RESUMO
A plasmonic metasurface composed of a self-assembled monolayer of gold nanoparticles allows for fluorescence imaging with high spatial resolution, owing to the collective excitation of localized surface plasmon resonance. Taking advantage of fluorescence imaging confined to the nano-interface, we examined actin organization in breast cancer cell lines with different metastatic potentials during cell adhesion. Live-cell fluorescence imaging confined within tens of nanometers from the substrate shows a high actin density spanning < 1 µm from the cell edge. Live-cell imaging revealed that the breast cancer cell lines exhibited different actin patterns during the initial phase of cell adhesion (â¼ 1â¯h). Non-tumorous MCF10A cells exhibited symmetric actin localization at the cell edge, whereas highly metastatic MDA-MB-231 cells showed asymmetric actin localization, demonstrating rapid polarization of MDA-MB-231 cells upon adhesion. The rapid actin organization observed by our plasmonic metasurface-based fluorescence imaging provides information on how quickly cancer cells sense the underlying substrate.
Assuntos
Neoplasias da Mama , Adesão Celular , Movimento Celular , Ouro , Nanopartículas Metálicas , Humanos , Neoplasias da Mama/patologia , Ouro/química , Linhagem Celular Tumoral , Feminino , Nanopartículas Metálicas/química , Ressonância de Plasmônio de Superfície , Actinas/metabolismo , Imagem Óptica , Propriedades de SuperfícieRESUMO
This study proposes a methodology for the fabrication of two-dimensional assembled colloidal nanocrystals based on the classical theory for the surface excess of a short-chain alcohol (butanol) in an aqueous mixture and Rayleigh-Bénard-Marangoni convection caused by temperature and/or surface tension gradients due to the volatilization of butanol at the air-water interface. When polyvinylpyrrolidone (PVP)-modified anisotropic silver nanoprisms dispersed in butanol were added into the water phase, the nanoprisms were guided to the air-water interface via adsorbed butanol together with free butanol and formed dense two-dimensional assemblies through the lateral attraction between nanoprisms as the adsorbed butanol was volatilized. The obtained dense film composed of silver nanoprisms exhibited surface-enhanced Raman scattering (SERS) activity, and in particular, the activity was largely enhanced by low-pressure plasma treatment. A SERS-based invisible printing platform that could only be recognized by x-y SERS mapping was demonstrated with the patterned nanoprism films.
RESUMO
Elevated concentrations of IL-6 (interleukin-6) and sIL-6r (soluble IL-6 receptor) in the synovial fluid and serum of patients with arthritis have been implicated in joint cartilage destruction. This study examined the effects of IL-6 and sIL-6r on the expression of MMPs (matrix metalloproteinases), TIMPs (tissue inhibitor of metalloproteinases), the plasminogen activation system including tPA (tissue-type PA), uPA (urokinase-type PA) and PAI-1 (PA inhibitor type 1) using chondrocytes derived from normal human femur cartilage. The cells were cultured with or without 50 ng/ml IL-6 and/or 30 ng/ml sIL-6r in the presence or absence of the JAK3 (Janus kinase 3) inhibitor WHI-P131 or the MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular signal protein kinase) kinase] inhibitor PD98059 for up to 28 days. The expression of MMPs, TIMPs, uPA, tPA and PAI-1 was investigated at the mRNA and protein levels. MMP protein expression and pSTAT3 (phosphorylation of signal transducer and activator of transcription 3) and pERK (phosphorylation of ERK) were also measured. Treatment with both IL-6 and sIL-6r markedly increased the expression of MMP-1, MMP-13, TIMP-1 and PAI-1, while significantly decreasing the expression of tPA and uPA and stimulating pSTAT3 and pERK. Adding WHI-P131 or PD98059 decreased IL-6 and sIL-6r enhancement of MMP-1, -3 and -13. The results suggest that IL-6 and sIL-6r stimulate the production of MMPs and their inhibitor via JAK-STAT and ERK-MAPK signalling in chondrocytes.
Assuntos
Condrócitos/efeitos dos fármacos , Interleucina-6/farmacologia , Metaloproteinases da Matriz/biossíntese , Receptores de Interleucina-6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Cartilagem/citologia , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Condrócitos/citologia , Condrócitos/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Metaloproteinases da Matriz/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Solubilidade , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Interleukin (IL)-17, a proinflammatory cytokine, is produced primarily by activated Th17 cells. IL-17 consists of six ligands that signal through five receptors (IL-17Rs); IL-17A and IL-17F share the highest homology in the family. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling whereas tissue inhibitor of metalloproteinases (TIMPs) inhibit the action of MMPs. In the present study, we examined the effect of IL-17F on the degradation and synthesis of the extracellular matrix in cartilage using human articular chondrocytes. We examined the effect of IL-17F on the expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and cyclooxygenases (COXs), as well as on prostaglandin E2 (PGE2) production. We also examined the indirect effect of PGE2 on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2. Cells were cultured with or without IL-17F in the presence or absence of either an IL-17R antibody or NS-398 for up to 28 days. Expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and COXs at mRNA and protein levels was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. PGE2 production was determined by ELISA. The expression of all types of IL-17Rs was detected in chondrocytes. However, IL-17RE expression was extremely low, compared with other IL-17Rs. The expression of MMP-1, MMP-3, MMP-13, and COX-2 as well as PGE2 production were increased by addition of IL-17F, whereas the expression of IL-17RD, TIMP-2, TIMP-4, type II collagen, aggrecan, link protein, and COX-1 was decreased. The expression of IL-17RA, IL-17RB, IL-17RC, MMP-2, MMP-14, TIMP-1, and TIMP-3 was unaffected by addition of IL-17F. The IL-17R antibody blocked the stimulating/reducing effect of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, aggrecan, and link protein. NS-398 blocked the reducing effect of IL-17F on aggrecan expression, whereas it did not completely block the stimulating/reducing effects of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, and link protein. Our results suggest that IL-17F stimulates cartilage degradation by increasing the expression of collagenases (MMP-1 and -13) and stromelysin-1 (MMP-3) and by decreasing expression of their inhibitors (TIMP-2 and -4), type II collagen, aggrecan, and link protein in chondrocytes. Furthermore, our results suggest that the expression of aggrecan, link protein, and TIMP-4 decrease through the autocrine action of PGE2 in chondrocytes.
Assuntos
Cartilagem/metabolismo , Colagenases/metabolismo , Interleucina-17/fisiologia , Metaloproteinase 3 da Matriz/metabolismo , Sequência de Bases , Cartilagem/citologia , Cartilagem/enzimologia , Proliferação de Células , Células Cultivadas , Condrócitos/citologia , Condrócitos/enzimologia , Condrócitos/metabolismo , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-17/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Prostaglandin (PG) E(2), which exerts its actions via the PG receptors EP1-4, is produced from arachidonic acid by cyclooxygenase (COX)-1 and COX-2. The aim of this study was to investigate the mechanisms by which interleukin (IL)-1beta induces the expression of PG receptors in cultured human chondrocytes and to explore the role of PGE(2) in this process. The cells were cultured with 0, 10, or 100 U/mL IL-1beta with or without 1 muM celecoxib, a specific inhibitor of COX-2, for up to 28 days. Expression of the genes encoding COX-1, COX-2, and EP1-4 was quantified using real-time PCR, and expression of the corresponding proteins was examined using immunohistochemical staining. PGE(2) production was determined using ELISA. IL-1beta treatment caused a marked dose- and time-dependent increase in the levels of PGE(2), COX-2, and EP4 as compared with the untreated control. It did not affect the expression of COX-1, and it decreased the expression of EP1 and EP2. EP3 expression was not detected in either the absence or the presence of IL-1beta. When celecoxib was also present, IL-1beta failed to stimulate PGE(2) production and EP4 expression, but its stimulatory effect on COX-2 expression and its inhibitory effect on EP1 and EP2 expression were unchanged. IL-1beta increases the production of PGE(2), COX-2, and the PG receptor EP4 in cultured human chondrocytes. The increase in EP4 expression appears to be a result of the increased PGE(2) production.
Assuntos
Condrócitos/metabolismo , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Interleucina-1beta/fisiologia , Receptores de Prostaglandina E/biossíntese , Celecoxib , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Ciclo-Oxigenase 1/efeitos dos fármacos , Ciclo-Oxigenase 2/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Dinoprostona/agonistas , Humanos , Interleucina-1beta/farmacologia , Pirazóis/farmacologia , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E Subtipo EP4 , Sulfonamidas/farmacologiaRESUMO
Elevated interleukin (IL)-1 concentrations in synovial fluid have been implicated in joint bone and cartilage destruction. Previously, we showed that IL-1beta stimulated the expression of prostaglandin (PG) receptor EP4 via increased PGE(2) production. However, the effect of IL-1beta on osteoclast formation via chondrocytes is unclear. Therefore, we examined the effect of IL-1beta and/or celecoxib on the expression of macrophage colony-stimulating factor (M-CSF), receptor activator of NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) in human chondrocytes, and the indirect effect of IL-1beta on osteoclast-like cell formation using RAW264.7 cells. OPG and RANKL expression increased with IL-1beta; whereas M-CSF expression decreased. Celecoxib blocked the stimulatory effect of IL-1beta. Conditioned medium from IL-1beta-treated chondrocytes decreased TRAP staining in RAW264.7 cells. These results suggest that IL-1beta suppresses the formation of osteoclast-like cells via increased OPG production and decreased M-CSF production in chondrocytes, and OPG production may increase through an autocrine mechanism involving celecoxib-related PGs.
Assuntos
Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Interleucina-1beta/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoprotegerina/biossíntese , Prostaglandinas/metabolismo , Pirazóis/farmacologia , Sulfonamidas/farmacologia , Animais , Comunicação Autócrina/efeitos dos fármacos , Sequência de Bases , Celecoxib , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Condrócitos/citologia , Meios de Cultivo Condicionados , Primers do DNA/genética , Dinoprostona/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/genética , Camundongos , Osteoclastos/citologia , Osteoprotegerina/genética , Ligante RANK/biossíntese , Ligante RANK/genéticaRESUMO
Elevated concentrations of interleukin (IL)-6 and soluble IL-6 receptor (sIL-6Ralpha) in synovial fluid have been implicated in joint cartilage destruction. We examined the effect of IL-6 and sIL-6Ralpha on cell growth, alkaline phosphatase (ALPase) activity, and the expression of Sox-9, type II collagen, aggrecan core, link protein, BMP-7, and BMP receptors in human chondrocytes. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Ralpha, whereas ALPase activity decreased markedly. The expression of Sox-9 and aggrecan core did not change in the presence or absence of IL-6 and sIL-6Ralpha, whereas the expression of type II collagen, link protein, BMP-7, and BMP receptors increased in the presence of both IL-6 and sIL-6Ralpha. These results suggest that IL-6 and sIL-6Ralpha suppress the differentiation of chondrocytes and induce the repair of arthrodial cartilage through an increase in the expression of cartilage matrix proteins, BMP-7, and BMP receptors in the cells.
Assuntos
Condrócitos/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Interleucina-6/farmacologia , Receptores de Interleucina-6/metabolismo , Fosfatase Alcalina/biossíntese , Proteína Morfogenética Óssea 7 , Receptores de Proteínas Morfogenéticas Ósseas/biossíntese , Proteínas Morfogenéticas Ósseas/biossíntese , Linhagem Celular , Proliferação de Células , Condrócitos/citologia , Proteínas de Grupo de Alta Mobilidade/biossíntese , Humanos , Fatores de Transcrição SOX9 , Fatores de Transcrição/biossínteseRESUMO
Cytokines released at sites of inflammation and infection can alter the normal processes of cartilage turnover, resulting in pathologic destruction or formation. Interleukin (IL)-1beta plays a central role in the pathophysiology of cartilage damage and degradation in arthritis. In the present study, we examined the effect of IL-1beta on the expression of IL-1beta, IL-6, IL-8, IL-11, tumor necrosis factor-alpha (TNF-alpha), and their receptors in human chondrocytes. The cells were cultured either with or without 100 U/ml of IL-1beta for up to 28 days. The level of expression of the cytokines and their receptors was estimated by determining mRNA levels using real-time PCR or by determining protein levels using ELISA. The expression of IL-1beta, IL-8, and TNF-alpha markedly increased in the presence of IL-1beta after day 14 of culture. The expression of IL-6 and IL-11 increased greatly in the presence of IL-1beta on day 1 and after day 14 of culture. The expression of IL-1beta, IL-8, IL-11, and TNF-alpha receptors significantly decreased in the presence of IL-1beta after day 14 of culture, whereas the expression of IL-6 receptor significantly increased. The expression of these cytokines, except for IL-6, decreased with the addition of human IL-1 receptor antagonist. These results suggest that IL-1beta promotes the resolution system of cartilage matrix turnover through an increase in inflammatory cytokine production by chondrocytes and that it also may promote the autocrine action of IL-6 through an increase in IL-6 receptor expression in the cells.
Assuntos
Condrócitos/química , Citocinas/genética , Interleucina-1/farmacologia , Receptores de Citocinas/genética , Comunicação Autócrina , Forma Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Citocinas/biossíntese , Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-6/genética , Interleucina-6/fisiologia , Receptores de Citocinas/biossíntese , Sialoglicoproteínas/farmacologiaRESUMO
This clinical report describes the fabrication and bonding of porcelain laminate veneer restorations in a patient with anterior open spaces. Laminate veneer restorations made of feldspathic porcelain were etched with 5% hydrofluoric acid, rinsed under tap water, ultrasonically cleaned with methanol, and primed with a chemically activated three-liquid silane bonding agent (Clearfil Porcelain Bond). The enamel surfaces were etched with 40% phosphoric acid, rinsed with water, and primed with a two-liquid bonding agent (Clearfil New Bond) that contained a hydrophobic phosphate (10-methacryloyloxydecyl dihydrogen phosphate; MDP). The restorations were bonded with a dual-activated luting composite (Clapearl DC). The veneers have been functioning satisfactorily for an observation period of one year. Combined use of the Clearfil bonding agents and Clapearl DC luting composite is an alternative to conventional materials for seating porcelain laminate veneer restorations, although the system is inapplicable to dentin bonding.
Assuntos
Colagem Dentária/métodos , Porcelana Dentária , Facetas Dentárias , Diastema/terapia , Condicionamento Ácido do Dente , Adulto , Cimentos Dentários , Humanos , Masculino , Compostos de Organossilício , Cimentos de Resina , SilanosRESUMO
Interleukin-1 (IL-1) plays key roles in altering cartilage matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). In the present study, we examined the effect of IL-1beta on cell proliferation, alkaline phosphatase (ALPase) activity, and the expression of MMPs, and TIMPs in chondrocytes derived from normal human femoral cartilage. The cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and 0, 1, 10, or 100 U/ml of IL-1beta for up to 28 days. The level of expression of MMPs and TIMPs was estimated by determining mRNA levels using real-time PCR and by determining protein levels using an enzyme-linked immunosorbent assay. Cell proliferation decreased in the presence of IL-1beta after day 21 of culture. ALPase activity decreased significantly in the presence of IL-1beta after day 10 of culture. The expression of MMP-1, -2, and -3 increased markedly in the presence of IL-1beta after day 21 of culture. MMP-13 expression increased markedly in the presence of IL-1beta on day 1 of culture, but decreased markedly after day 7. The expression of TIMP-1 increased significantly after day 14 of culture. The expression of TIMP-2 decreased significantly on day 1, but increased significantly from day 3 to day 14 of culture. These results suggest that IL-1beta may stimulate cartilage matrix turnover by increasing mainly MMP-13 production by the cells.
Assuntos
Condrócitos/efeitos dos fármacos , Interleucina-1/farmacologia , Metaloproteinases da Matriz/biossíntese , Inibidores Teciduais de Metaloproteinases/biossíntese , Cartilagem/citologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinases da Matriz/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidores Teciduais de Metaloproteinases/genéticaRESUMO
We examined the effect of the inflammatory mediator interleukin-1alpha (IL-1alpha) on cell proliferation, alkaline phosphatase (ALPase) activity, and the expressions of cartilage matrix proteins, bone morphogenetic protein-2 (BMP-2), and BMP-2 receptors in human chondrosarcoma cell line OUMS-27 (chondrocytes). The cells were cultured with Dulbecco's modified Eagle's medium containing 15% fetal bovine serum with 0, 1, 10, or 100 units/ml of IL-1alpha for up to 14 days. The expressions of cartilage matrix proteins, BMP-2, and BMP-2 receptors were estimated by determining mRNA levels using semiquantitative or real-time PCR and/or by determining protein levels using Enzyme-linked immunosorbent assay. Cell proliferation was decreased after 5 days in culture with IL-1alpha. The ALPase activity was decreased significantly in the presence of IL-1alpha until day 10 of culture. The expression of type II collagen was significantly decreased after 7 days in culture with IL-1alpha. The expressions of aggrecan and link protein were significantly decreased through day 14 of culture with IL-1alpha. The expression of BMP-2 was increased at days 3, 7, and 14 of culture with IL-1alpha, while the expression of type II receptor for BMP-2 was significantly decreased in the samples. These results suggest that IL-1alpha suppresses the expression of cartilage matrix proteins through a suppression of the autocrine action of BMP-2, brought about by the decrease in BMP-2 receptor expression in chondrocytes.