Apoptosis in the epithelial cells of the rests of Malassez of the periodontium of rat molars

Background and Objectives: Epithelial rests of Malassez are clusters of cells derived from Hertwig's root sheath that remain in the periodontal ligament throughout life. Although it is known that the cells of Malassez proliferate, there are no studies showing that they undergo programmed cell death, i.e. apoptosis. In most tissues, proliferation is balanced by apoptosis. Thus we examined regions of the periodontium of young and adult rat molars in the hope of detecting apoptosis.Methods: Wistar rats aged 29, 45 and 120 days were killed with chloral hydrate (600 mg/kg). Fragments containing maxillary molars were removed and fixed in formaldehyde, decalcified, and embedded in paraffin and glycol methacrylate. Sections were stained with hematoxylin/eosin and the Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) method for detection of apoptosis. Specimens were also fixed in glutaraldehyde-formaldehyde, decalcified and processed for transmission electron microscopy.Results: Epithelial rests of Malassez containing round/ovoid basophilic dense bodies and TUNEL-positive structures were found in all specimens examined. Ultrastructural examination revealed that some cells of Malassez contained masses of condensed peripheral chromatin and a shrunken cytoplasm exhibiting intact organelles - images typical of apoptosis. Moreover, round/ovoid electron-opaque structures appeared to be in the process of being engulfed by neighboring epithelial cells of Malassez.Conclusions: Our results demonstrate that epithelial cells of Malassez's rests undergo apoptosis in the developing and adult periodontium. Apoptosis may, together with proliferation, be part of the mechanism of turnover/remodelling of the cells of Malassez.

Monoclonal antibodies against the 43,000 da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis

The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin, Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo. We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen. MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction, Results suggest that P. brasiliensis propagules may start infection in man by strongly adhering to human lung cells, Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity, Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM. Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD 147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis. (C) 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Ultrastructural changes on the epithelial cells of uterine tubes of Wistar rats after chronic ethanol ingestion

The present paper describes the morphological alterations of the epithelial layer of the uterine tubes of rats submitted to experimental chronic alcoholism using anatomical, histological, ultrastructural and morphometric methods. Sixty adult rats (Rattus norvegicus albinus) at the same age (3 months) and with a mean body weight of 228 g were divided into two groups. The control group received solid diet (Purina rat chow) and tap water ad libitum. The alcoholic group received the same solid diet and was allowed to drink only sugar cane brandy dissolved in 30° Gay Lussac (v/v). After periods of 90, 180 and 270 days of treatment animals at normal estrus were anaesthetised with ethyl ether, weighed and sacrificed. Subsequently, the uterine tubes were dissected, weighed and prepared for TEM and SEM methods. The final mean body weights were similar in the control and alcoholic groups. The morphometric analysis showed no difference between control and alcoholic epithelial height. The alcoholic animals showed ultrastructural alterations: intense lipid droplet and lysosomes accumulation, dilated rough endoplasmic reticulum cisternae and vacuolization in both periods of treatment. It was concluded that alcohol acts as a toxin on the epithelial layer of the uterine tubes of rats.

Biostimulatory effects of low-level laser therapy on epithelial cells and gingival fibroblasts treated with zoledronic acid

Low-level laser therapy (LLLT) has been considered as an adjuvant treatment for bisphosphonate-related osteonecrosis, presenting positive clinical outcomes. However, there are no data regarding the effect of LLLT on oral tissue cells exposed to bisphosphonates. This study aimed to evaluate the effects of LLLT on epithelial cells and gingival fibroblasts exposed to a nitrogen-containing bisphosphonate - zoledronic acid (ZA). Cells were seeded in wells of 24-well plates, incubated for 48 h and then exposed to ZA at 5 μM for an additional 48 h. LLLT was performed with a diode laser prototype - LaserTABLE (InGaAsP - 780 nm ± 3 nm, 25 mW), at selected energy doses of 0.5, 1.5, 3, 5, and 7 J cm-2 in three irradiation sessions, every 24 h. Cell metabolism, total protein production, gene expression of vascular endothelial growth factor (VEGF) and collagen type I (Col-I), and cell morphology were evaluated 24 h after the last irradiation. Data were statistically analyzed by Kruskal-Wallis and Mann-Whitney tests at 5% significance. Selected LLLT parameters increased the functions of epithelial cells and gingival fibroblasts treated with ZA. Gene expression of VEGF and Col-I was also increased. Specific parameters of LLLT biostimulated fibroblasts and epithelial cells treated with ZA. Analysis of these in vitro data may explain the positive in vivo effects of LLLT applied to osteonecrosis lesions. © 2013 Astro Ltd.

Zoledronic acid decreases gene expression of vascular endothelial growth factor and basic fibroblast growth factor by human epithelial cells

Delayed wound healing in patients taking bisphosphonates could result from decreased expression of growth factors, which are directly related to cell proliferation and migration. In this study, we evaluated the gene expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by epithelial cells exposed to zoledronic acid 5 μmol for 48 h using real-time polymerase chain reaction. The gene expression of VEGF and bFGF by epithelial cells exposed to zoledronic acid decreased by 34% and 51%, respectively (p = 0.0001 and p = 0.0001). We conclude that zoledronic acid can decrease the expression of growth factors by epithelial cells. © 2013 The British Association of Oral and Maxillofacial Surgeons.

Phenotypic and metabolic aspects of prostatic epithelial cells in aged gerbils after antisteroidal therapy: Turnover in the state of chromatin condensation and androgen-independent cell replacement

The gerbil is a rodent considered a good model for studies of prostatic morphophysiology under different experimental conditions. Studies involving castration and steroidal blockers of aged gerbils showed that the glandular epithelium persists after long-term therapy, preventing the organ atrophy. Thus, the objective of this study was to evaluate the phenotypic characteristics and behavior of prostatic epithelial cells that remained after different periods of hormone ablation in aged gerbils. The identification of elements that influenced the survival of this cell type was performed by morphometric, nuclear phenotypes, ultrastructural and immune histochemical analysis. The most significant responses to treatment, by analyzing morphometric features, were observed during the first three time points (day 1, day 3, and day 7), after which there appeared to be an adjustment of the gland to the hormone ablation. All treatments led to changes in the state of chromatin condensation, DNA methylation pattern and phenotypic changes indicated cell senescence. Additionally, an increase in the basal cells seemed to guarantee self-renewal properties to the epithelium. These data indicate that changes occur at many levels, including gene expression and nuclear architecture in the epithelial cells, when aging and steroidal blockade are associated. These aspects are important when considering castration-resistant prostate cancer, a malignant tumor posing difficult therapeutic intervention. © 2013 Elsevier GmbH. All rights reserved.

Candida spp. adherence to oral epithelial cells and levels of IgA in children with orthodontic appliances

Adhesion and colonization of the oral cavity by Candida albicans is an initial step in candidosis. Orthodontic and other oral appliances seem to favor candidal presence. The aim of this work was to compare the presence of Candida species in saliva, their adherence to oral epithelial cells, and the levels of anti-C. albicans IgA in children with or without orthodontic appliances. This study included 30 children 5 to 12 years old (9.1 ± 1.7 years old) who were users of removable orthodontic devices for at least 6 months and 30 control children of similar ages (7.7 ± 1.5 years old). The presence of yeast species in the saliva was evaluated by microbiological methods. Candida species were identified using phenotypic methods. Anti-C. albicans IgA levels in saliva were analyzed by ELISA. The yeasts adhering to oral epithelial cells were assessed by exfoliative cytology. No statistically significant differences were observed for saliva yeast counts and anti-C. albicans IgA levels between the studied groups. Children with orthodontic devices exhibited more yeast cells adhering to oral epithelial cells and a higher percentage of non-albicans species relative to the control group. In conclusion, orthodontic appliances may favor the adherence of Candida to epithelial cells but do not influence the presence of these yeasts in saliva, and the levels of anti-C. albicans IgA do not correlate with yeast adherence or presence of Candida in the oral cavity

AKT and AMPK Activation after High-Fat and High-Glucose In Vitro Treatment of Prostate Epithelial Cells

Considering the increasing consumption of saturated fat and glucose in diets worldwide and its possible association to carcinogenesis, this investigation analysed the proliferation profile of nonmalignant human prostate epithelial cells after exposure to elevated levels of fat and glucose. PNT1A cells were cultured with palmitate (100 or 200 mu M) and/or glucose (450mg/dl) for 24 or 48 h. Treated cells were evaluated for viability test and cell proliferation (MTS assay). AKT and AMPK phosphorylation status were analysed by Western blotting. After 24 h of high-fat alone or associated with high-glucose treatment, there was an increase in AMPK and AKT activation associated to unchanged MTS-cell proliferation. Following 48 h of high-fat but not high-glucose alone, cells decreased AMPK activation and maintained elevated AKT levels. These data were associated to increased cell proliferation after further high-fat treatment. After longer high-fat exposure, MTS revealed that cells remained proliferating. High-glucose alone or associated to high-fat treatment was not able to increase cell proliferation and AKT activation. A high-fat medium containing 100 mu M of palmitate stimulates proliferation in PNT1A cells by decreasing the activation of AMPK and increasing activation of AKT after longer exposure time. These findings improve the knowledge about the negative effect of high levels of this saturated fatty acid on proliferative disorders of prostate.

Adhesion of Candida biofilm cells to human epithelial cells and polystyrene after treatment with silver nanoparticles

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Subinhibitory Concentrations of Triclosan Promote Streptococcus mutans Biofilm Formation and Adherence to Oral Epithelial Cells

Triclosan is a general membrane-active agent with a broad-spectrum antimicrobial activity that is commonly used in oral care products. In this study, we investigated the effect of sub-minimum inhibitory concentrations (MICs) of triclosan on the capacity of the cariogenic bacterium Streptococcus mutans to form biofilm and adhere to oral epithelial cells. As quantified by crystal violet staining, biofilm formation by two reference strains of S. mutans was dose-dependently promoted, in the range of 2.2- to 6.2-fold, by 1/2 and 1/4 MIC of triclosan. Observations by scanning electron microscopy revealed the presence of a dense biofilm attached to the polystyrene surface. Growth of S. mutans in the presence of triclosan at subMICs also increased its capacity to adhere to a monolayer of gingival epithelial cells. The expression of several genes involved in adherence and biofilm formation in S. mutans was investigated by quantitative RT-PCR. It was found that subMICs of triclosan significantly increased the expression of comD, gtfC, and luxS, and to a lesser extent of gtfB and atlA genes. These findings stress the importance of maintaining effective bactericidal concentrations of therapeutic triclosan since subMICs may promote colonization of the oral cavity by S. mutans.

Synergistic Anti-Inflammatory Activity of the Antimicrobial Peptides Human Beta-Defensin-3 (hBD-3) and Cathelicidin (LL-37) in a Three-Dimensional Co-Culture Model of Gingival Epithelial Cells and Fibroblasts

Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 mu M) and LL-37 (0.1 and 0.2 mu M) individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.