RESUMO
AIMS: This study aimed to investigate the effect of synthetic antimicrobial peptide dermaseptin-S1 (DS1) (ALWKTMLKKLGTMALHAGKAALGAADTISQGTQ) on the growth of Candida albicans, its transition from blastospore to hyphae, and its biofilm formation. We also analysed the expression of different genes (HWP1 and SAPs) involved in C. albicans virulence. METHODS AND RESULTS: Using cell count we showed that in addition to decreasing C. albicans growth, peptide DS1 inhibited its transition from blastospore to hyphal form. These effects are comparable to those obtained with amphotericin B (AmB). Electron microscopy analyses showed that C. albicans cells treated with either DS1 or AmB displayed a distorted cell wall surface, suggesting that the effect of DS1 was similar to that of AmB on C. albicans cell membrane structure. These observations were confirmed by our results with biofilms showing that both DS1 peptide and AmB significantly inhibited biofilm formation after 2 and 4 days. The effect of DS1 on C. albicans growth, transition and biofilm formation may occur through gene modulation, as the expression of HWP1, SAP1, SAP2, SAP3, SAP9 and SAP10 genes involved in C. albicans pathogenesis were all downregulated when C. albicans was treated with DS1. CONCLUSIONS: DS1 inhibits the growth and hyphal transition of C. albicans. DS1 was also able to decrease the expression of and gene expression of hyphal wall protein 1 and aspartic proteases genes by C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: These data provide new insight into the efficacy of DS1 against C. albicans and its potential for use as an antifungal therapy.
Assuntos
Proteínas de Anfíbios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Anfotericina B/farmacologia , Antibacterianos/farmacologia , Ácido Aspártico Proteases/genética , Biofilmes/crescimento & desenvolvimento , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/ultraestrutura , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Virulência/genéticaRESUMO
BACKGROUND: Bronchial fibroblasts play a key role in airway remodelling in asthma. They regulate epithelial cell functions such as proliferation through growth factors, cytokines, chemokines and exosomes. The role of exosomes in the communication between epithelial cells and fibroblasts by vehiculing these mediators in asthma remains to be determined. OBJECTIVE: To evaluate the role of exosomes released by bronchial fibroblasts on epithelial cell proliferation in severe asthma. METHODS: Exosomes were obtained from culture media of primary bronchial fibroblasts and characterized using Western blot, electron microscopy and flow cytometry. Uptake profile of fluorescent-labelled exosomes in epithelial cells was assessed by flow cytometry. Exosome cytokine content was analysed by Cytokine Arrays. Bronchial epithelial cell proliferation was evaluated by BrdU incorporation test. Exosome biogenesis/release was blocked using sphingomyelinase inhibitor. Plasmid transfection was used to modulate transforming growth factor beta 2 (TGF-ß2) gene expression. RESULTS: We showed that bronchial fibroblasts secreted exosomes, which were internalized by bronchial epithelial cells. Exosomes of severe asthmatic subjects' fibroblasts showed a lower level of TGF-ß2 and significantly increased the epithelial cell proliferation of both healthy and severe asthmatic subjects compared to healthy controls' exosomes. Overexpression of TGF-ß2 in severe asthmatics' fibroblasts induced enhanced TGF-ß2 in exosomes leading to a reduced proliferation of epithelial cells, whereas knockdown of TGF-ß2 enhanced epithelial cell proliferation. CONCLUSION: Our study shows that exosomes are involved in fine-tuning intercellular communication in asthma. Exosomes of severe eosinophilic asthmatics' fibroblasts can contribute to airway remodelling, at least in part, by modulating epithelial cell proliferation observed in severe asthma.
Assuntos
Asma/diagnóstico , Asma/metabolismo , Exossomos/metabolismo , Fibroblastos/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Adulto , Idoso , Asma/imunologia , Biomarcadores , Estudos de Casos e Controles , Proliferação de Células , Citocinas/metabolismo , Eosinófilos/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Índice de Gravidade de Doença , Adulto JovemRESUMO
BACKGROUND AND OBJECTIVE: The gingiva is the first oral tissue directly exposed to cigarette smoke (CS). Exposure to CS compromises the structure and function of gingival tissue. Damaging or altering the gingival epithelium leads to a compromised protective barrier of the periodontium, resulting in several diseases. The aim of this study was to assess the effect of repeated exposure to CS on gingival epithelial cell growth and on expression of apoptotic protein and keratin. MATERIAL AND METHODS: Primary human gingival epithelial cells were seeded on a collagen scaffold for 5 d to allow growth and stratification. The cells were then exposed for 5 min to whole CS for 3, 6 and 9 d. At the end of each exposure period, cell proliferation [using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays], gene expression [by quantitative reverse transcription polymerase chain reaction (qRT-PCR)] and protein production (by western blot analysis) were investigated. RESULTS: Higher metabolic activity was found in the CS-exposed cells than in the nonexposed cells, specifically after 3 and 6 d of exposure to CS. At 9 d there was no significant difference between CS-exposed and nonexposed cells. Metabolic activity was supported by the BrdU cell-proliferation analyses, which showed increased cell growth at 3 d compared with the control. However, at 6 and 9 d, cell proliferation in the CS-exposed culture was comparable to that in the nonexposed culture. Interestingly, the Bax/Bcl-2 protein ratios decreased with increased CS exposure, suggesting cell resistance. Moreover, protein analyses showed that CS decreased expression of keratin(K) 5 at 3, 6 and 9 d, and increased expression of K14 at 6 and 9 d. Finally, mRNA analyses showed significant decreases of K1, K6, K10 and K16 in CS-exposed cultures, correlating, at times, with a decrease of protein production. CONCLUSION: CS was shown to increase epithelial cell proliferation, which may involve cell resistance to apoptosis. This is supported by the modulation of expression of different keratin genes and proteins. Altogether, these data may explain the hyperplasia reported in gingival tissue, as well as periodontal disease, in smokers.
Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Gengiva/citologia , Gengiva/crescimento & desenvolvimento , Queratinas/metabolismo , Nicotiana/efeitos adversos , Fumaça/efeitos adversos , Adolescente , Adulto , Apoptose/efeitos dos fármacos , Bromodesoxiuridina , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/análise , Sais de Tetrazólio , Tiazóis , Fatores de Tempo , Adulto Jovem , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND AND OBJECTIVE: Periodontitis, a disease associated with chronic inflammation, results in significant destruction of periodontal tissues. Uncontrolled, periodontal disease negatively affects general patient health. We sought to evaluate the effect of α-tocopherol on gingival fibroblast behavior following exposure to Porphyromonas gingivalis lipopolysaccharide (LPS). MATERIAL AND METHODS: Primary human gingival fibroblasts were cultured for 24 and 48 h with α-tocopherol at various concentrations (0, 50, 100 and 200 µm) in the presence or absence of 1 µg/mL of LPS. At the end of each time point, cell adhesion and growth were evaluated by means of optical microscope observations and MTT assay. The secretion levels of cytokines interleukin (IL)-1ß and IL-6 and human ß-defensins 1 and 2 were measured by specific enzyme-linked immunosorbent assay. Finally, an in vitro scratch wound assay was performed to investigate the effect of α-tocopherol on fibroblast migration. RESULTS: α-tocopherol alone had no adverse effect on cell adhesion and morphology. Fibroblast proliferation increased in the presence of α-tocopherol with and without LPS. α-tocopherol alone had no effect on inflammatory cytokine (IL-1ß and IL-6) secretion. Interestingly, following cell exposure to P. gingivalis LPS, α-tocopherol significantly (p < 0.01) decreased the secretion of these two cytokines and increased human ß-defensin-1 and -2 secretion. Finally, α-tocopherol increased the healing rate of the gingival fibroblasts from 12 h up to 48 h. CONCLUSION: These results suggest that α-tocopherol may play an active role in countering the damaging effect of LPS by reducing inflammatory cytokines, increasing ß-defensins and promoting fibroblast growth, migration and wound healing.
Assuntos
Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Interleucina-1beta/efeitos dos fármacos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Porphyromonas gingivalis/metabolismo , alfa-Tocoferol/farmacologia , beta-Defensinas/metabolismo , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Fibroblastos/citologia , Gengiva/citologia , Gengiva/metabolismo , Humanos , Doenças Periodontais/metabolismo , Doenças Periodontais/microbiologia , Periodontite/metabolismo , Periodontite/microbiologia , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVE: Smoking cigarettes increases the risk of oral tissue damage leading to periodontal disease. Gingival fibroblasts, the predominant cell type inhabiting gingival connective tissue, play a critical role in remodeling and maintaining gingival structure. The objective of this study was to investigate the effect of long-term exposure to cigarette smoke on human gingival fibroblast survival/apoptosis and the molecular pathways involved in these cell responses. MATERIAL AND METHODS: Human gingival fibroblasts were extracted from healthy non-smokers and cultured in the presence of cigarette smoke condensate (CSC). At the end of each time point, cell growth was evaluated by means of MTT assay. Apoptotic and necrotic gene's expression was investigated by polymerase chain reaction array and by annexin V/propidium iodide staining and cell cycle assays. Western blot was used to investigate Bax and p53 proteins. These tests were supported by caspase 3 activity analyses. RESULTS: High levels of CSC decreased cell growth and deregulated cell cycle progression by increasing the G(0)/G(1) and reducing the S and G(2)/M phases of the gingival fibroblasts. Polymerase chain reaction arrays revealed the activation of several apoptotic genes by CSC, including TNF receptors, caspases, Bax and p53. This was supported by increases in the Bax and p53 protein levels as well as by an elevated activity of caspase-3 in the CSC-exposed cells. Immunofluorescence staining demonstrated that both Bax and caspase-3 displayed a cytosolic and mitochondrial distribution in the CSC-exposed gingival fibroblasts, compared to controls. The damaging effect of CSC on gingival fibroblast growth was also supported by the decrease in interleukin 6 and 8 secretion by the gingival fibroblasts. CONCLUSION: These results suggest that CSC may contribute to deregulating fibroblast functions. This can compromise fibroblast-epithelial cell interactions, which ultimately increases the risk of gingival tissue damage and the onset of periodontitis.
Assuntos
Caspase 3/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína X Associada a bcl-2/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Contagem de Células , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citosol/efeitos dos fármacos , Gengiva/citologia , Humanos , Interleucina-6/análise , Interleucina-8/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Receptores do Fator de Necrose Tumoral/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND AND OBJECTIVE: Smoking cigarettes increases the risk of developing various types of human diseases, including cancers and periodontitis. As gingival epithelial cells are known to play an active role in innate immunity via the secretion of a wide variety of mediators, and as these cells are the first ones exposed to environmental stimuli such as cigarette smoke, we sought to investigate the effects of whole cigarette smoke on normal human gingival epithelial cells and tissue. MATERIAL AND METHODS: Human gingival epithelial cells were extracted from healthy nonsmokers and used either as a monolayer or as an engineered human oral mucosa to investigate the effect of whole cigarette smoke on cell growth, apoptosis and wound repair/migration. RESULTS: Our findings show that when gingival epithelial cells were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell growth through an apoptotic pathway, as confirmed by an increase of Bax and a decrease of Bcl-xL and caspase-3 activity. Cigarette smoke also inhibited epithelial cell migration. These effects may explain the disorganization of the engineered human oral mucosa tissue when exposed to whole cigarette smoke. CONCLUSION: Exposure to whole cigarette smoke markedly inhibits epithelial cell growth through an apoptosis/necrosis pathway that involves Bax and Bcl-xL proteins and caspase-3 activity. Cigarette smoke also disrupts epithelial cell migration, which may negatively affect periodontal wound healing.
Assuntos
Apoptose , Gengiva/efeitos dos fármacos , Mucosa Bucal/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Cicatrização/efeitos dos fármacos , Anexinas/biossíntese , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Humanos , Necrose , Propídio/metabolismo , Estatísticas não Paramétricas , Proteína X Associada a bcl-2/biossíntese , Proteína bcl-X/biossínteseRESUMO
We investigated the toxicity of synthetic antimicrobial decapeptide KSL-W on normal human gingival epithelial cell cultures, its effect on Candida albicans adhesion and growth, and the activation of epithelial cell innate immunity. Our results indicate that KSL-W had no toxic effect on cell adhesion or growth, suggesting its safe use with human cells. Pre-treating C. albicans with KSL-W attenuated the yeast's virulence as demonstrated by its reduced adhesion and growth on engineered human oral mucosa epithelium and the subsequent decreased expression of some innate defense molecules by targeted epithelial cells. Indeed, the expression of Toll-like receptors and human ß-defensins was reduced in tissues infected with KSL-W-treated Candida. Proinflammatory cytokine secretion (IL-1ß and IL-6) by the epithelial cells was also regulated by KSL-W in a manner similar to that of antifungal molecule amphotericin B. These findings therefore show that KSL-W is safe for use with human cells and is able to attenuate Candida virulence by modulating its effects on host innate immunity. This study proposes the potential application of KSL-W peptide as an alternative antifungal agent.
Assuntos
Anti-Infecciosos/farmacologia , Candida albicans/efeitos dos fármacos , Citocinas/metabolismo , Mucosa Bucal/efeitos dos fármacos , Mucosa Bucal/metabolismo , Oligopeptídeos/farmacologia , Receptores Toll-Like/metabolismo , beta-Defensinas/metabolismo , Anti-Infecciosos/efeitos adversos , Western Blotting , Candida albicans/patogenicidade , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Gengiva/citologia , Humanos , Oligopeptídeos/efeitos adversos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Engenharia TecidualRESUMO
BACKGROUND: Bronchial epithelium is considered a key player in coordinating airway wall remodelling. The function of epithelial cells can be modulated by the underlying fibroblasts through autocrine and paracrine mechanisms. OBJECTIVE: To investigate the effect of phenotypic changes in bronchial fibroblasts from asthmatic subjects on epithelial cell proliferation. METHODS: Epithelial cells and fibroblasts derived from bronchial biopsies of asthmatic and healthy controls were cultured in an engineered model. Proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid (MTT). Epidermal growth factor receptor (EGFR), cyclin-dependent kinase inhibitors p21 and p27 were measured by western blots. Total and active forms of transforming growth factor (TGF)-ß1 were measured using ELISA and bioassay. TGF-ß was inhibited using a recombinant TGF-ß soluble receptor II protein. RESULTS: Proliferation of epithelial cells from asthmatics (AE) is increased when cells were cultured with fibroblasts from normal controls (NF). Fibroblasts from asthmatics (AF) significantly decreased the proliferation of epithelial cells from healthy subjects (NE). Activation of p21, p27, EGFR and TGF-ß1 reflects the proliferation data by decreasing in AE cultured with NF and increasing in NE cultured with AF. Neutralization of TGF-ß increased proliferation of epithelial cells cultured in the asthmatic model. CONCLUSION: Fibroblasts from asthmatic subjects regulate epithelial cell prolifearation, and TGF-ß signalling may represent one of the pathway involved in these interactions.
Assuntos
Asma/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais/fisiologia , Asma/imunologia , Asma/patologia , Western Blotting , Brônquios/imunologia , Brônquios/metabolismo , Brônquios/patologia , Comunicação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Proteínas Inibidoras de Quinase Dependente de Ciclina/biossíntese , Proteínas Inibidoras de Quinase Dependente de Ciclina/imunologia , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Células Epiteliais/ultraestrutura , Receptores ErbB/metabolismo , Fibroblastos/imunologia , Fibroblastos/ultraestrutura , Humanos , Microscopia Eletrônica de Varredura , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Engenharia Tecidual , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/imunologiaRESUMO
AIM: To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form. METHODS AND RESULTS: Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD(50) showed that the fibroblasts were able to tolerate up to 80 microg ml(-1) for 24 h, dropping thereafter to 62 mug ml(-1) after 72 h of contact, compared to 160 microg ml(-1) after 24 h, and 80 microg ml(-1) after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25 microg ml(-1)) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition. CONCLUSION: These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.
Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Nisina/análogos & derivados , Candida albicans/crescimento & desenvolvimento , Candida albicans/ultraestrutura , Candidíase Bucal/microbiologia , Candidíase Bucal/patologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Gengiva/citologia , Gengiva/crescimento & desenvolvimento , Humanos , Hifas/crescimento & desenvolvimento , Dose Letal Mediana , Testes de Sensibilidade Microbiana , Nisina/farmacologiaRESUMO
This study investigated the presence and origin of hydrocarbon pollution in industrial waste water sediments found near the Jarzouna (Bizerte, Tunisia) oil refinery. Analyses of surface sediments (layer 1) and deep sediments (layer 2) showed that Total Hydrocarbon (TH) concentrations ranged from 602 +/- 7.638 microg/g in layer-1 to 1270 +/- 2.176 microg/g in layer-2. The results suggest that the deeper the sediment, the higher the level of total hydrocarbon found. The sedimentary Non Aromatic Hydrocarbon (NAH) and Aromatic Hydrocarbon (AH) concentrations ranged from 66.22 +/- 1.516 to 211.82 +/- 10.670 microg/g for NAH, and from 13.84 +/- 0.180 to 115.60 +/- 2.479 microg/g for AH. The high variability of these concentrations was associated with the location of the sediment collection sites. Aliphatic biomarker analysis revealed petroleum contamination close to the refinery rejection site, and biogenic sources further away. Petroleum contamination may be associated with increased industrial activity in the area of Jarzouna-Bizerte in the Mediterranean Sea.
Assuntos
Indústrias Extrativas e de Processamento , Sedimentos Geológicos/química , Hidrocarbonetos/análise , Resíduos Industriais/análise , Petróleo , Poluentes Químicos da Água/análise , Mar Mediterrâneo , Água do Mar/químicaRESUMO
AIMS: To investigate the efficacy of nisin Z, an antimicrobial peptide produced by certain strains of Lactococcus lactis against Candida albicans growth and transition. METHODS AND RESULTS: Candida albicans was cultured in the presence of various concentrations of nisin Z (1000, 500, and 100 microg ml(-1)) for different time points. Candida albicans growth was determined using the Alamar Blue assay. The yeast's transition from blastospore to hyphal form was assessed through optical microscope observations. The effect of nisin Z on C. albicans ultrastructure was followed by scanning and transmission electron microscopy. Our results show that nisin Z inhibited C. albicans growth beginning at 500 microg ml(-1). This inhibition was both time- and dose-dependent. Nisin Z was also active against C. albicans transition by significantly inhibiting the transformation of C. albicans from the blastospore to hyphal form. Treatments with nisin Z lead to ultrastructural disturbances of C. albicans. CONCLUSION: Our findings indicate that nisin Z significantly reduced C. albicans growth and transition. These effects may have occurred through ultrastructural modifications of this yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, effect of nisin Z on C. albicans was investigated. These results therefore suggest that nisin Z may have antifungal properties, and could be used as an antifungal molecule.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Nisina/análogos & derivados , Candida albicans/crescimento & desenvolvimento , Candida albicans/ultraestrutura , Células Cultivadas/efeitos dos fármacos , Contagem de Colônia Microbiana , Relação Dose-Resposta a Droga , Hifas/crescimento & desenvolvimento , Microscopia Eletrônica de Transmissão e Varredura , Nisina/farmacologia , Fatores de TempoRESUMO
The goal of this study was to optimize key processes in recreating functional and viable palatal mucosa-like tissue that would be easy to handle and would promote wound healing. Normal human gingival fibroblasts and epithelial cells and a clinically useful biomaterial, CollaTape, were used. Structural and ultrastructural analyses showed that the gingival fibroblasts and epithelial cells adhered to the biomaterial and proliferated. Following a 6-day culture, using 10(5) fibroblasts and 10(6) epithelial cells, a well-organized palatal mucosa-like tissue was engineered. The engineered epithelium displayed various layers, including a stratum corneum, and contained cytokeratin 16-positive cells located in the supra-basal layer. This palatal mucosa-like engineered tissue was designed to meet a variety of surgical needs. The biodegradable collagen membrane (CollaTape) contributed to the flexibility of the engineered tissue. This engineered innovative tissue may contribute to the reconstruction of oral soft-tissue defects secondary to trauma, congenital defects, and acquired diseases.
Assuntos
Materiais Biocompatíveis , Gengiva/citologia , Mucosa Bucal/crescimento & desenvolvimento , Palato/citologia , Engenharia Tecidual/métodos , Colágeno/química , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Membranas Artificiais , Mucosa Bucal/química , Mucosa Bucal/citologiaRESUMO
Emerging data on the consequences of the interactions between invasive oral bacteria and host cells have provided new insights into the pathogenesis of periodontal disease. Indeed, modulation of the mucosal epithelial barrier by pathogenic bacteria appears to be a critical step in the initiation and progression of periodontal disease. Periodontopathogens such as Porphyromonas gingivalis have developed different strategies to perturb the structural and functional integrity of the gingival epithelium. P. gingivalis adheres to, invades, and replicates within human epithelial cells. Adhesion of P. gingivalis to host cells is multimodal and involves the interaction of bacterial cell-surface adhesins with receptors expressed on the surfaces of epithelial cells. Internalization of P. gingivalis within host cells is rapid and requires both bacterial contact-dependent components and host-induced signaling pathways. P. gingivalis also subverts host responses to bacterial challenges by inactivating immune cells and molecules and by activating host processes leading to tissue destruction. The adaptive ability of these pathogens that allows them to survive within host cells and degrade periodontal tissue constituents may contribute to the initiation and progression of periodontitis. In this paper, we review current knowledge on the molecular cross-talk between P. gingivalis and gingival epithelial cells in the development of periodontitis.
Assuntos
Células Epiteliais/microbiologia , Periodontite/microbiologia , Porphyromonas gingivalis/patogenicidade , Animais , Aderência Bacteriana , Células Epiteliais/metabolismo , Gengiva/citologia , Humanos , Mediadores da Inflamação/metabolismo , Porphyromonas gingivalis/fisiologia , Receptores de Superfície Celular/fisiologia , Transdução de SinaisRESUMO
Porphyromonas gingivalis gingipains are thought to be critical virulence factors in periodontitis. Increased serum levels of the soluble ectodomains of surface effectors have been reported to occur during bacterial infections. In the present study, we show that the cell surface proteoglycan syndecan-1 was highly expressed on human gingival epithelial cells. Treatments with P. gingivalis culture supernatants consistently mediated the shedding of syndecan-1 from the surface of epithelial cells. Concomitantly, the amount of soluble syndecan-1 detected in the culture medium increased significantly in a time-dependent manner. However, neither a heat-inactivated supernatant nor a supernatant from a gingipain-deficient mutant had a significant effect on syndecan-1 shedding. Such a shedding process may play an important role in the bacterial invasion of periodontal tissue and the modulation of host defences.
Assuntos
Adesinas Bacterianas/farmacologia , Cisteína Endopeptidases/farmacologia , Gengiva/efeitos dos fármacos , Hemaglutininas/farmacologia , Glicoproteínas de Membrana/efeitos dos fármacos , Porphyromonas gingivalis/fisiologia , Proteoglicanas/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Cisteína Endopeptidases Gingipaínas , Gengiva/patologia , Humanos , Glicoproteínas de Membrana/análise , Proteoglicanas/análise , Sindecana-1 , Sindecanas , Fatores de Tempo , Fatores de Virulência/farmacologiaRESUMO
Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.
Assuntos
Dímeros de Pirimidina/metabolismo , Pele/efeitos da radiação , Adulto , Divisão Celular/efeitos da radiação , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Neoplasias Induzidas por Radiação/metabolismo , Neoplasias Induzidas por Radiação/patologia , Radioimunoensaio , Pele/citologia , Pele/ultraestrutura , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Raios UltravioletaRESUMO
The mechanisms of fibrillar collagen accumulation in asthmatic bronchi remain unclear, an imbalance between synthesis and degradation of collagen may be implicated in this process. The aim of this study was to compare the capacities of normal (BNF) and asthmatic (BAF) bronchial fibroblasts to degrade collagen. Metalloproteinases and their inhibitors were measured by ELISA, types I and III procollagen synthesis was determined by liquid RIA and, finally, zymography was used to assess the presence of active and latent forms of MMPs. The capacity of fibroblasts to degrade collagen coated onto latex beads was evaluated by flow cytometry. Our results showed that MMP-2 secretion was significantly higher in BNF when compared to BAF and this was confirmed by gelatin zymography. In BNF culture, TIMP-1 and MMP-1 secretions positively correlated with types I and III procollagen synthesis. However, in BAF, this correlation was negative. This suggests that a balance exists between collagen synthesis and degradation in BNF and that this balance is compromised in BAF. On the other hand, BAF did show significantly reduced capacity to degrade collagen when compared to that of BNF. This reduced phagocytic activity was not associated with a decrease in collagen receptor expression. This study establishes for the first time that a relationship exists between metalloproteinases enzyme dysregulation and the reduced capacity of asthmatic bronchial fibroblast to degrade collagen. These events may shed light on why accumulation of collagen can be observed in asthmatic airways.
Assuntos
Asma/metabolismo , Brônquios/metabolismo , Colágeno/metabolismo , Asma/patologia , Brônquios/citologia , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Metaloproteinase 1 da Matriz/biossíntese , Metaloproteinase 2 da Matriz/biossíntese , Fagocitose , Pró-Colágeno/biossíntese , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-2/biossínteseRESUMO
BACKGROUND: The use of fiberoptic bronchial biopsies has improved our understanding of the immunopathology of asthma. However, this approach offers a limited ability to perform mechanistic studies observing cell-cell and cell-matrix interactions, which are a key issue in the study of airway remodeling. Tissue engineering is a technique that combines the use of biology and engineering expertise to generate a limitless amount of tissue from small samples. This technology allows for the study of cell interactions under conditions as close as possible to the natural environment. OBJECTIVE: The aim of this study was to evaluate the feasibility of an engineered human bronchial mucosa as a model to study cellular interactions in asthma. METHODS: Human bronchial fibroblasts from normal and asthmatic donors were incorporated into collagen gel. Bronchial epithelial cells were seeded over this gel and then cultured in an air-liquid interface in the presence or the absence of T lymphocytes. Biopsy specimens from these engineered mucosa were taken for structural and ultrastructural analysis, and T lymphocytes were harvested and used to localize IL-5. RESULTS: Histologic analysis showed that engineered mucosa with normal bronchial cells presented a pseudostratified ciliated epithelium with the presence of mucus secretory cells. The electron microscopy analysis confirmed these histologic results. These features were comparable with those observed in normal bronchial tissues. However, in engineered mucosa from asthmatic subjects, the tissue structure was disorganized, particularly the epithelial cell arrangement. The percentage of IL-5(+) lymphocytes was significantly (P =.03) higher in engineered bronchial mucosa from asthmatic subjects (87% +/- 2%) compared with mucosa from normal volunteers (2% +/- 0.3%). CONCLUSION: Using tissue engineering, we produced an in vitro model of bronchial mucosa from normal and asthmatic subjects. These models could be a valuable tool to better understand key mechanisms involved in inflammation and airway repair.
Assuntos
Engenharia Biomédica/métodos , Brônquios , Asma/patologia , Biópsia , Brônquios/patologia , Comunicação Celular , Células Cultivadas , Técnicas de Cocultura , Células Epiteliais/patologia , Fibroblastos/patologia , Imunofluorescência , Humanos , Imuno-Histoquímica , MucosaRESUMO
Sunscreens are known to protect against sunlight-induced erythema and sunburn, but their efficiency at protecting against skin cancer is still a matter of debate. Specifically, the capacity of sunscreens to prevent or reduce tissue and DNA damage has not been thoroughly investigated. The present study was undertaken to assess the ability of a chemical broad-spectrum sunscreen to protect human skin against tissue and DNA damage after solar UV radiation. Engineered human skin was generated and either treated or not with a broad-spectrum SPF 30 sunscreen and exposed to increasing doses of simulated sunlight (SSL). Immediately after irradiation, histological, immunohistochemical, and molecular quantitative analyses were performed. The unprotected irradiated engineered human skin showed significant epidermal disorganization accompanied by a complete absence of laminin deposition. The sunscreen prevented SSL-induced epidermal damage at low doses and allowed laminin deposition at almost all SSL doses tested. The frequencies of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts, and photooxidative lesions measured by alkaline gel electrophoresis and radioimmunoassay were significantly reduced by the sunscreen. Thus, tissue and DNA damage may provide excellent quantitative end points for assessing the photoprotective efficacy of sunscreens.
Assuntos
Dano ao DNA , Pele/efeitos dos fármacos , Pele/efeitos da radiação , Protetores Solares/uso terapêutico , Raios Ultravioleta , Adolescente , Adulto , Células Cultivadas , Ciclobutanos/metabolismo , Dimerização , Relação Dose-Resposta à Radiação , Feminino , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , Laminina/metabolismo , Pirimidinas/metabolismo , Pirimidinonas/metabolismo , RadioimunoensaioRESUMO
The alkaline and neutral comet assays have been widely used to assess DNA damage and repair in individual cells after in vivo or in vitro exposure to chemical or physical genotoxins. Cells processed under neutral conditions generate comets primarily from DNA double strand breaks, whereas under alkaline conditions, comets arise from DNA single and double strand breaks and alkali-labile lesions. A modified version of the alkaline comet assay, as described here, used silver stain to visualize the comets and a Gelbond base to facilitate the manipulation and processing of samples. To demonstrate how these modifications improve the assay, fibroblasts derived from both normal and Xeroderma pigmentosum (Xp) individuals were exposed to simulated solar radiation and the resulting DNA damage and repair evaluated and compared with results from the relevant literature. Comets from normal fibroblasts reached their maximum length at about an hour after irradiation. Dose-dependent increases in comet length were observed up to at least 360 mJ/cm2. In contrast, comet lengths from repair deficient Xp fibroblasts were shorter than normal cells reflecting their reduced capacity to generate single strand breaks by the excision of DNA dimers. For incubation times of more than 1 h, comet lengths from normal fibroblasts underwent a time-dependent decrease, supporting the contention that this change was related to the ligation step in the DNA repair process. These changes were compatible with the model of DNA damage and repair established by others for ultraviolet radiation.
Assuntos
Dano ao DNA , Reparo do DNA , Luz Solar , Xeroderma Pigmentoso/genética , Linhagem Celular , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Coloração pela Prata , Xeroderma Pigmentoso/patologiaRESUMO
Retinoids are frequently used for treatment of photodamaged skin. We wished to find out whether photodamage could be attenuated by applying all-trans retinoic acid (RA) during repetitive irradiation. For this purpose, we used human cutaneous cells and tissue: pure monolayer cultures containing either keratinocytes or fibroblasts, and human skin substitute (SS) containing both cell types. All cultures were exposed to 8 mJ/cm(2) of UVB and were immediately treated with RA (0, 1.5, or 3 microM). The irradiation and RA treatment protocol was repeated until the cells of the nonirradiated culture had reached confluence. In the irradiated SS, RA preserved the structure (epidermal stratification and differentiation) and ultrastructure (well-organized intermediate filaments and desmosomes) in a state comparable to that observed in nonirradiated SS. As well RA maintained secretion of basement membrane components (laminin and type-IV collagen). Following irradiation, cutaneous cells also displayed more proliferative capacity when SS was treated. In the irradiated monolayer cultures, RA maintained the proliferative capacity of fibroblasts and decreased their differentiation whereas the opposite effect was seen on keratinocytes. In conclusion, RA clearly helps protect human skin against photodamage induced by repeated exposure to UVB.