A macroscopic and histological analysis of radiographically well-defined deep and extremely deep carious lesions: carious lesion characteristics as indicators of the level of bacterial penetration and pulp response.

AIM: To investigate the relationship between radiographically and macroscopically well-defined carious lesions and the dentine-pulp complex with regard to: (i) level of bacterial penetration; (ii) inflammatory status including the presence of hyperplastic pulp stroma; and (iii) formation of hard and/or ectopic connective tissue. METHODOLOGY: The material comprised 68 untreated cavitated permanent teeth divided into well-defined radiographic categories based on the lesion penetration depth: (i) deep lesions ( ≥3/4 of the dentine thickness with a radio-dense zone separating the lesion from the pulp) and (ii) extremely deep lesions (the carious lesion penetrated the entire thickness of the dentine, without a radio-dense zone). After extraction, the teeth were processed for histology. The material was scored with regard to coronal breakdown, macroscopic variables describing caries activity and histological variables describing the dentine-pulp complex (bacteria, inflammatory infiltrate, partial pulp necrosis, hyperplastic changes and hard tissue/ectopic presence of connective tissue). Interrater agreement was assessed using Cohen's kappa. Associations between variables were assessed using Pearson's chi-squared or Fisher's exact test. The effect size was reported by odds ratio (OR) and associated 95% confidence interval (CI). Level of significance was set to 5%. RESULTS: There were significant associations between a closed environment (1-2 surfaces involved) and the presence of biofilm, retrograde demineralization and light-coloured demineralized dentine. Whereas radiographically defined deep lesions tended to have bacteria only in the primary dentine (P < 0.001, OR = 20.55, 95% CI [4.44, 107.89]), extremely deep carious lesions tended to have bacteria in contact with the pulpal tissue (P = 0.007, OR = 6.84, 95% CI [2.00, 62.83]), presence of an inflammatory infiltrate (Fisher's exact; P < 0.001) and partial pulp necrosis. Hyperplastic pulps were seen only in extremely deep lesions. CONCLUSIONS: Unlike deep lesions, extremely deep carious lesions were often associated with severe pulp inflammation and infection. A radiographic threshold between deep and extremely deep lesions is suggested as indicator of the bacterial penetration level and the severity of the pulpal response prior to intervention.

Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV). METHODS: Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature. RESULTS: One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%. CONCLUSION: MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.

Consumer available permanent hair dye products cause major allergic immune activation in an animal model.

Background p-Phenylenediamine (PPD) and related substances are ingredients of more than two-thirds of oxidative (permanent) hair dyes currently used. Although PPD is a potent skin sensitizer in predictive assays, the extent to which permanent hair dyes sensitize humans has been questioned due to the in-use conditions, e.g. the presence of couplers in the hair dye gel and rapid oxidation using a developer. Objectives To study the skin sensitizing potential of permanent hair dyes in mice. Methods Two different permanent hair dye products containing PPD were studied in CBA mice using a modified version of the local lymph node assay. The colour gel and developer (oxidant) were tested separately and in combination. Response was measured by ear swelling and cytokine production in ear tissue and serum by enzyme-linked immunosorbent assay. The immune cellular response in the draining lymph nodes was analysed by flow cytometry. Results Application of the colour gel both alone and mixed with the developer induced skin production of interleukin (IL)-1beta, tumour necrosis factor-alpha and IL-6 as well as systemic IL-6 release. Both treatments induced B- and T-cell infiltration as well as T-cell proliferation within the draining lymph nodes. Treatment with the mixture induced at least 20% more skin inflammation, cytokine production and CD4+ T-cell activation compared with the colour gel alone. Conclusions Consumer available PPD-containing permanent hair dyes can be potent and rapid immune activators. Mixing the colour gel and developer (oxidant) increased the induction of skin inflammation compared with application of the colour gel alone.

Repeated exposure to hair dye induces regulatory T cells in mice.

BACKGROUND: We have recently shown that commercial p-phenylenediamine (PPD)-containing hair dyes are potent immune activators that lead to severe contact hypersensitivity in an animal model. However, only a minority of people exposed to permanent hair dyes develops symptomatic contact hypersensitivity. This suggests that the majority of people exposed to hair dyes does not become sensitized or develop immunological tolerance. OBJECTIVES: To study the immune response in mice repeatedly exposed to PPD-containing hair dye in a consumer-like manner. METHODS: A commercial hair dye containing PPD was tested in C57BL/6 mice. The local immune response was measured by ear swelling and by histological examinations. The immune response in the draining lymph nodes was analysed by flow cytometry. RESULTS: The hair dye induced local inflammation as seen by swelling and cell infiltration of the treated ears. In addition, exposure to hair dye caused T-cell activation as seen by T-cell proliferation and production of interferon-γ and interleukin (IL)-17 within the draining lymph nodes. The inflammatory response peaked at the fourth exposure to hair dye. From this point on, an upregulation of regulatory T cells and IL-10-producing cells was seen. CONCLUSIONS: This study shows that PPD-containing hair dyes strongly affect the immune system. In addition to being potent skin sensitizers that activate inflammatory T cells, hair dyes also induce anti-inflammatory mechanisms. This might explain why many consumers can use hair dyes repeatedly without developing noticeable allergies, but it also raises the question whether the immune modulatory effects of hair dyes might influence the development of autoimmune diseases and cancers.

HGF is released from buccal fibroblasts after smokeless tobacco stimulation.

To investigate the effect of smokeless tobacco (ST) on (1) HGF, KGF and GM-CSF expression by buccal fibroblasts and (2) on keratinocyte and fibroblast proliferation. Buccal fibroblasts were stimulated with different concentrations of ST extracts in a double dilution from 0.50% w/v to 0.03% w/v. Supernatant was collected after 24/48/72/96 h and assayed for HGF, KGF, and GM-CSF by ELISA. The amount of RNA was used as an indicator of fibroblast cell number. Buccal epithelial cell proliferation was determined by CyQUANT proliferation assay. The amount of HGF and KGF in the supernatant was dependent on exposure time and on concentration of the tobacco extract. High concentration increased production of HGF 4-fold. KGF production was doubled when high concentration of tobacco was used, low concentration did not stimulate cells. GM-CSF production was low in both stimulated and non-stimulated cells. Keratinocytes and fibroblasts showed no increase in proliferation after stimulation with increased concentrations of ST. The results suggest that HGF and KGF may play an important role as a paracrine growth factor in epithelial hyperplasia in ST lesions.

Proliferation and mitogenic response to PDGF-BB of fibroblasts isolated from chronic venous leg ulcers is ulcer-age dependent.

Several pathophysiologic mechanisms have been proposed to explain slow-healing leg ulcers, but little is known about the growth behavior of cells in these wounds. Platelet-derived growth factor-BB applied topically to chronic wounds has shown beneficial effects, although the effects have been less pronounced than would have been expected based on studies on acute wounds. The objective of this study was to compare fibroblasts in culture obtained from chronic wounds (non-healing chronic venous leg ulcers), acute wounds and normal dermis regarding growth, mitogenic response to platelet-derived growth factor-BB and levels ofplatelet-derived growth factor alpha-receptor and beta-receptor. Fibroblasts were obtained by an explant technique and expanded in vitro using fibroblast growth medium supplemented with 10% fetal bovine serum and used for the assays at their third passage. Growth of chronic wound fibroblasts (n = 8) was significantly (p < 0.05) decreased compared with those from acute wounds (n = 10) and normal dermis (n = 5). Fibroblasts from ulcers older than 3 y grew significantly (p < 0.01) slower than those from ulcers that had been present for less than 3 y. Morphology and size of fibroblasts from the oldest chronic wounds deviated substantially from those of acute wounds and normal dermis, and resembled in vitro aged or senescent fibroblasts. Mitogenic response of chronic wound fibroblasts to human recombinant platelet-derived growth factor-BB was also reduced with ulcer age. No significant differences were found in the amount of either platelet-derived growth factor alpha-receptor or beta-receptor among the three groups. The features decreased growth related to ulcer age, altered morphology, and reduced response to platelet-derived growth factor, indicating that fibroblasts in some chronic wounds have approached or even reached the end of their lifespan (phase III). This might provide one explanation for the non-healing state and therapy resistance to topical platelet-derived growth factor-BB of some venous leg ulcers.

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts.

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa, from gingiva, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal nature of fibroblasts. Cells were first cultured in DMEM with 0.5% fetal calf serum (FCS) and then incubated for 8 h, and 72 h in fresh DMEM with 10% FCS. Total RNA was isolated and used for Northern blotting with a P32-labeled KGF cDNA probe. Total RNA from cultured keratinocytes was used as negative controls. KGF mRNA was found in all cultured fibroblasts. Upon addition of 10% FCS to the cell cultures, an increase in KGF mRNA levels was noticed especially after 72 h. Furthermore, RT-PCR analysis of material scraped from the tooth root surface indicated the presence of KGF mRNA even in noncultured periodontal ligament cells.