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 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.

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.

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.

Bcl-2 expression in sequential biopsies of potentially malignant oral mucosal lesions assessed by immunocytochemistry.

OBJECTIVE: To examine, for the first time Bcl-2 expression in sequential (autogenous) oral mucosal biopsies taken from the same sites in a gender, risk-factor matched, Caucasoid sample, over a 21-year period. DESIGN: Retrospective immunocytochemical longitudinal study of archival serial biopsies. MATERIALS AND METHODS: Computer records were used to identify biopsy specimens derived from 12 patients. These were divided into four groups: (1) Histologically innocuous lesions which remained histologically innocuous. (2) Dysplastic lesions which remained dysplastic. (3) Histologically innocuous lesions which later progressed to squamous cell carcinoma (SCC). (4) Dysplastic lesions which later progressed to SCC. This represented 65 biopsies in total. Bcl-2 expression was studied using mouse antihuman BCL-2 oncoprotein clone 124 (Dako, Denmark). RESULTS: Generally, there was a lack of Bcl-2 immunoreactivity in the epithelium, with one exception in dysplastic epithelium from a group (3) patient. CONCLUSION: These findings suggest that in our series, Bcl-2 is not expressed early in oral premalignant lesions and appears to contradict previous reports. Possible explanations for this disparity are considered.

TFF1 gene expression in human medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC) is an uncommon tumour of calcitonin-secreting C-cells of the thyroid gland. This cancer represents an important potential model for the study of mechanisms of human epithelial cell transformation. Although recent studies have identified the gene involved in familial forms of MTC, little is known about the molecular pathogenesis of the sporadic variants of this tumour. The biological and prognostic significance of TFF1 expression, particularly in diverse human malignancies, suggests that the TFF1 protein could have a role in human neoplasia. Furthermore, in prostate cancer it has been demonstrated that TFF1 expression is closely associated with premalignant changes and neuroendocrine differentiation. In the present study, the expression of TFF1 was analysed in 18 human MTCs, comprising sporadic and familial tumours, C-cell hyperplasia, and one case of lymph gland metastasis. TFF1 expression was also examined in the cultures of a human MTC-derived tumour cell line (TT cell line). The results showed that ten sporadic tumours, three hereditary tumours (including C-cell hyperplasia), and one lymph gland metastasis displayed TFF1 immunoreactivity. Indirect fluorescence immunocytochemistry and Western blotting revealed that the TFF1 protein was strongly expressed in the TT cells. Northern analysis revealed that tumours and TT cells expressed the TFF1 transcript. Although the function of TFF1 protein in the carcinogenesis of MTC remains to be elucidated, its expression in the majority of cases of both sporadic and hereditary tumours, metastatic tumours, and in C-cell hyperplasia suggests that it may contribute to the pathogenesis of MTC.

Evaluation of major parotid glycoproteins in patients with burning mouth syndrome.

OBJECTIVE: This study was to investigate the potential role of salivary glycoproteins in burning mouth syndrome. STUDY DESIGN: This study compared major parotid glycoproteins in a group of patients with burning mouth syndrome and age-, sex-, race-matched healthy controls. RESULTS: By use of a glycoprotein detection kit, saliva from both patients and controls exhibited three major parotid glycoprotein banding patterns consisting of either one or two bands, molecular weights 58 kDa and 77 kDa. The strong lectin reactivity of major parotid glycoproteins with Ricinus communis agglutinin suggests that galactose is the most prevalent terminal sugar. In addition, major parotid glycoproteins were shown to express blood group antigen H. On the basis of metachromatic characteristics and immunologic reactivity, major parotid glycoproteins appear to be members of the proline rich protein multigene family, proline rich glycoprotein, genetic polymorphism G1. No qualitative difference was observed in major parotid glycoprotein banding patterns between patients and controls. CONCLUSION: These findings do not support a role for major parotid glycoproteins in burning mouth syndrome.

The determination of asialoglycoforms of serum glycoproteins by lectin blotting with Ricinus communis agglutinin.

Serum proteins were fractionated by polyacrylamide gel electrophoresis under denaturing conditions and transferred to nitrocellulose membranes. The blotted polypeptides were probed with biotinylated Ricinus communis lectin (RCA120) followed by streptavidin/alkaline phosphatase. This procedure detected five asialoglycoproteins (alpha 2-macroglobulin, transferrin, alpha 1-antitrypsin, alpha 1-antichymotrypsin and haptoglobin beta chain). The asialoform of the alpha 1-trypsin inhibitor was found to be decreased in inflammation.