RESUMO
Studies on the pathogen-host interaction are crucial for the understanding of the mechanisms involved in the establishment, maintenance, and spread of infection. In recent years, our research group has observed that the P. brasiliensis species interact with integrin family receptors and increase the expression of α3 integrin in lung epithelial cells within 5 h of infection. Interestingly, α3 integrin levels were reduced by approximately 99% after 24 h of infection with P. brasiliensis compared to non-infected cells. In this work, we show that, during infection with this fungus, α3 integrin is increased in the late endosomes of A549 lung epithelial cells. We also observed that the inhibitor of the lysosomal activity bafilomycin A1 was able to inhibit the decrease in α3 integrin levels. In addition, the silencing of the charged multivesicular body protein 3 (CHMP3) inhibited the reduction in α3 integrin levels induced by P. brasiliensis in A549 cells. Thus, together, these results indicate that this fungus induces the degradation of α3 integrin in A549 lung epithelial cells by hijacking the host cell endolysosomal pathway.
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Prediction of pulmonary metabolites following inhalation of a locally acting pulmonary drug is essential to the successful development of novel inhaled medicines. The lungs present metabolic enzymes, therefore they influence drug disposal and toxicity. The present review provides an overview of alternative methods to evaluate the pulmonary metabolism for the safety and efficacy of pulmonary delivery systems. In vitro approaches for investigating pulmonary drug metabolism were described, including subcellular fractions, cell culture models and lung slices as the main available in vitro methods. In addition, in silico studies are promising alternatives that use specific software to predict pulmonary drug metabolism, determine whether a molecule will react with a metabolic enzyme, the site of metabolism (SoM) and the result of this interaction. They can be used in an integrated approach to delineate the major cytochrome P450 (CYP) isoforms to rationalize the use of in vivo methods. A case study about a combination of experimental and computational approaches was done using fluticasone propionate as an example. The results of three tested software, RSWebPredictor, SMARTCyp and XenoSite, demonstrated greater probability of the fluticasone propionate being metabolized by CYPs 3A4 at the S1 atom of 5-S-fluoromethyl carbothioate group. As the in vitro studies were not able to directly detect pulmonary metabolites, those alternatives in silico methods may reduce animal testing efforts, following the principle of 3Rs (Replacement, Reduction and Refinement), and contribute to the evaluation of pharmacological efficacy and safety profiles of new drugs in development.
Assuntos
Sistema Enzimático do Citocromo P-450 , Pulmão , Animais , Preparações Farmacêuticas/metabolismo , Pulmão/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Administração por Inalação , FluticasonaRESUMO
The respiratory epithelium is highly complex, and its composition varies along the conducting airways and alveoli. In addition to their primary function in maintaining the respiratory barrier and lung homeostasis for gas exchange, epithelial cells interact with inhaled pathogens, which can manipulate cell signaling pathways, promoting adhesion to these cells or hosting tissue invasion. Moreover, pathogens (or their products) can induce the secretion of chemokines and cytokines by epithelial cells, and in this way, these host cells communicate with the immune system, modulating host defenses and inflammatory outcomes. This review will focus on the response of respiratory epithelial cells to two human fungal pathogens that cause systemic mycoses: Aspergillus and Paracoccidioides. Some of the host epithelial cell receptors and signaling pathways, in addition to fungal adhesins or other molecules that are responsible for fungal adhesion, invasion, or induction of cytokine secretion will be addressed in this review.
RESUMO
The respiratory epithelium is highly complex, and its composition varies along the conducting airways and alveoli. In addition to their primary function in maintaining the respiratory barrier and lung homeostasis for gas exchange, epithelial cells interact with inhaled pathogens, which can manipulate cell signaling pathways, promoting adhesion to these cells or hosting tissue invasion. Moreover, pathogens (or their products) can induce the secretion of chemokines and cytokines by epithelial cells, and in this way, these host cells communicate with the immune system, modulating host defenses and inflammatory outcomes. This review will focus on the response of respiratory epithelial cells to two human fungal pathogens that cause systemic mycoses: Aspergillus and Paracoccidioides. Some of the host epithelial cell receptors and signaling pathways, in addition to fungal adhesins or other molecules that are responsible for fungal adhesion, invasion, or induction of cytokine secretion will be addressed in this review.
RESUMO
Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and may be caused by the species Paracoccidioides brasiliensis. In the lungs, this fungus interacts with epithelial cells, activating host cell signalling pathways, resulting in the production of inflammatory mediators. This event may be initiated through the activation of Pattern-Recognition Receptors such as Toll-like Receptors (TLRs). By interacting with cell wall components, TLR2 is frequently related to fungal infections. In this work, we show that, after 24 h post-infection with P. brasiliensis, A549 lung epithelial cells presented higher TLR2 levels, which is important for IL-8 secretion. Besides, integrins may also participate in pathogen recognition by host cells. We verified that P. brasiliensis increased α3 integrin levels in A549 cells after 5 h of infection and promoted interaction between this receptor and TLR2. However, after 24 h, surprisingly, we verified a decrease of α3 integrin levels, which was dependent on direct contact between fungi and epithelial cells. Likewise, we observed that TLR2 is important to downmodulate α3 integrin levels after 24 h of infection. Thus, P. brasiliensis can modulate the host inflammatory response by exploiting host cell receptors and cell signalling pathways.
Assuntos
Células Epiteliais/metabolismo , Integrina alfa3/metabolismo , Pulmão/metabolismo , Receptor 2 Toll-Like/metabolismo , Células A549 , Western Blotting , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Pulmão/microbiologia , Pulmão/patologia , Paracoccidioides/fisiologiaRESUMO
In this study we aimed to develop a roflumilast (R) and formoterol fumarate (F) dry powder inhaler formulation (DPI) incorporating HPßCD by spray drying and evaluated if it attenuates the inflammatory process and improves lung function in a murine model of ovalbumin induced allergic asthma. The DPI was characterized by powder X-ray diffraction, thermal analysis, scanning electron microscopy, particle size, density, specific surface area and dynamic vapor sorption analyses. In vitro deposition studies were performed using a NGI, while transepithelial permeability and in vivo effects on lung mechanics and inflammation in a model of allergic asthma were also assessed. The R:F formulation was amorphous with high glass transition temperatures, comprised of wrinkled particles, had low bulk and tapped densities, high surface area, suitable particle size for pulmonary delivery and exhibited no recrystallization even at high relative humidities. MMAD were statistically similar of 4.22 ± 0.19 and 4.32 ± 0.13 µm for F and R, respectively. Fine particle fractions (<5 µm) were of more than 50% of the emitted dose. The R:F formulation led to reduced eosinophil infiltration and airway collagen fiber content, yielding decreased airway hyperresponsiveness. In the current asthma model, the R:F formulation combination decreased inflammation and remodeling, thus improving lung mechanics.
Assuntos
Asma , Inaladores de Pó Seco , Administração por Inalação , Aminopiridinas , Animais , Asma/tratamento farmacológico , Benzamidas , Ciclopropanos , Fumarato de Formoterol/uso terapêutico , Camundongos , Tamanho da Partícula , Pós/uso terapêuticoRESUMO
The cell wall is one of the most important structures of pathogenic fungi, enabling initial interaction with the host and consequent modulation of immunological responses. Over the years, some researchers have shown that cell wall components of Histoplasma capsulatum vary among fungal isolates, and one of the major differences is the presence or absence of α-(1,3)-glucan, classifying wild-type fungi as chemotypes II or I, respectively. The present work shows that an isolate of H. capsulatum chemotype I induced lower levels of interleukin (IL)-8 secretion by the lung epithelial cell line A549, when compared to chemotype II yeasts. Thus, we expected that the absence of α-glucan in spontaneous variant yeasts, which were isolated from chemotype II cultures, would modify IL-8 secretion by A549 cells, but surprisingly, these fungi promoted similar levels of IL-8 secretion as their wild-type counterpart. Furthermore, when using a specific inhibitor for Syk activation, we observed that this inhibitor reduced IL-8 levels in A549 cell cultures infected with wild type chemotype I fungi. This inhibitor failed to reduce this cytokine levels in A549 cell cultures infected with chemotype II and their spontaneous variant yeasts, which also do not present α-glucan on their surface. The importance of SFKs and PKC δ in this event was also analyzed. Our results show that different isolates of H. capsulatum modulate distinct cell signaling pathways to promote cytokine secretion in host epithelial cells, emphasizing the existence of various mechanisms for Histoplasma pathogenicity.
Assuntos
Células Epiteliais Alveolares/metabolismo , Histoplasma/metabolismo , Interleucina-8/metabolismo , Células A549 , Células Epiteliais Alveolares/microbiologia , Parede Celular/metabolismo , Glucanos/metabolismo , Histoplasma/isolamento & purificação , Interações Hospedeiro-Patógeno , Humanos , Pulmão/patologia , Proteína Quinase C-delta/metabolismo , Transdução de Sinais , Especificidade da Espécie , Quinase Syk/metabolismo , Quinases da Família src/metabolismoRESUMO
Fungi that belong to the genus Paracoccidioides are the etiologic agents of paracoccidioidomycosis, a human systemic mycosis, which occurs in Latin America. Epithelial cell is one of the first cells that interact with these fungi and responds by secreting inflammatory mediators such as cytokines. In the present study, we demonstrate that yeasts of different isolates of Paracoccidioides brasiliensis (Pb18 and Pb03) and Paracoccidioides lutzii (Pb01) distinctly promoted interleukin (IL)-8 secretion by the lung epithelial cell line A549. Depending on the isolate, this cytokine release may rely on the epithelial cell interaction with fungal secreted components or direct contact with the pathogen. In addition, adhesion of yeasts to the pulmonary epithelial cells was also different among Paracoccidioides isolates, and the highest percentage of A549 cells with adhered fungi was observed with P. lutzii. All Paracoccidioides isolates induced an expression increase of α3 and α5 integrins in A549 cells and, using small interfering RNA, we observed that the integrin silencing promoted a reduction of P. lutzii adhesion, which suggests the involvement of integrins in this event. Together, these results indicate that host epithelial cell response may depend on the isolate of Paracoccidioides.
Assuntos
Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/microbiologia , Interleucina-8/biossíntese , Paracoccidioides/fisiologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , Células A549 , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Inativação Gênica , Humanos , Integrinas/genéticaRESUMO
The aim of this study was to develop roflumilast dry powder inhaler (DPI) formulations by spray drying using hydroxypropyl-ß-cyclodextrin (HPßCD) and to determine their suitability for pulmonary delivery. Different feed solution concentrations, solvent systems and spray drying parameters were used to obtain the formulations which were characterized using X-ray powder diffraction, thermal analysis, scanning electron microscopy, particle size distribution, bulk and tapped density, specific surface area, dynamic vapour sorption, in vitro deposition properties using a Next Generation Impactor (NGI) and transepithelial permeability. Microparticles spray dried from ethanol were wrinkled and amorphous, exhibiting high glass transition temperatures while those from methanol:n-butyl acetate consisted of irregularly shaped porous particles partially crystalline. All formulations presented low density, particle size and residual solvent content exhibiting high depositon in the lower stages of the NGI. Mass median aerodynamic diameters (MMADs) were in the range of 3.32-4.49⯵m, with high fine particle fractions (FPFâ¯<â¯5⯵m). Stability studies demonstrated no significant modifications in the solid-state nature and in the aerolisation performance of the selected formulation which presented a Papp of 8.73â¯×â¯10-6⯱â¯4.70â¯×â¯10-7â¯cm/s. The developed roflumilast DPI formulations have potential therapeutic applications in the treatment of lung diseases.
Assuntos
Aminopiridinas/química , Benzamidas/química , Composição de Medicamentos , Inaladores de Pó Seco , Administração por Inalação , Aminopiridinas/administração & dosagem , Aminopiridinas/farmacocinética , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacocinética , Benzamidas/administração & dosagem , Benzamidas/farmacocinética , Linhagem Celular Tumoral , Ciclopropanos/administração & dosagem , Ciclopropanos/química , Ciclopropanos/farmacocinética , Sistemas de Liberação de Medicamentos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , PósRESUMO
In its hyphal form, Candida albicans invades epithelial and endothelial cells by two distinct mechanisms: active penetration and induced endocytosis. The latter is dependent on a reorganization of the host cytoskeleton (actin/cortactin recruitment), whilst active penetration does not rely on the host's cellular machinery. The first obstacle for the fungus to reach deep tissues is the epithelial barrier and this interaction is crucial for commensal growth, fungal pathogenicity and host defense. This study aimed to characterize in vitro epithelial HeLa cell invasion by four different isolates of C. albicans with distinct clinical backgrounds, including a C. albicans SC5314 reference strain. All isolates invaded HeLa cells, recruited actin and cortactin, and induced the phosphorylation of both Src-family kinases (SFK) and cortactin. Curiously, L3881 isolated from blood culture of a patient exhibited the highest resistance to oxidative stress, although this isolate showed reduced hyphal length and displayed the lowest cell damage and invasion rates. Collectively, these data suggest that the ability of C. albicans to invade HeLa cells, and to reach and adapt to the host's blood, including resistance to oxidative stress, may be independent of hyphal length.
RESUMO
Paracoccidioides brasiliensis is one of the etiological agents of the human systemic mycosis paracoccidioidomycosis. Protease-activated receptors (PARs) are expressed in many cell types and comprise a family of G protein-coupled receptors (PAR-1, PAR-2, and PAR-4), which may be activated by proteases secreted by several pathogens. In the present study, we showed that the pathogenic fungus P. brasiliensis secretes components that promote interleukin (IL)-6 and IL-8 secretion by the lung epithelial cell line A549. Cytokine secretion was reduced by antagonistic peptides for PAR-1 and PAR-2, but not for PAR-4. P. brasiliensis proteases were isolated from fungal culture supernatants in a p-aminomethylbenzamidine-Sepharose column. The obtained fractions were tested for enzymatic activity against fluorescence resonance energy transfer (FRET) peptides derived from sequences that spanned the activation sites of human PARs. The eluted fraction, termed PbP, contained protease activities that were able to hydrolyze the FRET peptides. PbP also induced IL-6 and IL-8 secretion in A549 epithelial cells, which was reduced upon heat inactivation of PbP, incubation with antagonistic peptides for PAR-1 and PAR-2, and the protease inhibitors aprotinin, leupeptin, and E-64. Together, these results show for the first time that P. brasiliensis yeasts secrete proteases that activate PARs in lung epithelial cells, leading to cytokine secretion.
Assuntos
Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Paracoccidioides , Receptores Ativados por Proteinase/metabolismo , Células A549 , Linhagem Celular , Sobrevivência Celular/imunologia , Endopeptidases/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Paracoccidioides/enzimologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/metabolismo , Paracoccidioidomicose/microbiologia , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacosRESUMO
Introduction: the recent enhancement of interest in green consumerism has given rise to a renewed scientiï¬c awareness towards essential oils. Essential oil from Baccharis trimera (Less.) DC. (B. trimera) (Asteraceae) is cited as one of the ten most consumed oils by the cosmetic and other industries in Brazil. Objective: to investigate the antimicrobial activity of the essential oil from the leaves of B. trimera against Staphylococcus epidermidis ATCC 12228, Proteus vulgaris ATCC 13315, Micrococcus luteus ATCC 7468 and Corynebacterium xerosis IAL105, which are the main bacteria responsible for bad perspiration odor. Methods: the gas chromatography (GC) analysis was performed and the antimicrobial activity was evaluated by means of the turbidimetric method, using a microdilution assay. Results: ywenty constituents were identified, being that ß-pinene (23.4 percent) was the major compound found. The minimum inhibitory concentration (MIC) values of the essential oil ranged from 500 µg/mL to 1,000 µg/mL. A detrimental effect of the essential oil was observed on the morphology of cell membranes of the bacteria studied by scanning electron microscopy (SEM). Conclusions: the results demonstrate the essential oil of B. trimera has potential in the application of antimicrobial agents in personal care products(AU)
Introducción: el reciente aumento del interés por el consumo verde ha dado lugar a una renovada conciencia científica hacia a los aceites esenciales. El aceite esencial de Baccharis trimera (Less.) DC. (B. trimera) (Asteraceae) es considerado uno de los diez aceites más consumidos por la industria cosmética del Brasil. Objetivos: valorar la actividad antimicrobiana del aceite esencial de hojas de B. trimera frente al Staphylococcus epidermidis ATCC 12228, Proteus vulgaris ATCC 13315, Micrococcus luteus ATCC 7468 y Corynebacterium xerosis IAL105, que son las principales bacterias responsables del mal olor que es consecuencia de la transpiración. Métodos: se realizó la cromatografía de gases (GG) y la actividad antimicrobiana fué valorada por el método turbidimétrico, usando el ensayo de microdilución. Resultados: se identificarón veinte constituyentes, siendo el ß-pineno (23,4 por ciento) el principal compuesto encontrado. Los valores de la concentración mínima inhibitoria (CMI) del aceite esencial variaron de 500 µg/mL a 1,000 µg/mL. Se observó un efecto perjudicial del aceite esencial en la morfología de las membranas celulares de las bacterias estudiadas por microscopía electrónica de barrido (SEM). Conclusión: los resultados demuestran que el aceite esencial de B. trimera tiene potencial en la aplicación de los agentes antimicrobianos en productos de higiene personal(AU)
Assuntos
Humanos , Plantas Medicinais , Óleos Voláteis/uso terapêutico , Cromatografia Gasosa/métodos , Baccharis , Produtos com Ação Antimicrobiana , Microscopia Eletroquímica de Varredura/métodos , BrasilRESUMO
Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-ß-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.
RESUMO
Paracoccidioides brasiliensis is one of the etiological agents of paracoccidioidomycosis, a human systemic mycosis, highly prevalent in Latin America. In the present work, we demonstrate that P. brasiliensis yeasts promote IL-6 and IL-8 secretion by the human lung epithelial cell line A549 in an integrin-dependent manner. In fact, small interfering RNA directed to α3 and α5 integrins decreased IL-6 and IL-8 levels in P. brasiliensis-infected A549 cell cultures. This fungus also led to an increase in the expression of α3 and α5 integrins in this epithelial cell line. In addition, P. brasiliensis yeasts promoted α3 and α5 integrins clustering into A549 cell membrane rafts. Furthermore, epithelial cell membrane raft disruption with nystatin decreased IL-6 and IL-8 levels in P. brasiliensis-A549 cell cultures. Therefore, by increasing host α3 and α5 integrins levels and clustering these receptors into membrane rafts, P. brasiliensis yeasts may modulate host inflammation.
Assuntos
Membrana Celular/metabolismo , Células Epiteliais/imunologia , Integrina alfa3/metabolismo , Integrina alfa5/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Paracoccidioides/imunologia , Linhagem Celular , Membrana Celular/microbiologia , Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , HumanosRESUMO
In this study, we investigated the role of protein kinases C (PKCs) in interleukin (IL)-6 and IL-8 secretion by human lung epithelial A549 cells during infection with the fungal pathogen Paracoccidioides brasiliensis. Rottlerin and the broad spectrum PKC inhibitor Go 6983 reduced cytokine levels in A549 cell-P. brasiliensis cultures. Next, by western blot, we verified that infection with this fungus led to phosphorylation of PKC δ (Thr(505)). By using a peptide inhibitor for PKC δ or PKC δ short interfering RNA technique, IL-6 and IL-8 levels in A549-P. brasiliensis cultures were also reduced. Together, these results indicate that P. brasiliensis promotes IL-6 and IL-8 secretion by A549 cells in a PKC δ-dependent manner.
Assuntos
Células Epiteliais/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Pulmão/patologia , Paracoccidioides/imunologia , Paracoccidioidomicose/patologia , Proteína Quinase C/metabolismo , Linhagem Celular , Humanos , Pulmão/microbiologia , Modelos TeóricosRESUMO
BACKGROUND: Kallikreins play a pivotal role in establishing prostate cancer. RESULTS: In contrast to the classical Kunitz plant inhibitor SbTI, the recombinant kallikrein inhibitor (rBbKIm) led to prostate cancer cell death, whereas fibroblast viability was not affected. CONCLUSION: rBbKIm shows selective cytotoxic effect and angiogenesis inhibition against prostate cancer cells. SIGNIFICANCE: New actions of rBbKIm may contribute to understanding the mechanisms of prostate cancer. Prostate cancer is the most common type of cancer, and kallikreins play an important role in the establishment of this disease. rBbKIm is the recombinant Bauhinia bauhinioides kallikreins inhibitor that was modified to include the RGD/RGE motifs of the inhibitor BrTI from Bauhinia rufa. This work reports the effects of rBbKIm on DU145 and PC3 prostate cancer cell lines. rBbKIm inhibited the cell viability of DU145 and PC3 cells but did not affect the viability of fibroblasts. rBbKIm caused an arrest of the PC3 cell cycle at the G0/G1 and G2/M phases but did not affect the DU145 cell cycle, although rBbKIm triggers apoptosis and cytochrome c release into the cytosol of both cell types. The differences in caspase activation were observed because rBbKIm treatment promoted activation of caspase-3 in DU145 cells, whereas caspase-9 but not caspase-3 was activated in PC3 cells. Because angiogenesis is important to the development of a tumor, the effect of rBbKIm in this process was also analyzed, and an inhibition of 49% was observed in in vitro endothelial cell capillary-like tube network formation. In summary, we demonstrated that different properties of the protease inhibitor rBbKIm may be explored for investigating the androgen-independent prostate cancer cell lines PC3 and DU145.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Calicreínas/antagonistas & inibidores , Proteínas de Plantas/farmacologia , Apoptose/efeitos dos fármacos , Sinalização do Cálcio , Caspase 3 , Caspase 9/metabolismo , Adesão Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Citocromos c/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipopolissacarídeos/farmacologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias da Próstata , Proteínas Recombinantes/farmacologia , Inibidor da Tripsina de Soja de Kunitz/farmacologiaRESUMO
In the present study we investigated the chemical composition of hexane fraction and essential oil of Stachytarpheta gesnerioides (Verbenaceae) by GC-MS, total phenol and flavonoid contents. The antioxidant capacity and antimicrobial activity were investigated in five extracts of leaves of S. gesnerioides. Aqueous and 100 percent ethanol extracts were prepared by dynamic maceration. Hexane, ethyl acetate and methanol extracts were prepared by Soxhlet extraction. The essential oil (EO) and hexane fraction (HF) are mainly composed by guaiol. Moreover, the HF is also rich in the monoterpene alpha-pinene. The total phenol content ranged from 0.85 to 22.74 mg gallic acid equivalent /100mg dry extract at FolinCiocalteus reagent method. The total flavonoid concentration ranged from 0.68 to 13.65 mg rutin equivalent /100mg dry extract, detected using 8 percent aluminium chloride. The ethyl acetate extract (IC50=9.41 ug/ml) showed the highest antioxidant activity. The extracts were found to be effective to inhibit the microorganisms tested.
Se han investigado la composición química de la fracción hexánica (FH) y aceite esencial (AE) de Stachytarpheta gesnerioides (Verbenaceae) por GC-MS, el contenido de fenoles totales y flavonoides. La capacidad antioxidante y actividad antimicrobiana fueron investigadas en cinco extractos de hojas de S gesnerioides. Extractos acuosos y etanolico fueron preparados por la maceración dinámica y extracción continua en Soxhlet con hexano, acetato de etilo y metanol. Las fracciones AE y FH están compuestas principalmente por guaiol. La fracción FH es también rica en alfa-pineno. El contenido de fenoles totales varió desde 0,85 hasta 22,74 mg de ácido gálico/100 mg de extracto seco (Folin-Ciocalteu). La concentración total de flavonoides varió desde 0,68 hasta 13,65 mg en equivalentes de rutina/100 mg de extracto seco, que se detectó mediante reacción con cloruro de aluminio al 8 por ciento. El extracto de acetato de etilo (CI50=9,41 ug/ml) enseño la más grande actividad antioxidante. Los extractos se encontraron eficaces para inhibir los microorganismos ensayados.
Assuntos
Óleos Voláteis/química , Antibacterianos/farmacologia , Antioxidantes/farmacologia , Verbenaceae/química , Fenóis/análise , Flavonoides/análise , Cromatografia Gasosa-Espectrometria de Massas , Hexanos/química , Testes de Sensibilidade Microbiana , Folhas de PlantaRESUMO
Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.
Assuntos
Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Paracoccidioides/imunologia , Linhagem Celular , Humanos , Interferon gama/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Analysis of membrane lipids of Histoplasma capsulatum showed that ~40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4°C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30kDa laminin-binding protein, and a 50kDa protein recognized by anti-α5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (mßCD). Removal of ergosterol by mßCD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. mßCD treatment did not displace/remove the 50kDa α5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with mßCD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30kDa laminin-binding protein; ergosterol and/or the 30kDa protein may be involved in macrophage infectivity.
Assuntos
Proteínas Fúngicas/metabolismo , Histoplasma/metabolismo , Histoplasma/patogenicidade , Histoplasmose/metabolismo , Macrófagos Alveolares/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Animais , Ergosterol/metabolismo , Histoplasmose/microbiologia , Histoplasmose/patologia , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos BALB C , beta-Ciclodextrinas/farmacologiaRESUMO
Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. The effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin ß1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways.