Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell death evaluation in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17

The incidence of apoptosis and nuclear instability, including the incidence of catastrophic death, were investigated in benzo[a]pyrene (BP)-transformed human breast epithelial cells (BP1-E cell line) after microcell-mediated transfer of chromosomes 11 and 17. Since the introduction of normal chromosomes 11 and 17 into tumorigenic human breast BP1-E cells reverts some of these cells' characteristics (especially those affected by microsatellite instabilities and loss of heterozygosity) those of parental non-transformed MCF-10F cells, it was expected that the cell death rates would also be affected by this treatment. The transfer of the mentioned chromosomes, especially chromosome 17, to tumorigenic BP1-E cells increased the apoptotic ratios and decreased the nuclear instability ratios, thus showing that the microsatellite instability and loss of heterozygosity induced by BP in these chromosomes of MCF-10F cells affect the control of cell death mechanisms. (C) 2003 Elsevier B.V. All rights reserved.

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.

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

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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.

Cytotoxic Effects of Zoledronic Acid on Human Epithelial Cells and Gingival Fibroblasts

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

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.

Salivary epithelial cells as model to study immune response against cutaneous pathogens

The human skin not only provides passive protection as a physical barrier against external injury, but also mediates active surveillance via epidermal cell surface receptors that recognize and respond to potential invaders. Primary keratinocytes and immortalized cell lines, the commonly used sources to investigate immune responses of cutaneous epithelium are often difficult to obtain and/or potentially exhibit changes in cellular genetic make-up. Here we investigated the possibility of using salivary epithelial cells (SEC) to evaluate the host response to cutaneous microbes. Elevated secretion of IFN-γ and IL-12 was observed in the SEC stimulated with Staphylococcus aureus, a transient pathogen of the skin, as mono species biofilm as compared to SEC stimulated with a commensal microbe, the Staphylococcus epidermidis. Co-culture of the SEC with both microbes as dual species biofilm elicited maximum cytokine response. Stimulation with S. aureus alone but not with S. epidermidis alone induced maximum toll-like receptor-2 (TLR-2) expression in the SEC. Exposure to dual species biofilm induced a sustained upregulation of TLR-2 in the SEC for up to an hour. The data support novel application of the SEC as efficient biospecimen that may be used to investigate personalized response to cutaneous microflora. © 2013 Wiley Periodicals, Inc.

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.

Effect of LPS treatment on the viability and chemokine synthesis by epithelial cells and gingival fibroblasts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Green tea polyphenol epigallocatechin-3-gallate and cranberry proanthocyanidins act in synergy with cathelicidin (LL-37) to reduce the LPS-induced inflammatory response in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts

Objectives: The human antimicrobial peptide cathelicidin (LL-37) possesses anti-inflammatory properties that may contribute to attenuating the inflammatory process associated with chronic periodontitis. Plant polyphenols, including those from cranberry and green tea, have been reported to reduce inflammatory cytokine secretion by host cells. In the present study, we hypothesized that A-type cranberry proanthocyanidins (AC-PACs) and green tea epigallocatechin-3-gallate (EGCG) act in synergy with LL-37 to reduce the secretion of inflammatory mediators by oral mucosal cells. Methods: A three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts treated with non-cytotoxic concentrations of AC-PACs (25 and 50 mg/ml), EGCG (1 and 5 mg/ml), and LL-37 (0.1 and 0.2 mM) individually and in combination (AC-PACs + LL-37 and EGCG + LL-37) were stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). Multiplex ELISA assays were used to quantify the secretion of 54 host factors, including chemokines, cytokines, growth factors, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Results: LL-37, AC-PACs, and EGCG, individually or in combination, had no effect on the regulation of MMP and TIMP secretion but inhibited the secretion of several cytokines. ACPACs and LL-37 acted in synergy to reduce the secretion of CXC-chemokine ligand 1 (GRO-a), granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6), and had an additive effect on reducing the secretion of interleukin-8 (IL-8), interferon-g inducible protein 10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) in response to LPS stimulation. EGCG and LL-37 acted in synergy to reduce the secretion of GRO-a, G-CSF, IL-6, IL-8, and IP-10, and had an additive effect on MCP-1 secretion. Conclusion: The combination of LL-37 and natural polyphenols from cranberry and green tea acted in synergy to reduce the secretion of several cytokines by an LPS-stimulated 3D coculture model of oral mucosal cells. Such combinations show promising results as potential adjunctive therapies for treating inflammatory periodontitis.

Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus' transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.

MicroRNA-100 Acts as a Tumor Suppressor in Human Bladder Carcinoma 5637 Cells

Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. Better understanding of the factors involved in disease pathogenesis would provide insights for the development of more effective strategies in treatment. Recently, differential miRNA expression profiles in bladder urothelial carcinomas identified miR-100 down-regulation and miR-708 up-regulation among the most common alterations, although the possible influence of these miRNAs in the control of basic mechanisms in bladder tumors has not been addressed. In this context, the present study aimed to evaluate the in vitro effects of miR-100 forced expression and miR-708 inhibition in the bladder carcinoma cell line 5637. Our results showed that overexpression of miR-100 significantly inhibited growth when compared to controls at both times tested (72 and 96 hours, p<0.01) with a maximum effect at 72 hours reducing proliferation in 29.6 %. Conversely, no effects on cell growth were observed after inhibition of miR-708. MiR-100 also reduced colony formation capacity of 5637 cells by 24.4%. No alterations in cell cycle progression or apoptosis induction were observed. The effects of miR-100 on growth and clonogenicity capacity in 5637 cells evince a possible role of this miRNA in bladder carcinoma pathogenesis. Further studies are necessary to corroborate our findings and examine the potential use of this microRNA in future therapeutic interventions.

Bacteria-derived hydrogen sulfide promotes IL-8 production from epithelial cells

Hydrogen Sulfide (H(2)S) a volatile Sulfur compound, is implicated as a cause of inflammation. especially when it is produced by bacteria colonizing gastrointestinal organs However, It IS Unclear if H(2)S produced by periodontal pathogens affects the inflammatory responses mediated by oral/gingival epithelial cells Therefore. the aims of this Study were (1) to compare the in vitro production of H(2)S among. 14 strains of Oral bacteria and (2) to evaluate the effects of H(2)S on inflammatory response induced in host oral/gingival epithelial cells Porphyromonas gingivalis (Pg) produced the most H(2)S in Culture, Which, in turn resulted in the promotion of proinflammatory cytokine IL-8 from both gingival and Oral epithelial cells The up-regulation of IL-8 expression was reproduced by the exogenously applied H(2)S Furthermore. the Mutant Strains of Pg that do not produce major Soluble Virulent factors. ie gingival, still showed the Production of H(2)S. as well as the promotion of epithelial IL-8 production. which was abrogated by H(2)S scavenging reagents These results demonstrated that Pg produces a concentration of H(2)S capable of Up-regulating-IL-8 expression induced in gingival and oral epithelial cells, revealing a possible mechanism that may promote the inflammation in periodontal disease (C) 2009 Elsevier B.V. All rights reserved