The neutralising and stimulatory effects of antimicrobial peptide LL-37 in human gingival fibroblasts.

OBJECTIVES: To investigate the effects of LL-37, a broad spectrum antimicrobial peptide expressed in periodontal tissues, on human gingival fibroblast responsiveness to microbial challenge and to explore the direct effects of LL-37 on human gingival fibroblasts. DESIGN: The effect of LL-37 on bacterial lipopolysaccharide-induced expression of Interleukin (IL-6) and chemokine C-X-C motif ligand (CXCL) 8 was determined by enzyme linked immunosorbent assay (ELISA). LL-37's influence on bacterial lipopolysaccharide-induced IκBα degradation was investigated by western blot. DNA microarray analysis initially determined the direct effects of LL-37 on gene expression, these findings were subsequently confirmed by quantitative polymerase chain reaction and ELISA analysis of selected genes. RESULTS: Bacterial lipopolysaccharide-induced IL-6 and CXCL8 production by human gingival fibroblasts was significantly reduced in the presence of LL-37 at concentrations in the range of 1-10 µg/ml. LL-37 led to a reduction in lipopolysaccharide-induced IκBα degradation by Escherichia coli lipopolysaccharide and Porphyromonas gingivalis lipopolysaccharide (10 µg/ml). LL-37 (50 µg/ml) significantly altered the gene expression of 367 genes in human gingival fibroblasts by at least 2-fold. CXCL1, CXCL2, CXCL3, Interleukin-24 (IL-24), CXCL8, Chemokine (C-C motif) Ligand 2, and Suppressor of Cytokine Signalling 3 mRNA were significantly upregulated by LL-37. LL-37 also significantly stimulated expression of CXCL8, hepatocyte growth factor and CXCL1 at the protein level. CONCLUSION: LL-37 plays an important regulatory role in the immunomodulatory activity of gingival fibroblasts by inhibiting lipopolysaccharide -induced expression of inflammatory cytokines and directly stimulating the expression of an array of bioactive molecules involved in inflammation and repair.

A protocol for the Development of Core Outcome Sets for Endodontic Treatment modalities (COSET): an international consensus process.

BACKGROUND: The outcome of endodontic treatment is generally assessed using a range of patient and clinician-centred, non-standardised clinical and radiographic outcome measures. This makes it difficult to synthesise evidence for systematic analysis of the literature and the development of clinical guidelines. Core outcome sets (COS) represent a standardised list of outcomes that should be measured and reported in all clinical studies in a particular field. Recently, clinical researchers and guideline developers have focussed on the need for the integration of a patient-reported COS with clinician-centred measures. This study aims to develop a COS that includes both patient-reported outcomes and clinician-centred measures for various endodontic treatment modalities to be used in clinical research and practice. METHODS: To identify reported outcomes (including when and how they are measured), systematic reviews and their included clinical studies, which focus on the outcome of endodontic treatment and were published between 1990 and 2020 will be screened. The COSs will be defined by a consensus process involving key stakeholders using semi-structured interviews and an online Delphi methodology followed by an interactive virtual consensus meeting. A heterogeneous group of key 'stakeholders' including patients, general dental practitioners, endodontists, endodontic teachers, clinical researchers, students and policy-makers will be invited to participate. Patients will establish, via interactive interviews, which outcomes they value and feel should be included in a COS. In the Delphi process, other stakeholders will be asked to prioritise outcomes identified from the literature and patient interviews and will have the opportunity at the end of the first round to add outcomes that are not included, but which they consider relevant. Feedback will be provided in the second round, when participants will be asked to prioritise the list again. If consensus is reached, the remaining outcomes will be discussed at an online meeting and agreement established via defined consensus rules of outcome inclusion. If consensus is not reached after the second round, a third round will be conducted with feedback, followed by the online meeting. Following the identification of a COS, we will proceed to identify how and when these outcomes are measured. DISCUSSION: Using a rigorous methodology, the proposed consensus process aims to develop a COS for endodontic treatment that will be relevant to stakeholders. The results of the study will be shared with participants and COS users. To increase COS uptake, it will also be actively shared with clinical guideline developers, research funders and the editors of general dental and endodontology journals. TRIAL REGISTRATION: COMET 1879. 21 May 2021.

Odontoblast cell death induces NLRP3 inflammasome-dependent sterile inflammation and regulates dental pulp cell migration, proliferation and differentiation.

AIM: To investigate the ability of dead odontoblasts to initiate NLRP3 inflammasome-dependent sterile inflammation and to explore the effect on dental pulp cell (DPCs) migration, proliferation and odontogenic differentiation. METHODS: Odontoblast-like cells were subjected to freezing-thawing cycles to produce odontoblast necrotic cell lysate (ONCL). DPCs were treated with ONCL to assess proliferation and migration. THP-1 differentiated macrophages stimulated with ONCL and live cell imaging and western blotting were used to assess NLRP3 inflammasome activation. Cytokines were measured with multiplex arrays and ELISA. qPCR, alkaline phosphatase and Alizarin red assays were used to assess odontogenic differentiation of DPCs. Data were analysed using the t-test or anova followed by a Bonferroni post hoc test with the level of significance set at P ≤ 0.05. RESULTS: ONCL induced migration and proliferation of DPCs. Treatment of THP-1 macrophages with ONCL resulted in the release of the inflammatory cytokines IL-1ß, IL-6, IL-8, TNFα, IFN-γ, CCL2 and angiogenic growth factors, angiogenin and angiopoietin. This inflammatory response was associated with activation of NFκB, p38MAPK and NLRP3 inflammasome. To confirm that ONCL induced inflammatory response is NLRP3 inflammasome-dependent, treatment with a caspase-1 inhibitor and a specific NLRP3 inhibitor significantly reduced IL-1ß release in THP-1 macrophages (P = 0.01 and 0.001). Inflammasome activation product, IL-1ß, induced odontogenic differentiation of DPCS as evident by the increase in odontogenic genes expression DMP-1, RUNX-2, DSPP and SPP, alkaline phosphatase activity and mineralization. CONCLUSION: Dead odontoblasts induced NLRP3 inflammasome-dependent sterile inflammation and activated the migration, proliferation and differentiation of DPCs.

A connectivity mapping approach predicted acetylsalicylic acid (aspirin) to induce osteo/odontogenic differentiation of dental pulp cells.

AIM: To use connectivity mapping, a bioinformatics approach, to identify compounds that could induce odontogenic differentiation of dental pulp cells (DPCs) and to experimentally validate this effect. A subsidiary aim was to investigate the anti-inflammatory effect of any identified compound. METHODOLOGY: The Gene Expression Omnibus (GEO) database was searched for microarray data sets assessing odontogenic differentiation of human DPCs. An odontogenic gene expression signature was generated by differential expression analysis. The statistical significant connectivity map (ssCMap) method was used to identify compounds with a highly correlating gene expression pattern. DPCs were treated with the compound identified, and osteo/odontogenic differentiation was assessed by Alizarin red staining, alkaline phosphatase activity and expression of osteo/odontogenic genes ALPL, RUNX2, COL1A1, DSPP, DMP1 and SPP1 by RT-PCR. The anti-inflammatory effect of the compound was assessed using an ex vivo pulpitis model, and cytokine levels were measured with multiplex assay. Means were compared using the t-test or ANOVA followed by a Bonferroni post hoc test with the level of significance set at P ≤ 0.05. RESULTS: The GEO database search identified a specific gene expression signature for osteo/odontogenic differentiation. Analysis using ssCMap found that acetylsalicylic acid [(ASA)/aspirin] was the drug with the strongest correlation with that gene signature. The treatment of DPCs with 0.05 mmol L-1 ASA showed increased alkaline phosphatase activity (P < 0.001), mineralization (P < 0.05), and increased the expression of the osteo/odontogenic genes, DMP1 and DSPP (P < 0.05). Low concentration (0.05 mmol L-1 ) ASA reduced inflammatory cytokines IL-6 (P < 0.001), CCL21 (P < 0.05) and MMP-9 (P < 0.05) in an ex vivo pulpitis model. CONCLUSIONS: Connectivity mapping, a web-based informatics method, was successfully used to identify aspirin as a candidate drug that could modulate the differentiation of DPCs. Aspirin was shown to induce odontogenic differentiation in DPCs in vitro and this, together with its anti-inflammatory effects, makes it a potential candidate for vital pulp therapies.

Putative periodontal pathogens in the subgingival plaque of Sudanese subjects with aggressive periodontitis.

BACKGROUND AND OBJECTIVES: There has been limited study of the bacterial species associated with aggressive periodontitis (AgP) in high-risk populations in Africa. The aim of this study was to investigate and quantify the presence of four putative periodontal pathogens in the subgingival plaque of Sudanese subjects with AgP. A secondary aim was to investigate the effect of varying the detection threshold on the reported prevalence of the bacterial species investigated. MATERIALS AND METHODS: Subgingival plaque samples were collected from AgP cases (n=73) and healthy controls (n=71). Bacterial DNA was extracted and analyzed by quantitative polymerase chain reaction for the detection and quantification of four putative periodontal pathogens: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola and Tannerella forsythia. RESULTS: At the lowest detection threshold (>101 cells), P. gingivalis (p<0.0001) was more prevalent in AgP cases than controls. T. forsythia and T. denticola had a high prevalence (>70%) in AgP cases at all detection levels. While T. forsythia was significantly more frequently identified in AgP than in controls at all detection thresholds, this was only the case for T. denticola at the intermediate threshold (>102 cells). A. actinomycetemcomitans was identified less frequently than the other bacterial species with no difference in its prevalence between AgP cases and controls. CONCLUSION: The prevalence of the putative periodontal pathogens investigated varied considerably in Sudanese subjects with AgP and in periodontally healthy controls depending on the detection thresholds applied. T. forsythia was identified as having the strongest association with AgP.

Matrix metalloproteinase-8 activity in gingival crevicular fluid: development of a novel assay.

BACKGROUND AND OBJECTIVE: The matrix metalloproteinases (MMPs) play a role in regulating turnover and metabolism of connective tissues in health but they have also been implicated in a wide variety of pathological conditions, including periodontal disease. MMP-8 has been extensively studied in periodontal health and disease using ELISA, although this technique is limited by its inability to determine enzyme activity. The aim was to develop an assay specifically to measure MMP-8 activity and to demonstrate its use in the analysis of gingival crevicular fluid samples. MATERIAL AND METHODS: A specific antibody was used to coat black 96-well microtitre plates to capture MMP-8 selectively. The activity of bound MMP-8 was measured using a fluorogenic substrate. Gingival crevicular fluid samples, from healthy and periodontally diseased sites, were collected using PerioPaper strips and tested for MMP-8 activity. RESULTS: Significantly higher MMP-8 activity was demonstrated in gingival crevicular fluid from periodontally diseased sites compared with healthy sites that exhibited basal or no MMP-8 activity. No cross-reactivity with other MMPs was noted. CONCLUSION: We show, for the first time, that MMP-8 activity can be specifically detected and quantified in gingival crevicular fluid samples. Measurement of MMP-8 activity could prove to be useful in monitoring periodontal disease progression.

Interferon-γ stimulates CD14, TLR2 and TLR4 mRNA expression in gingival fibroblasts increasing responsiveness to bacterial challenge.

OBJECTIVE: To investigate the potential effects of IFN-03A5 on the responsiveness of human gingival fibroblasts to bacterial challenge. DESIGN: mRNA and protein expression of CD14, TLR2 and TLR4 in human gingival fibroblasts was detected by quantitative polymerase chain reaction (Q-PCR) and flow cytometry. The effect of preincubation with IFN-03A5 on subsequent bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. Bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot. RESULTS: Human gingival fibroblasts express CD14, TLR2 and TLR4 mRNAs. IFN-03A5, but not IL-103B2, induced mRNA expression of all three receptors and the expression of membrane bound CD14 protein. Pre-incubation of fibroblasts with IFN-03A5 and subsequent stimulation with Escherichia coli LPS or Porphyromonas gingivalis LPS led to increased production of IL-6 and IL-8. LPS-induced pro-inflammatory cytokine production was abrogated by a blocking antibody to CD14. Both E. coli LPS and P. gingivalis LPS induced IκBα degradation in human gingival fibroblasts. CONCLUSION: Our data indicate that IFN-03A5 primes human gingival fibroblasts, through the upregulation of CD14 expression, which results in increased responsiveness to bacterial LPS challenge, as determined by pro-inflammatory cytokine production.

A secretory leukocyte protease inhibitor variant with improved activity against lung infection.

Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI's proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.

Dental pulp fibroblasts express neuropeptide Y Y1 receptor but not neuropeptide Y.

AIM: To investigate whether dental pulp fibroblasts express neuropeptide Y (NPY) and NPY-Y1 in vitro and to determine the effects of the cytokines including interleukin-1ß (IL-1ß), TGF- ß(1) , substance P and NPY on the expression of NPY Y1. METHODOLOGY: Three primary fibroblast cell strains were obtained from freshly extracted human third molar teeth. RT-PCR was utilized to detect expression of NPY and mRNA expression. Membrane protein samples were isolated, and protein expression was determined by Western blotting. Radioimmunoassay was used to quantify NPY expression in healthy (n = 35) and carious (n = 39) whole pulp samples, and the student's t-test was used to test for statistical significance. In addition, the 3-(4,5-Dimethylthiazol,2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assay fibroblast cell growth. RESULTS: mRNA transcripts were found in all three fibroblast cell populations with the cytokines having a stimulatory effect on its expression (P < 0.05). NPY mRNA was not detected in the cell strains. NPY-Y1 receptor protein expression was visualized by Western blotting, and there was no effect of IL-1ß or TGF- ß(1) on its expression. The mean concentration of NPY-Ir determined by radioimmunoassay in non-carious teeth was 19.40 ng x g(-1) (±17.03 SD) compared to 29.95 ng x g(-1) (±20.99 SD) in carious teeth (P < 0.05). CONCLUSION: Human dental pulp fibroblasts express, but do not synthesize, NPY, demonstrating that the fibroblast is a target cell for NPY. The effect of proinflammatory cytokines suggests that fibroblasts play a neuroimmunomodulatory role in the pulpal response to dental caries and injury.

PAR-2 regulates dental pulp inflammation associated with caries.

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated enzymatically by proteolysis of an N-terminal domain. The cleavage and activation of PARs by serine proteases represent a novel mechanism by which such enzymes could influence the host inflammatory response. The aim of this study was to determine whether PAR-2 expression and activation were increased in dental caries. Using immunohistochemistry, we showed PAR-2 to be localized to pulp cells subjacent to caries lesions, but minimally expressed by healthy pulp tissue. Trypsin and the PAR-2 agonist (PAR2-AP) activated PAR-2 in an in vitro functional assay. Endogenous molecules present in pulp cell lysates from carious teeth specifically activated PAR-2, but those from healthy teeth failed to do so. The activation of PAR-2 in vitro was shown to increase the expression of the pro-inflammatory mediator cyclo-oxygenase-2 (COX-2), providing a mechanism whereby PAR-2 could modulate pulpal inflammation.

Neuropeptide Y Y1 receptor in human dental pulp cells of noncarious and carious teeth.

AIM: To determine the distribution of the NPY Y1 receptor in carious and noncarious human dental pulp tissue using immunohistochemistry. A subsidiary aim was to confirm the presence of the NPY Y1 protein product in membrane fractions of dental pulp tissue from carious and noncarious teeth using western blotting. METHODOLOGY: Twenty two dental pulp samples were collected from carious and noncarious extracted teeth. Ten samples were processed for immunohistochemistry using a specific antibody to the NPY Y1 receptor. Twelve samples were used to obtain membrane extracts which were electrophoresed, blotted onto nitrocellulose and probed with NPY Y1 receptor antibody. Kruskal-Wallis one-way analysis of variance was employed to test for overall statistical differences between NPY Y1 levels in noncarious, moderately carious and grossly carious teeth. RESULTS: Neuropeptide Y Y1 receptor immunoreactivity was detected on the walls of blood vessels in pulp tissue from noncarious teeth. In carious teeth NPY Y1 immunoreactivity was observed on nerve fibres, blood vessels and inflammatory cells. Western blotting indicated the presence and confirmed the variability of NPY Y1 receptor protein expression in solubilised membrane preparations of human dental pulp tissue from carious and noncarious teeth. CONCLUSIONS: Neuropeptide Y Y1 is expressed in human dental pulp tissue with evidence of increased expression in carious compared with noncarious teeth, suggesting a role for NPY Y1 in modulation of caries induced pulpal inflammation.

Effect of incremental urea supplementation of a conventional corn silage-based diet on ruminal ammonia concentration and synthesis of microbial protein.

One primiparous and 3 multiparous lactating Holstein cows fitted with ruminal and duodenal cannulas were used in a 4 x 4 Latin square design to determine the efficacy of adding urea to a corn silage-based diet on ruminal fermentation and microbial protein synthesis. Dietary treatments were 0, 0.3, 0.6, and 0.9% urea in diet dry matter (DM); urea was manually top dressed and incorporated into the ration. The basal diet contained (DM basis) 52% forage (with 61% of forage provided as corn silage) and 48% concentrate ingredients. The basal diet was formulated to meet National Research Council (NRC, 2001) requirements for energy and all nutrients except rumen-degradable protein (RDP) and metabolizable protein. Experimental periods lasted 14 d with the first 9 d for adaptation. The basal diet, without urea addition, contained 9.2% RDP in DM and had a predicted RDP balance of -167 g/d (NRC, 2001). There were no effects of dietary treatment on ruminal true digestibility of organic matter or ruminal apparent digestibility of neutral detergent fiber and acid detergent fiber. Total ruminal volatile fatty acid concentrations increased linearly with increasing urea level. Feeding increasing amounts of urea quadratically increased rumen ammonia N concentrations (9.0, 11.9, 12.8, and 17.4 mg/dL at 0, 0.3, 0.6, and 0.9% urea supplementation, respectively), passage of microbial N, and microbial N in duodenal digesta as a percentage of nonammonia N. The results of this study indicate that there were some positive effects of adding urea to the described lactating dairy cow diet, and that microbial protein synthesis was maximized at an average ruminal ammonia N concentration of 12.8 mg/dL when urea was added at 0.6% in diet DM.

Matrix metalloproteinase-3 differences in oral and skin fibroblasts.

While skin wounds heal by scarring, wounds of oral mucosa show privileged healing with minimal scar formation. Our hypothesis was that phenotypic differences between oral and skin fibroblasts underlie these differences in healing. The aims of this study were to compare MMP-3 expression by oral and skin fibroblasts and investigate a role for MMP-3 in mediating collagen gel contraction. Oral fibroblasts induced significantly greater gel contraction than did paired skin cells. Inhibition of MMP activity significantly inhibited gel contraction by both cell types. Specific inhibition of MMP-3 activity reduced gel contraction by oral, but not skin, fibroblasts. Oral fibroblasts produced significantly higher levels of MMP-3 than did skin fibroblasts at all levels studied. TGF-beta1 and -beta3 isoforms stimulated MMP-3 expression at mRNA, protein, and activity levels by both fibroblast populations. Results suggest that increased MMP-3 production by oral fibroblasts may underlie the differences in wound-healing outcome seen in skin and oral mucosa.

Detection of individual human neutrophil alpha-defensins (human neutrophil peptides 1, 2 and 3) in unfractionated gingival crevicular fluid--a MALDI-MS approach.

The role of antimicrobial peptides is particularly important in the oral cavity where there is constant challenge by microorganisms. The alpha-defensins are a group of cationic peptides that comprise 30-50% of the total protein in azurophilic granules of human neutrophils. They include the human neutrophil peptides (HNP) 1, 2 and 3 which have almost identical amino acid sequences but differ in their biological activities. The amino acid sequence similarities of the defensins have made it difficult to unequivocally determine the presence of individual defensins using antibody-based techniques. However, by virtue of their cationic nature we postulated that the defensins would fly particularly well in mass spectrometry and that this characteristic would allow facile identification of individual HNPs in unfractionated gingival crevicular fluid (GCF) from periodontitis patients and healthy controls. Although there was variability in levels of defensins detected in periodontal health and disease, HNP-1 was always identified as the major peak in the triad and HNP-3 as the minor peak, lending support to the hypothesis that HNP-2 may arise by post-translational proteoyltic cleavage of HNP-3 rather than HNP-1. The finding that the defensins were more abundant in a higher proportion of the healthy sites studied could be linked to a more intact defensin barrier in periodontal health.

Ruminal biohydrogenation in Holstein cows fed soybean fatty acids as amides or calcium salts.

Fatty amides of high oleate fats and calcium salts of palm oil were reported to resist biohydrogenation by ruminal microorganisms. This study was conducted to determine whether converting polyunsaturated fat sources to amides and calcium salts had equal ability to resist biohydrogenation. A total mixed ration consisting of forage and concentrate contained (dry basis): 1) 2.45% soybean oil (SBO), 2) 2.75% calcium salt of SBO, 3) 2.75% amide of SBO, or 4) 2.75% of a mixture of the calcium salt and amide (80:20, wt/wt) of SBO. The 4 diets were fed ad libitum to 4 multiparous lactating Holstein cows fitted with ruminal cannulas in a 4 x 4 Latin square with 21-d periods. Omasal samples were taken to measure postruminal fatty acid content and determine the extent of ruminal biohydrogenation. Adding SBO to the diets as either calcium salts or amides increased omasal flow of C18:2 (n-6) from 25 to 39 g/d. Omasal flow of C18:1 increased from 36 to 49 g/d when SBO was fed to cows as calcium salts, but increased to 86 g/d when SBO was fed as amides. Adding the soybean amide to the diet more than doubled the delivery of C18:1 (n-9) to the omasum of lactating cows, but it also increased trans fatty acid production in the rumen accompanied by milk fat depression. In this study, calcium salts and amide derivatives of fatty acids were both effective in enhancing omasal flow of unsaturataed fatty acids in lactating dairy cows. Amides were more effective than calcium salts for increasing the postruminal flow of oleic acid.

Sjögren's syndrome: a condition with features of chronic graft-versus-host disease: does duct cell adhesion or permeability play a role in pathogenesis?

Primary and Secondary Sjögren's syndrome are disease complexes characterized by periductal inflammatory cell infiltration of the salivary and lacrimal glands and manifest as dry mouth and dry eyes. Secondary Sjögren's syndrome may be associated with a connective tissue disorder. Additional extraglandular features in Sjögren's syndrome include a generalized inflammatory exocrinopathy that might be associated with abnormalities of both humoral and cellular mediated immunity. Similar inflammatory changes and extraglandular features, including an altered immune response, have been reported in patients developing graft-versus-host disease after bone-marrow transplantation and in patients with primary biliary cirrhosis. The periductal nature of the inflammatory response involving minor salivary and other glands raises the possibility of altered duct cell adhesion or permeability in playing a role in the aetiopathogenesis of Sjögren's syndrome. The paper pulls together evidence that could be interpreted in this light. Evidence for bacterial or viral factor(s) altering the antigenicity of the histocompartibility (HC) complex on ductal cells in Sjögren's syndrome patients is also described. A hypothesis is proposed for Sjögren's syndrome in which the principal feature is an alteration in salivary gland duct cell adhesion or permeability. A re-evaluation of current knowledge of these two conditions from a clinical and experimental context are interpreted in this light.

NEUROPEPTIDES AND NEUROGENIC MECHANISMS IN ORAL AND PERIODONTAL INFLAMMATION.

It is generally accepted that the nervous system contributes to the pathophysiology of peripheral inflammation, and a neurogenic component has been implicated in many inflammatory diseases, including periodontitis. Neurogenic inflammation should be regarded as a protective mechanism, which forms the first line of defense and protects tissue integrity. However, severe or prolonged noxious stimulation may result in the inflammatory response mediating injury rather than facilitating repair. This review focuses on the accumulating evidence suggesting that neuropeptides have a pivotal role in the complex cascade of chemical activity associated with periodontal inflammation. An overview of neuropeptide synthesis and release introduces the role of neuropeptides and their interactions with other inflammatory factors, which ultimately lead to neurogenic inflammation. The biological effects of the neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY) are summarized, and evidence for their involvement in the localized inflammatory lesions which characterize periodontitis is presented. In this context, the role of CGRP in bone metabolism is described in more detail. Recent research highlighting the role of the nervous system in suppressing pain and inflammation is also discussed.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue.

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted.

Quantitative analysis of substance P, neurokinin A and calcitonin gene-related peptide in pulp tissue from painful and healthy human teeth.

AIM: The purpose of this study was to investigate the levels of substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) in painful and healthy human dental pulps. METHODOLOGY: Forty-six samples of pulp tissue were collected from extracted or endodontically treated painful teeth and 20 from clinically healthy teeth extracted for orthodontic reasons. All pulp samples were boiled in 0.5 m acetic acid for 10 min, centrifuged and the supernatant collected. SP, NKA and CGRP levels were measured using radioimmunoassay. RESULTS: Substance P and CGRP were present in all samples and NKA was detected in 96% of the pulps. CGRP was present in much higher concentrations than SP and NKA in both painful and non-painful teeth. The painful teeth had significantly higher concentrations of SP (P = 0.02), NKA (P < 0.001) and CGRP (P = 0.03) than non-painful teeth. The concentration of CGRP was significantly higher in the pulps of smokers compared with non-smokers (P = 0.02). CONCLUSIONS: Elevated levels of these neuropeptides in pulps from painful teeth indicate that they may play an important role in the process of pulpal inflammation and pain. Further investigation of the association between these neuropeptides and pulpal status may help to improve our understanding of pulpal inflammation and dental pain.

Quantitative analysis of MRP-8 in gingival crevicular fluid in periodontal health and disease using microbore HPLC.

BACKGROUND: The protein components of GCF can be separated by reverse-phase microbore HPLC on a C18 column with detection on the basis of 214 nm absorbance. A single major symmetrical protein peak eluting with a retention time of 26 min (50% acetonitrile) was evident in gingival crevicular fluid (GCF) from periodontitis patients but not in healthy GCF. This protein was identified as human MRP-8 by N-terminal amino acid sequencing and liquid chromatography quadropole mass spectrometry. AIMS: To quantify the amount of MRP-8 detectable in GCF from individual healthy, gingivitis and periodontitis affected sites and to study the relationship, if any, between the levels of this responsive protein and periodontal health and disease. METHODS: GCF was sampled (30 s) from healthy, gingivitis, and periodontitis sites in peridontitis subjects (n=15) and from controls (n=5) with clinically healthy gingiva and no periodontitis. Purified MRP-8 was sequenced by Edmann degradation and the phenylthiohydantoin (PTH) amino acid yield determined (by comparison of peak area with external PTH amino acid standards). This value was subsequently used to calculate the relative amount of protein in the peak eluting with a retention time of 26.0 min (MRP-8) in individual GCF chromatograms. RESULTS: Higher levels of MRP-8 were detected in inflammatory sites: periodontitis 457.0 (281.0) ng; gingivitis 413.5 (394.5) ng compared with periodontally healthy sites in diseased subjects 14.6 (14.3) ng and in controls 18.6 (18.5) ng, p=0.003. There was at least 20-fold more MRP-8 in the inflammatory compared with the healthy sites studied. CONCLUSIONS: The preliminary data indicate that MRP-8 is present in GCF, with significantly greater amounts present at diseased than healthy sites. A systematic study of the relationship of this protein to periodontal disease could prove useful in further clarifying whether MRP-8 could be a reliable GCF biomarker of gingivitis and periodontitis.