Neutrophil elastase and endogenous inhibitors in Behçet's disease saliva.

Behçet's disease (BD) is a vasculitis of unknown aetiology typified by chronic recurrent oral ulcers and systemic inflammatory manifestations. Neutrophils, and specifically their protease neutrophil elastase (NE), have been implicated in its pathology. Although NE is an effective anti-microbial, excessive NE can damage host tissue. Recurrent oral ulceration is a primary BD symptom, therefore we hypothesized that excessive neutrophil infiltration evidenced by increased NE and a reduction in specific endogenous inhibitors, secretory leucocyte protease inhibitor (SLPI) and alpha1-anti-trypsin (α1AT) contributes to BD mucosal instability. NE, SLPI and α1AT were quantified in saliva from BD patients with active oral ulcers (BDa) and quiet without ulcers (BDq), recurrent aphthous stomatitis (RASa; RASq) and healthy controls (HC). Although BDq saliva had marginally higher median NE levels (1112 ng/ml) compared to both RASq (1043 ng/ml) and HC (999 ng/ml), SLPI was significantly reduced in BDq (P < 0·01). Despite decreased SLPI protein, mRNA expression was significantly increased in BDq buccal epithelial swabs compared to RASq and HC (P < 0·05, P < 0·001). NE remained enzymatically active, although α1AT levels were at least eight times higher than SLPI in all groups, suggesting that α1AT does not have a primary role in counteracting NE in saliva. Furthermore, NE levels in BDa patients medicated with both azathioprine (AZA) and colchicine (COLC) were significantly lower than those on COLC (P = 0·0008) or neither (P = 0·02), indicating that combining AZA + COLC may help to regulate excessive NE during ulceration. This study showed that enzymatically active NE coupled with reduced SLPI in BD saliva may contribute to recurrent oral ulcerations.

Characterizing the immunological effects of oral healthcare ingredients using an in vitro reconstructed human epithelial model.

Oral healthcare products are well tolerated and while adverse occurrences are rare there is still a need to explore the interaction between these products and the oral mucosa. This study assessed the effects of oral healthcare ingredients: sodium lauryl sulphate (SLS), a detergent; cinnamic aldehyde (CA), a flavouring agent; and cetylpyridinium chloride (CPC), an antiseptic, using a reconstructed human oral mucosal model (OMM). Differential release of inflammatory cytokines IL-1α, IL-8 and cytotoxicity was compared with other known irritants and sensitizers to identify a signature response profile that could be associated with oral mucosal irritation. Response profiles differed with irritants being more cytotoxic. CA and control sensitizers nickel sulphate (NiSO4) and 1-chloro-2,4-dinitrochlorobenzene (DNCB) released lower levels of IL-1α than CPC and control irritant benzalkonium chloride (BC), whereas the opposite was observed for IL-8. Significant levels of IL-8 and IL-1α were released with 5-15 mg/ml (0.5-1.5% w/v) SLS. Quantitative PCR indicated that cytokine release at lower SLS concentrations is not entirely due to cell necrosis but in part due to de novo synthesis. These findings suggest that the OMM can be used to predict oral irritation thus making it a potentially valuable model for screening new oral healthcare ingredients prior to clinical release.

Differential expression of suppressor of cytokine signalling proteins in Behçet's disease.

Behçet's disease (BD) is a chronic, multisystemic, recurrent vasculitis disease of unknown aetiology. Proinflammatory cytokines are a key feature of the disease, but the triggers for their induction are not well understood and/or controversial. Suppressor of cytokine signalling (SOCS) proteins which negatively regulate the JAK-STAT signalling pathway of cytokine induction may be dysregulated in BD. The expression of SOCS1 and 3 mRNA and protein was studied in peripheral blood mononuclear cells (PBMCs) and neutrophils of patients with BD and compared with healthy controls (HCs) and patients with recurrent aphthous stomatitis (RAS) using RT-PCR, Western blot and immunohistochemistry. SOCS1 and 3 mRNA was also measured in buccal mucosal cells (BMC) of patients with BD and HCs. SOCS1 and 3 mRNA was significantly upregulated in PBMCs of patients with BD compared with HCs (P = 0.0149; P = 0.0007). In addition, there were subtle differences between expression in active and symptom-free BD (quiescent BD). SOCS1 and SOCS 3 were also significantly upregulated in BMC from oral ulcers of BD compared with HCs (both at P = 0.0001). A differential expression of both SOCS1 and 3 was observed between PBMCs and neutrophils in patients with BD. Immunohistochemical analysis revealed differential expression of SOCS proteins in the buccal mucosa with an increased expression at the ulcer surface of ulcers than in the non-ulcerated tissue. These observations suggest a dysregulation of the expression of these important regulators not only between patients with BD and healthy controls but also between mucosal and systemic tissues, which may reflect the nature of the aetiopathology of the disease.

Quantitative optical coherence tomography of fluid-filled oral mucosal lesions.

The decision of selecting the most representative site for the biopsy of fluid-filled lesions can be difficult. This may be attributed to the poor delineation of the correct lesional site by clinical observation alone. In this study, optical coherence tomography is used to quantify the contrast between solid- and fluid-filled lesions by measuring the light intensity change at the tissue-fluid interface (intensity drop). This parameter was measured from sequential axial scans (n ≈ 10(6) per sample) of 3D optical coherence tomography (OCT) datasets from control tissues (n = 14) and fluid-filled lesions (n = 7) and displayed as a 2D-scaled intensity drop (SID) image. The results of the SID image allowed for discrimination, characterisation and extent of a fluid filled region. The differentiation of normal and fluid-filled areas using individual SID values yielded both a sensitivity and specificity of approximately 80 %. OCT complemented by SID analysis provides a potential in vivo clinical tool that would enable non-invasive objective visualisation of the oral mucosa.

Oral health and related quality of life status in patients from UK and Turkey: a comparative study in Behcet's disease.

BACKGROUND: The aim of this study was to evaluate and compare oral health-related quality of life (oral QoL) in patients from UK and Turkey with Behcet's disease (BD). METHODS: Thirty-one BD patients from UK (F/M: 18/13, mean age: 41.8 +/- 11.5 years) and Turkey (F/M: 18/13, mean age: 41.5 +/- 10.3) who were matched according to age and gender were included in the study. All patients had active oral ulcers. Oral QoL was assessed by Oral Health Impact Profile-14 (OHIP-14). Oral health was evaluated by dental and periodontal indices. RESULTS: No significant difference was found in OHIP-14 scores between patients from UK (22.7 +/- 14.4) and Turkey (20.4 +/- 14.3) (P = 0.709). The OHIP-14 score correlated with the healing time of oral ulcers in UK (r = 0.4, P = 0.04) and the number of oral ulcers in Turkey (r = 0.4, P = 0.012). The number of oral ulcers per month was significantly higher in UK (3.3 +/- 2.8) compared with that in Turkey (1.5 +/- 2.5) (P = 0.014). However, the number of filled teeth and frequency of tooth brushing were significantly lower in patients from Turkey compared with those in UK (P = 0.000). Similarly, the duration since the last dental visit (5.1 +/- 7.2 months) was significantly lower in UK compared with that in Turkey (28.6 +/- 23.7 months) (P = 0.000). CONCLUSIONS: Oral QoL was similar in patients from UK and Turkey with active oral ulcers. However, the number of oral ulcers was observed to be higher in UK. As expected, a lower utilization rate of dental services might have led to a poorer oral health in patients from Turkey.

Fingerprinting genomic instability in oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a high-risk pre-cancerous condition where 7-13% of these patients develop head and neck squamous cell carcinoma (HNSCC). To date there is no cancer predictive markers for OSF patients. Genomic instability hallmarks early genetic events during malignant transformation causing loss of heterozygosity (LOH) and chromosomal copy number abnormality. However, to date there is no study on genomic instability in OSF. Although this condition is known as a high-risk pre-cancerous condition, there is no data regarding the genomic status of this disease in terms of genetic susceptibility to malignant transformation. METHODS: In this study, we investigated the existence of genetic signatures for carcinogenesis in OSF. We employed the high-resolution genome-wide Affymetrix Mapping single nucleotide polymorphism microarray technique to 'fingerprint' global genomic instability in the form of LOH in 15 patient-matched OSF-blood genomic DNA samples. RESULTS: This rapid high-resolution mapping technique has revealed for the first time that a small number of discrete hot-spot LOH loci appeared in 47-53% of the OSF tissues studied. Many of these LOH loci were previously identified regions of genomic instability associated with carcinogenesis of the HNSCC. CONCLUSION: To our knowledge, this is the first evidence that genomic instability in the form of LOH is present in OSF. We hypothesize that the genomic instability detected in OSF may play an important role in malignant transformation. Further functional association studies on these putative genes may reveal potential predictive oral cancer markers for OSF patients.

Upregulation of HIF-1alpha in malignant transformation of oral submucous fibrosis.

BACKGROUND: Oral submucous fibrosis (OSF) is a precancerous condition showing extensive fibrosis of the submucosa and affects most parts of the oral cavity, including pharynx and upper third of the oesophagus. The molecules involved in the biological pathways of the fibrotic process appeared to be either down- or upregulated at different stages of the disease. Despite the precancerous nature, malignant transformation of the epithelium in the background of fibrosis has not been studied in detail. HIF-1alpha is a known transcription factor that is induced by hypoxia. AIMS: To test the hypothesis that hypoxia plays a role in malignant transformation and progression of OSF. MATERIALS AND METHODS: We used both formalin-fixed and frozen samples of OSF and normal mucosa to investigate the relationship between HIF-1alpha and epithelial dysplasia using immunohistochemistry and RT-PCR. CONCLUSIONS: Our data indicate that HIF-1alpha is upregulated at both protein and mRNA levels in OSF and the correlation with epithelial dysplasia is statistically significant (P < 0.001). We propose that HIF-1alpha may play a role in malignant transformation of OSF. Further, over-expression of HIF-1alpha may contribute to the progression of fibrosis. It may be possible to use HIF-1alpha as a marker for malignant transformation of OSF.

Expression of adrenomedullin and its receptors in human salivary tissue.

Adrenomedullin is a multifunctional peptide produced by a wide range of different cells and tissues. This study was designed to investigate whether adrenomedullin is present in human saliva and in salivary glands. It was expected that saliva may contain high concentrations of adrenomedullin, which has antimicrobial activity in vitro, which may have functional implications in the oral cavity. Saliva from the submandibular and parotid glands contained higher concentrations of adrenomedullin than did the circulation, but lower concentrations than in whole saliva. This suggests that oral epithelium may contribute the majority of the adrenomedullin peptide found in saliva. Specific adrenomedullin receptors were found in cell lines from the submandibular (HSG) and parotid (HSY) salivary glands. These findings suggest a paracrine/autocrine role for adrenomedullin in these tissues; however, the concentration of adrenomedullin in saliva was insufficient to suggest a significant antimicrobial action in the healthy oral cavity.

Permeability barrier properties of oral keratinocyte cultures: a model of intact human oral mucosa.

OBJECTIVES: The aim of this study was to establish whether an in vitro model of human oral mucosa had similar permeability characteristics to normal oral mucosa. Such a model would have considerable value as an alternative to the use of mucosal biopsies in studies of transmucosal drug delivery. MATERIALS AND METHODS: Keratinocytes obtained from buccal mucosa, hard palate and abdominal skin were seeded onto inert collagen membranes (Cellagen Discs) or dead de-epidermised dermis (DDED) and grown either as submerged or air-liquid interface cultures. Subsequently the ultrastructural characteristics, permeability to water and barrier lipid content of the epithelial cultures were assessed and compared with samples of intact mucosa and skin. RESULTS: All the cultures stratified into multilayered epithelia and displayed features of differentiation including tonofilaments, desmosomes and membrane coating granules. The permeability characteristics and barrier lipid content of the oral mucosal cultures resembled those of intact mucosa. By contrast, epidermal keratinocytes failed to produce a permeability barrier comparable with that of skin and had low levels of barrier associated lipids. CONCLUSIONS: Cultures of human oral mucosal keratinocytes obtained from healthy adults develop similar permeability properties and barrier lipid composition to their site of origin. This model system may be useful for the evaluation of local and systemic oral mucosal drug delivery.

Adrenomedullin expression in pathogen-challenged oral epithelial cells.

Adrenomedullin, a multifunctional peptide, is expressed by many surface epithelial cells and, previously, we have demonstrated that adrenomedullin has antimicrobial activity. The oral cavity contains an epithelium that is permanently colonized by microflora, yet infections in a host are rare. We exposed oral keratinocytes to whole, live cells from four microorganisms commonly isolated from the oral cavity, Porphyromonas gingivalis, Streptococcus mutans, Candida albicans and Eikenella corrodens. There was upregulation of protein and gene expression in these cells in response to bacterial suspensions, but not with the yeast, Candida albicans. We propose there is a potential role for microbial products in enhancing mucosal defense mechanisms and that adrenomedullin participates in the prevention of local infection, thus contributing to host defense mechanisms.

Synthesis of a transition state analogue for the hydrolysis of cocaine: assistance to phosphonylation of a 3beta-hydroxytropane by a neighbouring amide group.

A simple synthesis of phenylphosphonate monoester analogues of the transition state for hydrolysis of the benzoyl ester group in cocaine is provided by the reaction of 2beta-amido-3beta-tropanols with phenylphosphonyl dichloride. Steric hindrance to phosphonylation of the hydroxyl is overcome because the neighbouring 2beta-amido group participates in the reaction. The intramolecular assistance by the amide to formation of the phosphonate ester is influenced by the electronic environment of the amide group.

Direct effects of corticotrophin on oral keratinocyte cell proliferation.

Corticotropin is produced by keratinocytes and may have an immunoregulatory role in oral mucosa and skin. We have investigated its effects on a human oral keratinocyte cell line and shown that corticotropin, acting via its specific receptor, stimulates a dose-dependent increase in DNA synthesis and induces cell proliferation. When cells were incubated in the presence of increasing concentrations of corticotropin, there were significant increases in intracellular cAMP levels. Corticotropin-stimulated mitogenesis and cell proliferation were attenuated by the adenylyl cyclase inhibitor SQ22,536, but were unaffected by inhibitors of protein kinase C or tyrosine kinase. These data identify corticotropin as a mitogenic regulatory peptide of keratinocytes acting via cAMP.

Vascular endothelial growth factor in normal human salivary glands and saliva: a possible role in the maintenance of mucosal homeostasis.

Saliva is an enriched milieu containing biologically active proteins, including several different growth factors and cytokines. This study documents that vascular endothelial growth factor (VEGF), a potent, multifunctional, angiogenic cytokine, is a component of normal human saliva. VEGF was measured by ELISA in whole saliva (median concentration, 460 pg/ml) and in ductal secretions obtained from the parotid (277 pg/ml) and the submandibular-sublingual (80 pg/ml) salivary glands. VEGF seems to be synthesized endogenously by the salivary glands because both VEGF mRNA and protein (as revealed by in situ reverse transcriptase-PCR and by immunohistochemistry, respectively) colocalized to serous acinar cells and ductal epithelial cells within the parotid, submandibular, and minor salivary glands. These findings point to the existence of a "salivary VEGF system." It is possible that salivary VEGF plays a role in regulating physiologic and pathologic angiogenic and other vascular responses in salivary and mucosal tissues. And in particular, the presence of VEGF in saliva may contribute to the remarkable healing capacity of the oral mucosa as well as other regions of the digestive tract.

Proadrenomedullin N-terminal 20-peptide (PAMP) receptors and signal transduction in the rat adrenal gland.

Using ligand binding techniques we have demonstrated specific saturable binding of proadrenomedullin N-terminal 20-peptide (PAMP) in the rat adrenal zona glomerulosa. Hill plot analysis revealed two classes of receptor (Kd1 1.9nmol/l Bmax 53fmol/mg protein: Kd2 10nmol/l, Bmax 225fmol/mg). [125 I]-PAMP was displaced by adrenomedullin at one of these receptors. In the adrenal medulla [125 I]-PAMP bound to a single class of specific receptor (Kd 4.9 nmol/l: Bmax 556fmol/mg). PAMP caused an increase in cAMP generation by zona glomerulosa cells, but not inner zone/medulla cells. These data suggest that PAMP acts through a specific receptor to elevate cAMP levels in the rat adrenal.

Characterization of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors.

The hydrolyses of 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate and of a new, more soluble, substrate, 4-nitrophenyl 4'-(3-aza-7-hydroxy-2-oxoheptyl)phenyl carbonate, each catalysed by a polyclonal antibody preparation elicited in a sheep by use of an analogous phosphate immunogen, were shown to adhere closely to the Michaelis-Menten equation, in accordance with the growing awareness that polyclonal catalytic antibodies may be much less heterogeneous than had been supposed. The particular value of studies on polyclonal catalytic antibodies is discussed briefly. Both the kcat and kcat/K(m) values were shown to increase with increase in pH across a pKa of approx. 9. Group-selective chemical modification studies established that the side chains of tyrosine and arginine residues are essential for catalytic activity, and provided no evidence for the involvement of side chains of lysine, histidine or cysteine residues. The combination of evidence from the kinetic and chemical modification studies and from studies on the pH-dependence of binding suggests that catalysis involves assistance to the reaction of the substrate with hydroxide ions by hydrogen-bond donation at the reaction centre by tyrosine and arginine side chains. This combination of hydrogen-bond donors appears to be a feature common to a number of other hydrolytic catalytic antibodies. High-pKa acidic side chains may be essential for the effectiveness of catalytic antibodies that utilize hydroxide ions.

Adrenomedullin has mitogenic effects on human oral keratinocytes: involvement of cyclic AMP.

The effects of the novel vasoactive regulatory peptide, adrenomedullin, on human oral keratinocytes was investigated. Adrenomedullin, acting via its specific receptor, stimulated a dose-dependent increase in DNA synthesis, and, in addition, stimulated further changes in the cell cycle resulting in the proliferation of keratinocytes. When cells were incubated in the presence of increasing concentrations of adrenomedullin, there was a rapid and dose-dependent rise in intracellular cyclic AMP levels. Stimulation of mitogenesis and cell proliferation in these cells were mimicked by the cell permeable cAMP analogue, dibutyryl cAMP. Adrenomedullin-stimulated mitogenesis was attenuated by the adenylyl cyclase inhibitor SQ22,536, but was unaffected by inhibitors of PKC, tyrosine kinase or the CGRP receptor antagonist, CGRP(8-37). These data identify adrenomedullin as a new mitogenic regulatory peptide of keratinocytes acting via the cAMP cascade.