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.

Porphyromonas gingivalis culture supernatants differentially regulate interleukin-1beta and interleukin-18 in human monocytic cells.

Porphyromonas gingivalis is a major bacterial species implicated in chornic periodontitis, a disease characterized by inflammatory destruction of the tooth supporting tissues. Its main virulence factors are lipopolysaccharide (LPS) and gingipains, a group of cysteine proteinases. Interleukin (IL)-18 is a potent pro-inflammatory cytokine with structural similarities to IL-1beta. This study aimed to investigate if P .gingivalis regulates IL-1beta and IL-18 in monocytic cells. Monomac-6 cells were challenged with P. gingivalis culture supernatants. Quantitative real-time PCR and ELISA were used to investigate IL-1beta and IL-18 mRNA expression and protein secretion, respectively. P. gingivalis enhanced IL-1beta and IL-18 mRNA expression, the former being induced earlier, but transiently. IL-18 up-regulation was not affected by P. gingivalis heat-inactivation or chemical inhibition of its gingipains, whereas both treatments resulted in 50% reduction of IL-1beta expression. Purified P. gingivalis LPS enhanced both IL-1beta and IL-18 expression. However, only IL-1beta, but not IL-18, secretion was detected, and was up-regulated by P. gingivalis. In conclusion, although IL-1beta and IL-18 belong to the same cytokine family, their gene expression and secretion are differentially regulated in human monocytic cells in response to P. gingivalis. Therefore, cytokines of the IL-1 family may participate via different pathways in the complex pathogenesis of periodontitis.

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.

A polymeric system for the intra-oral delivery of an anti-fungal agent.

Oral candidal infections are often persistent and intractable and thus the aim of this study was to develop a polymeric sustained release device to improve the topical treatment of these infections. A self curing system based on poly(ethyl methacrylate) and tetrahydrofurfuryl methacrylate (PEM/THFM) was used with chlorhexidine diacetate (CX) added at levels between 0 and 12% w/w. Water uptake by the device was assessed gravimetrically and CX release measured by UV spectrometry. Anti candidal activity was established by culturing azole sensitive and resistant strains of Candida albicans in the presence of the polymeric delivery device with and without CX. Candidal growth was measured by turbidimetry or surviving colony-forming unit (CFU) formation. There was an initial high release of CX over 24 h followed by a slow diffusion up to 7 days. CX inhibited candidal growth and survival markedly in vitro, with the test samples showing less than 0.5 x 10(-7) CFU/ml compared to controls (3-4 x 10(-7) CFU/ml). These results indicate the potential of a chlorhexidine containing PEM/THFM polymeric system in the treatment of persistent candidal infections.

Short-term exposure to alcohol increases the permeability of human oral mucosa.

OBJECTIVES: The aim of this study was to evaluate the effect of short-term exposure to ethanol on the permeability barrier properties of human oral mucosa. MATERIALS AND METHODS: Permeability constants (Kp x 10(-4) cm min(-1)) to tritiated water were determined, for untreated human ventral tongue, and following treatment with phosphate-buffered saline (PBS), 5, 15 or 40% ethanol using an in vitro perfusion chamber system. Some samples were also exposed to fluorescent-labelled albumin and examined by fluorescence microscopy. Permeability barrier lipid composition was assessed in treated and untreated mucosa by heat separation, solvent extraction and thin layer chromatography. RESULTS: Fifteen per cent ethanol significantly increased mucosal permeability (5.8 +/- 0.44; P < 0.05) compared with untreated, PBS and 5% ethanol treated mucosa (4.69 +/- 0.26, 4.48 +/- 0.3 and 4.13 +/- 0.27, respectively). Albumin was restricted to the epithelial surface in control tissue, but extended further through the epithelium and, in some cases, into the connective tissue after treatment with ethanol. Biochemical analysis revealed no significant difference in the epithelial lipid composition of treated and untreated mucosa. CONCLUSIONS: These results suggest that short-term exposure to ethanol may act as a permeability enhancer, possibly by causing molecular rearrangement of the permeability barrier, not as a result of lipid extraction.

The effect of sodium lauryl sulphate, triclosan and zinc on the permeability of normal oral mucosa.

OBJECTIVE: Sodium lauryl sulphate (SLS), an important component in many oral health products, is well established as a contact irritant in skin. Recent studies have suggested that it may also affect the structural integrity of oral mucosa. SLS is rarely used alone in dentifrices or mouthwashes and the aim of this study was to establish the effect of SLS both alone and in combination with Triclosan (TCN) and zinc (Zn) on the permeability barrier properties of normal human oral mucosa. METHOD: Ventral tongue mucosa was obtained from nine males and seven females within 60 h of death and stored frozen at -70 degrees C until use. The permeability of the tissue to tritiated water was measured after pretreatment for 15 min with SLS alone, SLS/TCN, SLS/Zn and a SLS/TCN/Zn mixture. Treatment with distilled water (DW) served as control. The histological appearance of the tissue before and after treatment was also examined by light microscopy. RESULTS: SLS treatment caused a significant increase in water permeability compared to control tissue (Kp = 11.7 +/- 1.00; 4.96 +/- 0.50 respectively; P < 0.005). Treatment with a SLS/TCN/Zn mixture, however, had no effect on the permeability to water (Kp = 5.5 +/- 0.56). Histological examination revealed that tissue exposed to SLS had a marked disruption of the epithelial surface whilst tissue treated with a SLS/TCN/Zn mixture was indistinguishable from controls. CONCLUSION: Although mucosa exposed to SLS alone showed an increase in permeability to water, the addition of TCN and Zn to SLS appeared to prevent this effect. As SLS is included in some dental products to solubilise compounds such as TCN, its presence may have no effect on the permeability barrier property of oral mucosa.

Regulation of rat adrenal neuropeptide Y (NPY) content: effects of ACTH, dexamethasone and hypophysectomy.

While the presence of neuropeptide Y (NPY) in the adrenal cortex is well established, little is known about its regulation. In the present study the involvement of the pituitary gland in the regulation of rat adrenal NPY content was investigated. Rats were subjected to one of the following treatments: hypophysectomy, sham operation, ACTH, the synthetic glucocorticoid, dexamethasone, dexamethasone plus ACTH, or saline control. The immunoreactive NPY (irNPY) content of both capsule/zona glomerulosa and inner zone/medulla fractions were estimated by radioimmunoassay. Treatment with ACTH caused a significant decrease in both the capsular/zona glomerulosa and the inner zone/medulla irNPY content compared with controls, while hypophysectomy resulted in a significant increase in adrenal irNPY. Dexamethasone treatment caused a significant increase in capsular irNPY, which was reversed by simultaneous administration of ACTH. In the medulla, however, dexamethasone treatment significantly decreased irNPY content. These results suggest that there is differential regulation of adrenal irNPY content, with irNPY in the zona glomerulosa regulated directly by ACTH, while the irNPY content of the inner zones/medulla is regulated by glucocorticoids.

Immunolocalization of protease-activated receptor-2 in skin: receptor activation stimulates interleukin-8 secretion by keratinocytes in vitro.

The protease-activated receptor-2 (PAR-2) is a seven transmembrane domain receptor related to the thrombin receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.

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.

Bromodeoxyuridine incorporation and Ki 67 expression in oral hairy leukoplakia.

OBJECTIVES: Oral hairy leukoplakia (HL) is an acanthotic, hyperparakeratotic lesion characterised by the presence of a replicative Epstein-Barr virus (EBV) infection in the superficial and adjoining layers of the epithelium. EBV or its gene products are capable of modifying epithelial differentiation. The aim of this study was to establish whether the presence of EBV was associated with an alteration in cell turnover by assessing bromodeoxyuridine (BrdU) incorporation and Ki 67 expression in lesional tissue and control mucosa. METHODS: Biopsies of HL together with age, site and sex matched controls (n = 7 and 8 respectively) were incubated in 200 microM BrdU in vitro, fixed in methacarn and processed to paraffin wax. Following acid hydrolysis, incorporated BrdU and Ki 67 were identified in serial 5 microns sections using a three-stage immunoperoxidase technique and cell density expressed as the number of positive cells per mm basement membrane length. RESULTS: Overall, there was no difference in the number of BrdU positive cells per mm basement membrane length between control and HL tissue. However, within HL alone, the presence of focal EBV replication was associated with a significant reduction in the number of basal cells incorporating BrdU compared to adjacent EBV free areas (P < 0.05). There was no significant difference between Ki 67 positive cells in control and HL tissue and no evidence of a reduction of Ki 67 positive cells in areas associated with EBV replication. CONCLUSIONS: These results suggest that there is no evidence of a generalised alteration of the proliferative capacity of basal cells in HL, although the focal reduction in BrdU incorporation may reflect subtle changes on cell turnover by EBV infection.

Receptors for calcitonin gene-related peptide (CGRP) in the rat adrenal cortex.

We previously demonstrated the presence of adrenomedullin receptors in the rat adrenal cortex. There is evidence, however, that the actions of adrenomedullin may also be mediated by the CGRP receptor. The present study was designed to determine whether specific CGRP receptors are present in the rat adrenal cortex. Adrenal glands were, sectioned and immunostained with a primary antibody raised against the first intracellular loop of the CGRP-I receptor. Staining was visualised using alkaline phosphatase and vector red. Immunostaining for the CGRP-I receptor was found in the zona glomerulosa and the adrenal medulla, but not in the inner adrenocortical zones. ACTH treatment caused an increase in staining intensity in the glomerulosa. Ligand binding studies suggested the existence of two populations of CGRP binding sites, one with a Kd of 0.1 nM, the second of 37 nM. Only CGRP-I and adrenomedullin displaced labeled CGRP binding. These results suggest that the CGRP-I receptor is expressed in the adrenal zona glomerulosa and that a second class of binding site is also present. The CGRP-I receptor appears to be regulated by ACTH.

Ki-67 antigen in ameloblastomas: correlation with clinical and histological parameters in 54 cases from Kenya.

The aim of this study was to assess the cell proliferation in ameloblastomas and to correlate this with clinical features and histology. Immunohistochemistry with Ki-67 monoclonal antibody was performed on fresh tissue from 54 ameloblastomas. A labelling index (LI) was calculated by expressing the percentage of Ki-67 positive cells. There was no significant correlation between LI and clinical features: age, sex or tumour size. Follicular ameloblastomas had significantly higher LI (5.0 +/- 0.5; mean +/- SEM) than plexiform tumours (3.2 +/- 0.6; P < 0.05). Plexiform ameloblastomas from the anterior mandible had a significantly lower LI (1.8 +/- 0.5) than those from the posterior (3.9 +/- 0.8; P < 0.05). LI was higher in squamous arcades (6.4 +/- 3.1%) than in epithelial cords and cysts (1.4 +/- 1.3%; P < 0.001). These results suggest that LI correlates most closely with the histological pattern of the epithelium of ameloblastoma, both within and between different tumours.

Human neutrophils secrete vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that plays a pivotal role in mediating neovascularization as well as other endothelial cell alterations during inflammation. In this study, we demonstrate that human neutrophils are a source of VEGF. We observed that isolated blood neutrophils released VEGF in response to different stimuli and we demonstrated by immunohistochemistry that neutrophils infiltrating inflamed tissues contain VEGF. These results indicate that neutrophil-derived VEGF may be instrumental in regulating vascular responses during acute and chronic inflammation.

Recombinant human bone morphogenetic protein-2 promotes wound healing in rat periodontal fenestration defects.

Although there is considerable interest in the use of bone morphogenetic protein (BMP) to promote periodontal regeneration, little is known of its effects on the early stages of wound healing. The aim of this study was to investigate the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) on an early stage of post-operative wound healing and following complete healing (10 and 38 days, respectively) in a rat model of periodontal regeneration. The buccal aspects of molar roots were carefully denuded of their periodontal ligament through a bony window created in the mandibles of Wistar rats under general anesthesia. After the root surfaces were acid-conditioned, a 10-microL quantity of 50 microg/mL rhBMP-2 in a collagen gel solution was placed into the surgically created defect in test animals; in controls, either a 10-microL quantity of only collagen gel was received, or the defect was untreated. Animals were killed 10 days or 38 days after surgery and the tissues processed for histological examination. Transverse 5-microm sections were stained for the identification of new bone, cementum, and collagen fiber formation. In the 10-day study groups, new bone formation over the second molar and beyond the defect was significantly increased in the test group (p < 0.02), although there was no evidence of increased ankylosis. RhBMP-2 stimulated more than twice the area of cementum growth coronally compared with controls (712 +/- 286 microm2 and 258 +/- 57 microm2, respectively). Connective tissue attachment, including the number and width of collagen bundles, was similar in both test and controls. Complete healing without any evidence of ankylosis had occurred in all animals 38 days post-operatively, and no significant differences were observed between test and control groups. In conclusion, a single dose of rhBMP-2 increased the rate of normal intramembranous bone formation and selectively enhanced cementum formation coronally during early wound healing. However, the finding that rhBMP-2 induced bone formation at some distance from the defect suggests the importance of developing a suitable delivery system to maintain the concentration of BMP-2 at the site of implantation for potential therapeutic use.

Epstein-Barr virus: biology and disease.

Epstein-Barr virus is a human herpes virus which, whilst found as a widespread asymptomatic infection, is also associated with certain tumours of lymphoid and epithelial origin including Burkitt's lymphoma (BL), immunoblastic lymphoma (IBL), Hodgkin's Disease (HD) and nasopharyngeal carcinoma (NPC). A unique characteristic of EBV is its ability to infect and transform primary resting B lymphocytes in vitro into permanently growing lymphoblastoid cell lines (LCLs); this effect is associated with constitutive expression of a limited set of viral genes. Interestingly, the pattern of EBV gene expression observed in LCLs in vitro is also a feature of IBLs, a tumour associated with immunosuppression. The other EBV associated tumours display a more restricted pattern of EBV latent protein expression. B cell lines can be activated in vitro into the virus replicative cycle, where a large number of viral genes associated with EBV DNA replication and virus assembly are synthesised. Whilst EBV can be detected in throat washings from seropositive individuals, the only in vivo situation where full virus replication can be reliably observed in hairy leukoplakia (HL), a benign lesion of lingual epithelium frequently found in AIDS patients. Thus, the relative contribution of lymphoid cells and epithelial cells to latent EBV infection/persistence vs replication in vivo remains controversial. Recent studies suggest that HL represents a focus of EBV replication in the absence of a truly latent infection and this supports the contention that EBV persistence resides in the lymphoid compartment. These aspects together with the role of EBV in oral diseases and the effect of certain EBV genes on the control of epithelial cell growth and differentiation will be discussed.

The expression of the Epstein-Barr virus nuclear antigen (EBNA-I) in oral hairy leukoplakia.

OBJECTIVE: Oral hairy leukoplakia (OHL) is characterised by the presence of a replicative Epstein-Barr virus (EBV) in the superficial layers of the epithelium. There is some doubt, however, whether this reflects activation of a latent infection of the basal epithelial cells. EBV latency is associated with the expression of the viral gene product EBNA-I and the aim of this study was to investigate EBNA-I expression in OHL. METHODS: 22 biopsies of clinically suspicious OHL and three cases of normal mucosa were available as fresh frozen or paraffin embedded material. EBNA-I was detected immunocytochemically using a rat monoclonal antibody (IH4-I) following microwave irradiation. Lytic EBV infection was confirmed by the identification of the BZLF-I protein. RESULTS: 16 of the 22 cases displayed focal replicative EBV meeting the criteria for OHL, and in 13 of these, EBNA-I expression was restricted to the nuclei of epithelial cells in the upper layers of the epithelium. EBNA-I expression was absent from the basal cells in all cases and in the nine BZLF-I negative mucosal biopsies. CONCLUSION: These findings suggest that lytic EBV infection in OHL is not the result of activation of a latent infection of basal cells and suggests a role for EBNA-I, not only in latent EBV infection, but also in virus replication.

Oral mucosal Langerhans' cells.

Langerhans' cells (LC) are dendritic, antigen-presenting cells present within the epithelium of skin and mucosa, including that of the oral cavity. This article reviews the literature on the phenotypic and functional features of oral mucosal Langerhans' cells, and speculates on other aspects by extrapolating from data on their epidermal counterparts.

Langerhans cell density in normal human oral mucosa and skin: relationship to age, smoking and alcohol consumption.

The immune function of skin has been studied extensively and it has been suggested that epidermal Langerhans cell (LC) density and function decreases with increasing age. Little is known, however, about the effect of age on oral mucosal LC. Cryostat sections from biopsies of buccal mucosa, lip, hard palate, lateral border of tongue, floor of mouth and abdominal skin, obtained from 91 subjects (aged 16-96 yr), were reacted immunocytochemically with a monoclonal antibody against CD1a and then LC density was expressed as LC/mm epithelial surface length. No significant effect of age on mucosal or skin LC density was found, whilst a history of smoking was associated with an increase in LC density in lateral border of tongue and in biopsies of labial mucosa taken from men (P < 0.05). There was no significant difference between LC density in men and women in oral mucosa. Oral mucosal LC may therefore form a relatively stable population in the adult and thus the increased incidence of mucosal disease in the elderly may be the result of subtle changes in cell mediated immune function rather than changes in LC density.

Langerhans cell distribution and keratinocyte expression of HLADR in oral lichen planus.

Keratinocyte expression of the Class II major histocompatibility complex antigen HLADR, is seen in several inflammatory disorders of skin and mucosa, including lichen planus. The purpose of this study is to determine whether the distribution of Langerhans cells and their expression of CD4 in oral lichen planus is related to keratinocyte HLADR. The numbers of CD1- and CD4-positive Langerhans cells were compared in areas of keratinocyte HLADR and areas showing no expression in oral lichen planus and with normal oral mucosa. Cells were identified using an immunoalkaline phosphatase technique and numbers were expressed per mm epithelial surface length. In lichen planus, an increase both in the number of Langerhans cells and the numbers expressing CD4 were found in areas of keratinocyte HLADR expression compared with HLADR negative areas and with normal oral mucosa. There was no difference in the numbers of Langerhans cells or their expression of CD4 between HLADR-negative areas in LP and normal oral mucosa. These results show that the distribution of Langerhans cells is related to keratinocyte expression of HLADR and suggest that Langerhans cell entry may be enhanced in these areas. Whilst it is possible this enhancement is mediated by CD4/HLADR interaction, other molecules are also likely to be important in controlling Langerhans cell entry into oral mucosa.