RESUMO
In this work we have studied the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on pancreatic stellate cells (PSC). Cell viability was analyzed by AlamarBlue test. Production of reactive oxygen species (ROS) was monitored following CM-H2DCFDA and MitoSOX Red-derived fluorescence. Total protein carbonyls and lipid peroxidation were analyzed by HPLC and spectrophotometric methods respectively. Mitochondrial membrane potential (ψm) was monitored by TMRM-derived fluorescence. Reduced (GSH) and oxidized (GSSG) levels of glutathione were determined by fluorescence techniques. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Determination of SOD activity and total antioxidant capacity (TAC) were carried out by colorimetric methods, whereas expression of SOD was analyzed by Western blotting and RT-qPCR. The results show that melatonin decreased PSC viability in a concentration-dependent manner. Melatonin evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Oxidation of proteins was detected in the presence of melatonin, whereas lipids oxidation was not observed. Depolarization of ψm was noted with 1 mM melatonin. A decrease in the GSH/GSSG ratio was observed, that depended on the concentration of melatonin used. A concentration-dependent increase in the expression of the antioxidant enzymes catalytic subunit of glutamate-cysteine ligase, catalase, NAD(P)H-quinone oxidoreductase 1 and heme oxygenase-1 was detected in cells incubated with melatonin. Finally, decreases in the expression and in the activity of superoxide dismutase were observed. We conclude that pharmacological concentrations melatonin modify the redox state of PSC, which might decrease cellular viability.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Melatonina/farmacologia , Oxirredução/efeitos dos fármacos , Células Estreladas do Pâncreas/metabolismo , Animais , Antioxidantes/metabolismo , Catalase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/genética , Dissulfeto de Glutationa/genética , Heme Oxigenase-1/genética , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/genética , Células Estreladas do Pâncreas/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genéticaRESUMO
In this study, the effects of pharmacological concentrations of melatonin (1 µM-1 mM) on human pancreatic stellate cells (HPSCs) have been examined. Cell type-specific markers and expression of melatonin receptors were analyzed by western blot analysis. Changes in intracellular free Ca2+ concentration were followed by fluorimetric analysis of fura-2-loaded cells. Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined by fluorescence techniques. Production of reactive oxygen species (ROS) was monitored following 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester and MitoSOX™ Red-derived fluorescence. Cell viability was studied using the AlamarBlue® test. Cultured cells expressed markers typical of stellate cells. However, cell membrane receptors for melatonin could not be detected. Thapsigargin, bradykinin, or melatonin induced changes in intracellular free Ca2+ concentration. In the presence of the indole, a decrease in the GSH/GSSG ratio was observed that depended on the concentration of melatonin used. Furthermore, the indole evoked a concentration-dependent increase in ROS production in the mitochondria and in the cytosol. Finally, melatonin decreased HPSC viability in a time and concentration-dependent manner. We conclude that melatonin, at pharmacological concentrations, induces changes in the oxidative state of HPSC. This might regulate cellular viability and could not involve specific plasma membrane receptors.
Assuntos
Glutationa/metabolismo , Melatonina/farmacologia , Células Estreladas do Pâncreas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dissulfeto de Glutationa/metabolismo , Humanos , Camundongos , Pâncreas/metabolismo , Células Estreladas do Pâncreas/citologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Ratos , Receptor MT1 de Melatonina/metabolismoRESUMO
Virus-like particles (VLPs), comprised of viral structural proteins devoid of genetic material, are tunable nanoparticles that can be chemically or genetically engineered, to be used as platforms for multimeric display of foreign antigens. Here, we report the engineering of chimeric VLPs, derived from rabbit hemorrhagic disease virus (RHDV) for presentation of foreign B-cell antigens to the immune system. The RHDV capsid comprises 180 copies of a single capsid subunit (VP60). To evaluate the ability of chimeric RHDV VLPs to elicit protective humoral responses against foreign antigens, we tested two B-cell epitopes: a novel neutralizing B-cell epitope, derived from feline calicivirus capsid protein, and a well characterized B-cell epitope from the extracellular domain of influenza A virus M2 protein (M2e). We generated sets of chimeric RHDV VLPs by insertion of the foreign B-cell epitopes at three different locations within VP60 protein (which involved different levels of surface accessibility) and in different copy numbers per site. The immunogenic potential of the chimeric VLPs was analyzed in the mouse model. The results presented here indicated that chimeric RHDV VLPs elicit potent protective humoral responses against displayed foreign B-cell epitopes, demonstrated by both, in vitro neutralization and in vivo protection against a lethal challenge.
Assuntos
Capsídeo/imunologia , Epitopos de Linfócito B/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Imunidade Humoral/imunologia , Animais , Linfócitos B/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Feminino , Vírus da Doença Hemorrágica de Coelhos/genética , Imunização , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Engenharia de Proteínas/métodos , Coelhos , Proteínas da Matriz Viral/imunologia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologiaRESUMO
Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD) vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154) linked to a T-cell epitope (3A 21 to 35) of FMD virus (FMDV) elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T) or four (B4T) copies of the B-cell epitope from type O FMDV (a widespread circulating serotype) were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFN γ . Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.
Assuntos
Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Feminino , Vírus da Febre Aftosa/classificação , Imunidade Humoral , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Interferon gama/biossíntese , Camundongos , Peptídeos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Gomez-Lopez-Hernandez syndrome, or cerebellotrigeminal-dermal dysplasia, is a rare neurocutaneous syndrome of trigeminal anesthesia, scalp alopecia and cerebellar anomalies. Other features include craniosynostosis, short stature, hypertelorism, down-slanting palpebral fissures, corneal opacities, mediofacial hypoplasia, and turri-brachycephaly. There have been 19 cases documented to date and we report on two additional male patients, 1 and 6 years of age, with typical features, mild mental retardation and dyspraxia. In both cases, MRI findings included rhombencephalosynapsis, a constant neuroimaging feature in this syndrome, comprising fusion of the cerebellar hemispheres with agenesis of the cerebellar vermis. Based on literature and our experience, we propose the presence of trigeminal anesthesia and/or partial alopecia of the scalp to complete the diagnosis of the syndrome.