Gingival fibroblasts respond to the TRPV1 agonist capsaicin

Background: The oral cavity is a frontline barrier which is often exposed to physical trauma and noxious substances, leading to pro-inflammatory responses designed to be protective in nature. The transient receptor potential (TRP) super family of ion channels is believed to play a critical role in sensory physiology, acting as transducers for thermal, mechanical and chemical stimuli. Our understanding of the role of TRP channel activation in gingival and periodontal inflammation is currently limited. Gingival fibroblasts are the most abundant structural cell in periodontal tissues and we hypothesised that they may have a role in the inflammatory response associated with TRP channel activation. Objectives: The present study was designed to determine whether the TRPV1 agonist capsaicin could elicit a pro-inflammatory response in gingival fibroblasts in vitro by up-regulation of interleukin-8 (IL-8) production. Methods: Gingival fibroblasts were derived by explant culture from surgical tissues following ethical approval. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal calf serum (FCS) in 5% CO2. Following treatment of gingival fibroblasts with capsaicin, IL-8 levels were measured by ELISA. The potential cytotoxicity of capsaicin was determined by the MTT assay. Results: In gingival fibroblasts treated with the TRPV1 agonist capsaicin (10µM), IL-8 production was significantly increased compared with untreated control cells. Capsaicin was shown not to be toxic to gingival fibroblasts at the concentrations studied. Conclusion: The identification of factors that modulate pro-inflammatory cytokine production is important for our understanding of gingival and periodontal inflammation. This study reports for the first time that gingival fibroblasts respond to the TRPV1 agonist capsaicin by increased production of IL-8. Activation of TRPV1 on gingival fibroblasts could therefore have an important role in initiating and sustaining the inflammatory response associated with periodontal diseases

Effects of LL-37 Peptide Mimetics on Gingival Fibroblast Function

Host defence peptides, including the cathelicidin LL-37, play an important role in mucosal immunity, functioning as both antimicrobial agents and modulators of the inflammatory response. In the current climate of antibiotic resistance, the idea of using naturally occurring antimicrobial peptides, or their synthetic mimetics, to combat oral infection is particularly appealing. Objectives: The aim of this study was to investigate the effects of parent LL-37, and two peptide mimetics (KR-12 and KE-18), on cytokine expression and response to bacterial challenge by gingival fibroblasts. Methods: KR-12 and KE-18 are peptide mimetics of the biologically active, mid-region sequence of LL-37. The effects of commercially available LL-37, KR-12 and KE-18 on gingival fibroblast response to E coli and P gingivalis LPS challenge, analysed by IL-6 and IL-8 expression, were determined in cell culture by ELISA. The direct effects of each peptide on IL-6, IL-8, CXCL-1 and HGF expression were also determined by ELISA. The MTT assay was used to evaluate peptide effects on fibroblast viability. Results: LL-37 and KE-18, but not KR-12, inhibited LPS induction of inflammatory cytokine expression and directly stimulated CXCL-1 production by fibroblasts. All 3 peptides stimulated production of IL-8 and HGF. Neither LL-37 nor KE-12 affected cell viability, while KE-18, at higher concentrations, induced cell death. Conclusions: Shorter, peptide mimetics of LL-37, in particular KE-18, retain the immunomodulatory effects of the parent molecule and possess excellent potential as therapeutic agents in the treatment of oral infections including periodontal disease.

Immunocytochemical Localization of Substance P Receptors (NK-1 Receptors) in Human Gingival Tissue

Substance P (SP) is a member of the structurally related family of neuropeptides known as the tachykinins. In addition to neurotransmitter roles, the tachykinins are also known to modulate local inflammation which depends on signalling between the neuropeptide molecules and target cells and tissues. SP mediates its effects through a specific receptor, known as the substance P receptor or the neurokinin 1 (NK-1) receptor. The NK-1 receptor is a G-protein associated integral membrane protein and although it has been studied in a wide range of tissues, to date there has been no published data on the localisation of the NK-1 receptor in human gingival tissue. Objective: The aim of this study was to examine the distribution of the NK-1 receptor in human gingival tissue using immunocytochemistry. Method: Gingival tissue was obtained from patients undergoing periodontal surgery. Tissue was fixed in paraformaldehyde and embedded in wax for sectioning. Sections were dewaxed in xylene and then rehydrated in alcohols and phosphate buffered saline. Rehydrated sections were probed with rabbit polyclonal antibody to human NK-1 receptor which was subsequently detected using anti-rabbit horseradish peroxidase conjugate and diaminobenzidine as substrate. Results: Immunocytochemistry revealed that the NK-1 receptor was distributed along nerve fibres and blood vessel endothelial cells, suggesting these areas are main targets for the actions of SP via the NK-1 receptor. Conclusion: This is the first immunocytochemical report of NK-1 receptors in human gingival tissue and provides evidence for possible NK-1 mediated biological effects of SP in human gingival tissue from periodontitis patients.

Design, characterisation and preliminary clinical evaluation of a novel mucoadhesive topical formulation containing tetracycline for the treatment of periodontal disease

This study describes the formulation, characterisation and preliminary clinical evaluation of mucoadhesive, semi-solid formulations containing hydroxyethylcellulose (HEC, 1-5%, w/w), polyvinylpyrrolidine (PVP, 2 or 3%, w/w), poly carbophil (PC, 1 or 3%, w/w) and tetracycline (5%, w/w, as the hydrochloride). Each formulation was characterised in terms of drug release, hardness, compressibility, adhesiveness (using a texture analyser in texture profile analysis mode), syringeability (using a texture analyser in compression mode) and adhesion to a mucin disc (measured as a detachment force using the texture analyser in tensile mode). The release exponent for the formulations ranged from 0.78+/-0.02 to 1.27+/-0.07, indicating that drug release was non-diffusion controlled. Increasing the concentrations of each polymeric component significantly increased the time required for 10 and 30% release of the original mass of tetracycline, due to both increased viscosity and, additionally, the unique swelling properties of the formulations. Increasing concentrations of each polymeric component also increased the hardness, compressibility, adhesiveness, syringeability and mucoadhesion of the formulations. The effects on product hardness, compressibility and syringeability may be due to increased product viscosity and, hence, increased resistance to compression. Similarly, the effects of these polymers on adhesiveness/mucoadhesion highlight their mucoadhesive nature and, importantly, the effects of polymer state (particularly PC) on these properties. Thus, in formulations where the neutralisation of PC was maximally suppressed, adhesiveness and mucoadhesion were also maximal. Interestingly, statistical interactions were primarily observed between the effects of HEC and PC on drug release, mechanical and mucoadhesive properties. These were explained by the effects of HEC on the physical state of PC, namely swollen or unswollen. In the preliminary clinical evaluation, a formulation was selected that offered an appropriate balance of the above physical properties and contained 3% HEC, 3% PVP and 1% PC, in addition to tetracycline 5% (as the hydrochloride). The clinical efficacy of this (test) formulation was compared to an identical tetracycline-devoid (control) formulation in nine periodontal pockets (greater than or equal to 5 mm depth). One week following administration of the test formulation, there was a significant improvement in periodontal health as identified by reduced numbers of sub-gingival microbial pathogens. Therefore, it can be concluded that, when used in combination with mechanical plaque removal, the tetracycline-containing semi-solid systems described in this study would augment such therapy by enhancing the removal of pathogens, thus improving periodontal health. (C) 2000 Elsevier Science B.V. All rights reserved.

Neuropeptide Y (NPY) and NPY Y1 receptor in periodontal health and disease

Abstract
OBJECTIVES:
Neuropeptide Y (NPY) coordinates inflammation and bone metabolism which are central to the pathogenesis of periodontitis. The present study was designed to determine whether NPY was quantifiable in human gingival crevicular fluid (GCF) and to test the null hypothesis that GCF levels of NPY were the same in periodontal health and disease. A subsidiary aim was to determine the potential functionality of released NPY by detecting the presence of NPY Y1 receptors in gingival tissue.
DESIGN:
The periodontitis group consisted of 20 subjects (10 females and 10 males) mean age 41.4 (S.D. 9.6 years). The control group comprised 20 subjects (10 females and 10 males) mean age 37.4 (S.D. 11.7 years). NPY levels in GCF were measured in periodontal health and disease by radioimmunoassay. NPY Y1 receptor expression in gingival tissue was determined by Western blotting of membrane protein extracts from healthy and inflamed gum.
RESULTS:
Healthy sites from control subjects had significantly higher levels of NPY than diseased sites from periodontitis subjects. NPY Y1 receptor protein was detected in both healthy and inflamed gingival tissue by Western blotting.
CONCLUSIONS:
The significantly elevated levels of NPY in GCF from healthy compared with periodontitis sites suggests a tonic role for NPY, the functionality of which is indicated by the presence of NPY Y1 receptors in local gingival tissue.

Serum Antibodies to Porphyromonas gingivalis Chaperone HtpG Predict Health in Periodontitis Susceptible Patients

Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis.

Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon β 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-alpha and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-alpha after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-alpha 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-kappa B activation, whereas TNF-alpha expression is blocked at the gene transcription level. Interferon beta 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 beta. 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 beta and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Identification of MRP-8 (calgranulin A) as a major responsive protein in chronic periodontitis

The purpose of the study was to analyse how the protein composition of the inflammatory exudate associated with chronic periodontitis differed from the exudate in periodontal health. Gingival crevicular fluid (GCF) was collected from sites with chronic periodontal inflammation and from non-diseased sites in healthy control subjects. Microbore HPLC analysis revealed one major difference in GCF protein profiles between healthy controls and periodontitis patients. The protein enhanced in periodontitis patients was identified as migration inhibitory factor-related protein-8 (MRP-8) by a combination of N-terminal amino acid sequencing, mass spectrometry, and SDS-PAGE. Together, these data demonstrate, for the first time, the presence of monomeric MRP-8 in an inflammatory exudate. Whether monomeric MRP-8 is a unique feature of chronic periodontal inflammation is not yet clear, but the chemotactic properties of this peptide support a functional role for MRP-8 in periodontal inflammation. Copyright (C) 2000 John Wiley & Sons, Ltd.

Mapping biological to clinical phenotypes during the development (21 days) and resolution (21 days) of experimental gingivitis

Aim: To characterize and map temporal changes in the biological and clinical phenotype during a 21-day experimental gingivitis study. Materials and Methods: Experimental gingivitis was induced over 21 days in healthy human volunteers (n = 56), after which normal brushing was resumed (resolution phase). Gingival and plaque indices were assessed. Gingival crevicular fluid was collected from four paired test and contra-lateral control sites in each volunteer during induction (Days 0, 7, 14 and 21) and resolution (Days 28 and 42) of experimental gingivitis. Fluid volumes were measured and a single analyte was quantified from each site-specific, 30s sample. Data were evaluated by analysis of repeated measurements and paired sample tests. Results: Clinical indices and gingival crevicular fluid volumes at test sites increased from Day 0, peaking at Day 21 (test/control differences all p

Physicochemical characterization and preliminary in vivo efficacy of bioadhesive, semisolid formulations containing flurbiprofen for the treatment of gingivitis

In this study, the physicochemical properties and preliminary in vivo clinical performance of formulations containing hydroxyethylcellulose (HEC; 3, 5, 10% w/w, poly(vinylpyrrolidone) (PVP; 3, 5% w/w), polycarbophil (PC; 1, 3, 5% w/w), and flurbiprofen (5% w/w) were examined. Flurbiprofen release into PBS pH 7.4 was performed at 37 degrees C. The mechanical properties (hardness, compressibility, adhesiveness, initial stress) and syringeability of formulations were determined using a texture analyzer in texture profile analysis (TPA) and compression modes, respectively. In general, the time required for release of 10 and 30% of the original mass of flurbiprofen (t(10%), t(30%)) increased as the concentration of each polymeric component increased. However, in the presence of either 5 or 10% HEC and 5% PC, increased PVP concentration decreased both t(10%), t(30%) due to excessive swelling land disintegration) of these formulations. Increased concentrations of HEC, PVP, and PC significantly increased formulation hardness, compressibility, work of syringe expression, and initial stress due to the effects of these polymers on formulation viscoelasticity. Similarly, increased concentrations of PC (primarily), HEC, and PVP increased formulation adhesiveness-due to the known bioadhesive properties of these polymers. Clinical efficacies of formulations containing 3% HEC, 3% PVP, 3% PC, and either 0% (control) of 5% (test) flurbiprofen, selected to offer optimal drug release and mechanical properties, were evaluated and clinically compared in an experimental gingivitis model. The test (flurbiprofen-containing) formulation significantly reduced gingival inflammation, as evaluated using the gingival index, and the gingival crevicular fluid volume, whereas, these clinical parameters were generally increased in volunteers who had received the control formulation. There were no observed differences in the plaque indices of the two subject groups, confirming that the observed differences in gingival inflammation could not be accredited to differences in plaque accummulation. This study has shown both the applicability of the in vitro methods used, particularly TPA, for the rational selection of formulations for clinical evaluation and, additionally, the clinical benefits of the topical application of a bioadhesive semisolid flurbiprofen-containing formulation for the treatment of experimental gingivitis.

Role of nitric oxide in maintenance of basal oral tissue blood flow in anesthetized cats

Experiments were undertaken to determine if nitric oxide (NO) plays a role in regulation of basal blood flow in the oral cavity of pentobarbital anesthetized cats and, if so, to quantify this effect using dose-response relationships. Blood flow was continuously measured from the surface of the tongue and mandibular gingiva (laser-Doppler flowmetry) and from the lingual artery (ultrasonic flowmetry). Cardiovascular parameters also were recorded. Administration of the nonselective inhibitor of nitric oxide synthase (NOS), L-NAME (0.08-20 mg/kg i.v.), produced a dose-related increase of blood pressure associated with decreases of blood flow at all three measurement sites. Maximal blood flow depression of 50-60% was seen 30-60 min after administration of 1.25 mg/kg of L-NAME. D-NAME (1.25 mg/kg i.v.) was inactive at all sites. Subsequent administration of L-arginine partially reversed effects of L-NAME in the lingual artery and tongue, but not in the gingival circulation. The neuronally selective NOS inhibitor, 7-nitroindazole (7-NI, 30 mg/kg i.p.), was devoid of effect on any of the measured parameters. These results suggest that endothelial (but not neuronally derived) NO plays an important role in control of basal blood flow in oral tissues of the cat.

Monitoring of saliva pH using miniaturized sensing tools as an indicator of oral health

The monitoring of oral disease is important, not alone for oral health, but for the detection and prevention of
systemic disease. The link between oral health and systemic disease is the focus of many studies, with
indications emerging of a causal link [1]. For disease diagnostics, blood has typically been the fluid of choice
for analysis, the retrieval of which is invasive and therefore unsuitable for wearable technology. Analysis of
saliva, however, is less invasive than that of blood, requires little or no pre-treatment and is abundantly
available. A strong correlation has been found between the analytes of blood and saliva [2] with saliva
containing biomarkers for diseases such as diabetes, oral cancer and cardiovascular disease. The development of
an implantable multi-parametric wireless sensor, to monitor both salivary analytes and changes in gingival
temperature, is the aim of this research project.
The aim of our current study is to detect changes in salivary pH, using a gold electrode with a pHsensitive
iridium oxide layer, and an Ion Sensitive Field Effect Transistor probe. Characterisation studies were
carried out in artificial saliva (AS). A salivary pH of between 4.5pH-7.5pH [3], and gingival temperature
between 35°C-38°C [4], were identified as the target range of interest for the human oral environment. Sensor
measurements were recorded in solutions of varying pH and temperature. An ISFET probe was then implanted
into a prototype denture and characterised in AS. This study demonstrates the suitability of ISFET and gold
electrode pH sensors for incorporation into implantable oral sensors.
[1] G. Taylor and W. Borgnakke, “Periodontal disease: associations with diabetes, glycemic control and
complications,” Oral Dis., vol. 14, no. 3, pp. 191–203, Apr. 2008.
[2] E. Tékus, M. Kaj, E. Szabó, N. L. Szénási, I. Kerepesi, M. Figler, R. Gábriel, and M. Wilhelm,
“Comparison of blood and saliva lactate level after maximum intensity exercise,” Acta Biol. Hung., vol. 63
Suppl 1, pp. 89–98, 2012.
[3] S. Naveen, M. L. Asha, G. Shubha, A. Bajoria, and A. Jose, “Salivary Flow Rate, pH and Buffering
Capacity in Pregnant and Non Pregnant Women - A Comparative Study,” JMED Res., pp. 1–8, Feb. 2014.
[4] A. F. Holthuis and F. S. Chebib, “Observations on temperature and temperature patterns of the gingiva. I.
The effect of arch, region and health,” J. Periodontol., vol. 54, no. 10, pp. 624–628, Oct. 1983

The role of antimicrobial peptides in periodontal disease.

Objectives Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

Function of host defence peptide LL-37 in human periodontal disease.

Objectives: Fibroblasts play a significant role as regulators of the host response in periodontal disease, responding to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. LL-37, a host defence peptide, inhibits LPS-induced cytokine signalling in macrophages, suggesting an immunomodulatory role. The objective was to investigate the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and its effect on fibroblast response to LPS activation. Methods: Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (10ng/ml) along with LL-37 (0-50 µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. Western blot was performed to determine the effect of LL-37 on LPS -induced IκBα degradation in HGFs following LPS stimulation over 2 hours. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Confirmation of LL-37 effects on specific gene expression was obtained by QPCR. Results: At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL-37 induced IL-8 production independent of LPS. Addition of LL-37 blocked LPS-induced IκBα degradation in HGFs. Microarray analysis revealed that LL-37 (50µg/ml) upregulated a significant number of cytokines and chemokines by > 5 fold. Upregulation of five of these, CXCL1, CXCL2, CXCL3, IL-24 and IL-8 was confirmed by Q-PCR. Conclusion: The host defence peptide LL-37, the only known human cathelicidin, appears to have pleiotrophic effects in innate immunity. At least some of these are mediated through cytokine and chemokine signalling networks. The ability of LL-37 to reduce bacterial LPS-induced cytokine production in gingival fibroblasts, at low concentrations, suggests a potential therapeutic role in the management of periodontal disease.

The role of the cathelicidin LL-37 in periodontal disease.

Abstract Background Fibroblasts respond to bacterial stimulation by producing an array of inflammatory cytokines and chemokines. As such fibroblasts play a significant role as regulators of the host response in periodontal disease. LL-37, an antimicrobial peptide, found in saliva and GCF, inhibits LPS-induced cytokine signalling in macrophages, suggesting a role in host defence in periodontal disease. This study investigated the interaction between LL-37 and gingival fibroblasts – both its direct regulation of fibroblast activity and also its effect on fibroblast response to LPS activation. Methods Human gingival fibroblasts (HGFs) were incubated for 24 hours in the presence of either P. gingivalis LPS (10µg/ml) or E. coli LPS (0.01µg/ml) along with LL-37 (0-50µg/ml). IL-6 and IL-8 production by HGFs in the conditioned medium was determined by ELISA. DNA microarray analysis was performed on cell populations incubated for 6 hr in the presence or absence of the peptide. Results At low concentrations (≤ 5 µg/ml) LL-37 significantly inhibited LPS-induced cytokine production by HGFs. At higher concentrations LL37 induced IL-8 production independent of LPS. Microarray analysis revealed that LL-37 upregulated a significant number of cytokines and chemokines by > 5 fold. The stimulatory effect on IL-8 mRNA expression was confirmed by Q-PCR. Conclusion LL-37 appears to have pleiotrophic effects in innate immunity. Its ability, at low concentrations, to reduce bacterial LPS-induced cytokine production in gingival fibroblasts suggests a potential therapeutic role in the management of periodontal disease.