells have both been found to have much higher spontaneous IL-8 production than naïve THP-1 cells.

Antimicrobial and immunomodulatory properties of PGLa-AM1, CPF-AM1, and magainin-AM1: potent activity against oral pathogens.

Cationic amphipathic α-helical peptides are intensively studied classes of host defence peptides (HDPs). Three peptides, peptide glycine-leucine-amide (PGLa-AM1), caerulein-precursor fragment (CPF-AM1) and magainin-AM1, originally isolated from norepinephrine-stimulated skin secretions of the African volcano frog Xenopus amieti (Pipidae), were studied for their antimicrobial and immunomodulatory activities against oral and respiratory pathogens. Minimal effective concentrations (MECs), determined by radial diffusion assay, were generally lower than minimal inhibitory concentrations (MICs) determined by microbroth dilution. PGLa-AM1 and CPF-AM1 were particularly active against Streptococcus mutans and all three peptides were effective against Fusobacterium nucleatum, whereas Enterococcus faecalis and Candida albicans proved to be relatively resistant micro-organisms. A type strain of Pseudomonas aeruginosa was shown to be more susceptible than the clinical isolate studied. PGLa-AM1 displayed the greatest propensity to bind lipopolysaccharide (LPS) from Escherichia coli, P. aeruginosa and Porphyromonas gingivalis. All three peptides showed less binding to P. gingivalis LPS than to LPS from the other species studied. Oral fibroblast viability was unaffected by 50 µM peptide treatments. Production of the pro-inflammatory cytokine IL-8 by oral fibroblasts was significantly increased following treatment with 1 or 10 µM magainin-AM1 but not following treatment with PGLa-AM1 or CPF-AM1. In conclusion, as well as possessing potent antimicrobial actions, the X. amieti peptides bound to LPS from three human pathogens and had no effect on oral fibroblast viability. CPF-AM1 and PGLa-AM1 show promise as templates for the design of novel analogues for the treatment of oral and dental diseases associated with bacteria or fungi.

Human dental pulp fibroblasts express the "cold-sensing" transient receptor potential channels TRPA1 and TRPM8.

INTRODUCTION: Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts. METHODS: Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca(2+) microfluorimetry. RESULTS: Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca(2+) microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca(2+)](i)) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists. CONCLUSIONS: Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

Invasive front grading: reliability and usefulness in the management of oral squamous cell carcinoma.

BACKGROUND: The value of histological grading was examined with emphasis on reliability of assessment in 102 cases of intraoral squamous cell carcinoma from Northern Ireland with known outcome. METHODS: Two pathologists independently graded the invasive tumour front blinded to the stage and outcome. RESULTS: Intraobserver agreement was acceptable but interobserver agreement was not satisfactory. The degree of keratinisation was assessed most consistently while nuclear polymorphism was the least reliable feature. Multivariate survival analysis showed that the total grading score was associated with overall survival while the pattern of tumour invasion was the most valuable feature in estimating regional lymph node involvement. The number of positive lymph nodes was strongly associated with regional relapse, while the treatment modality and status of the surgical margins correlated with local relapse. CONCLUSIONS: Grading of selected features in OSCC is reliable and can facilitate treatment planning.