Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 47
Filtrar
1.
Front Pharmacol ; 10: 935, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31551765

RESUMO

α-Hispanolol (α-H) is a labdane diterpenoid that has been shown to induce apoptosis in several human cancer cells. However, the effect of α-H in human glioblastoma cells has not been described. In the present work, we have investigated the effects of α-H on apoptosis, migration, and invasion of human glioblastoma cells with the aim of identifying the molecular targets underlying its mechanism of action. The results revealed that α-H showed significant cytotoxicity against human glioma cancer cell lines U87 and U373 in a concentration- and time-dependent manner. This effect was higher in U87 cells and linked to apoptosis, as revealed the increased percentage of sub-G1 population by cell cycle analysis and acquisition of typical features of apoptotic cell morphology. Apoptosis was also confirmed by significant presence of annexin V-positive cells and caspase activation. Pretreatment with caspase inhibitors diminishes the activities of caspase 8, 9, and 3 and maintains the percentage of viable glioblastoma cells, indicating that α-H induced cell apoptosis through both the extrinsic and the intrinsic pathways. Moreover, we also found that α-H downregulated the anti-apoptotic Bcl-2 and Bcl-xL proteins and activated the pro-apoptotic Bid and Bax proteins. On the other hand, α-H exhibited inhibitory effects on the migration and invasion of U87 cells in a concentration-dependent manner. Furthermore, additional experiments showed that α-H treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase MMP-2 and MMP-9 and increased the expression of TIMP-1 inhibitor, probably via p38MAPK regulation. Finally, xenograft assays confirmed the anti-glioma efficacy of α-H. Taken together, these findings suggest that α-H may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells. This research describes α-H as a new drug that may improve the therapeutic efficacy against glioblastoma tumors.

2.
Biochem Pharmacol ; 154: 373-383, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29870712

RESUMO

Macrophages are highly plastic cells that adopt different functional phenotypes in response to environmental signals. Classically activated macrophages (M1) exhibit a pro-inflammatory role, mediating host defense against microorganisms or tumor cells; whereas alternatively activated macrophages (M2) perform a range of physiological processes, including inflammation, wound repair and tissue remodeling. Interestingly, M2 macrophages have been involved in pathological settings such as tumor progression, parasitic infection and respiratory disorders. Consequently, the search of new agents able to control macrophage polarization is on the basis of new therapeutic strategies. In the present study, we have evaluated the effect of the hispanolone derivative 8,9-dehydrohispanolone-15,16-lactol (DHHL) on M2 macrophage polarization. Our results reveal that DHHL significantly inhibited IL-4- or IL-13-stimulated M2 macrophage activation, as showed by reduced expression of M2 markers. In addition, DHHL suppressed IL-4-induced STAT-6 and JAK-1 tyrosine phosphorylation, suggesting that this compound inhibited M2 polarization by suppressing the JAK-STAT signaling pathway. Finally, DHHL prevented eosinophil recruitment and the presence of F4/80+-CD206+ M2-like macrophages in an in vivo model of M2 polarization via administration of chitin. Collectively, these results confirm DHHL as a novel regulator of macrophage polarization suitable to design future therapies towards M2-macrophages mediated pathologies.


Assuntos
Polaridade Celular/efeitos dos fármacos , Quitina/toxicidade , Diterpenos/farmacologia , Janus Quinase 1/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Fator de Transcrição STAT6/antagonistas & inibidores , Animais , Polaridade Celular/fisiologia , Diterpenos/uso terapêutico , Relação Dose-Resposta a Droga , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Janus Quinase 1/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator de Transcrição STAT6/metabolismo
3.
Urol Oncol ; 36(5): 243.e21-243.e27, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29395956

RESUMO

BACKGROUND: Prostate cancer (PCa) is the most frequently diagnosed noncutaneous malignant tumor among males in the Western world. Prostate-specific antigen has been considered the most important biomarker for PCa detection; however, it lacks specificity, leading to the search for alternative biomarkers. Volatile organic compounds (VOCs) are released during cell metabolism and can be found in exhaled breath, urine, and other fluids. VOCs have been used in the diagnosis of lung, breast, ovarian, and colorectal cancers, among others. The objective of this study was to identify urinary VOCs that may be sensitive and specific biomarkers for PCa. METHODS: The study included 29 patients with PCa and 21 with benign prostatic hyperplasia. Urine samples were obtained from all participants before and after prostate massage. VOCs were identified by gas chromatography-mass spectrometry. IBM SPSS Statistics v.20 was used for statistical analysis. Sample normality and homogeneity of variances were studied and, according to the distribution normality, ANOVA or the Kruskal-Wallis test was applied to evaluate significant differences between groups. The Pearson test was used to establish correlations. RESULTS: Fifty-seven VOCs were identified. Samples gathered before prostate massage showed significant between-group differences in urinary levels of furan (P≤ 0.001), 2-ethylhexanol (P = 0.032), 3,5-dimethylbenzaldehyde (P = 0.027), santolin triene (P = 0.032), and 2,6-dimethyl-7-octen-2-ol (P = 0.003). Samples gathered after prostate massage showed significant differences in urinary levels of furan (P≤ 0.001), 3- methylphenol (P = 0.014), p-xylene (P = 0.002), phenol (P≤ 0.001), and 2-butanone (P = 0.001). CONCLUSIONS: Significant differences between PCa and BPH patients were found in urinary levels of certain VOCs both before and after prostate massage, supporting the proposal that VOCs may serve as PCa-specific biomarkers.


Assuntos
Biomarcadores Tumorais/urina , Furanos/urina , Hiperplasia Prostática/diagnóstico , Neoplasias da Próstata/diagnóstico , Compostos Orgânicos Voláteis/urina , Xilenos/urina , Idoso , Estudos de Casos e Controles , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Hiperplasia Prostática/urina , Neoplasias da Próstata/urina
4.
J Vis Exp ; (127)2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28994756

RESUMO

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Inflamação/imunologia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Humanos , Inflamação/sangue , Inflamação/patologia
5.
Sci Rep ; 6: 30004, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27481356

RESUMO

Although originally identified as a B cell differentiation factor, it is now known that mammalian interleukin-6 (IL-6) only regulates B cells committed to plasma cells in response to T-dependent (TD) antigens within germinal centers (GCs). Even though adaptive immunity is present in teleost fish, these species lack lymph nodes and GCs. Thus, the aim of the present study was to establish the role of trout IL-6 on B cells, comparing its effects to those induced by bacterial lipopolysaccharide (LPS). We demonstrate that the effects of teleost IL-6 on naïve spleen B cells include proliferation, activation of NF-κB, increased IgM secretion, up-regulation of Blimp1 transcription and decreased MHC-II surface expression that point to trout IL-6 as a differentiation factor for IgM antibody-secreting cells (ASCs). However, LPS induced the secretion of IgM without up-regulating Blimp1, driving the cells towards an intermediate activation state in which antigen presenting mechanisms are elicited together with antibody secretion and expression of pro-inflammatory genes. Our results reveal that, in trout, IL-6 is a differentiation factor for B cells, stimulating IgM responses in the absence of follicular structures, and suggest that it was after follicular structures appeared that this cytokine evolved to modulate TD responses within the GC.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunoglobulina M/biossíntese , Interleucina-6/farmacologia , Lipopolissacarídeos/farmacologia , Oncorhynchus mykiss/imunologia , Animais , Linfócitos B/citologia , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Imunoglobulina M/imunologia , Interleucina-6/imunologia , Lipopolissacarídeos/imunologia , NF-kappa B/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/biossíntese , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
6.
J Biol Chem ; 291(41): 21363-21374, 2016 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-27551041

RESUMO

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder characterized by insensitivity to noxious stimuli and variable intellectual disability (ID) due to mutations in the NTRK1 gene encoding the NGF receptor TrkA. To get an insight in the effect of NTRK1 mutations in the cognitive phenotype we biochemically characterized three TrkA mutations identified in children diagnosed of CIPA with variable ID. These mutations are located in different domains of the protein; L213P in the extracellular domain, Δ736 in the kinase domain, and C300stop in the extracellular domain, a new mutation causing CIPA diagnosed in a Spanish teenager. We found that TrkA mutations induce misfolding, retention in the endoplasmic reticulum (ER), and aggregation in a mutation-dependent manner. The distinct mutations are degraded with a different kinetics by different ER quality control mechanisms; although C300stop is rapidly disposed by autophagy, Δ736 degradation is sensitive to the proteasome and to autophagy inhibitors, and L213P is a long-lived protein refractory to degradation. In addition L213P enhances the formation of autophagic vesicles triggering an increase in the autophagic flux with deleterious consequences. Mouse cortical neurons expressing L213P showed the accumulation of LC3-GFP positive puncta and dystrophic neurites. Our data suggest that TrkA misfolding and aggregation induced by some CIPA mutations disrupt the autophagy homeostasis causing neurodegeneration. We propose that distinct disease-causing mutations of TrkA generate different levels of cell toxicity, which may provide an explanation of the variable intellectual disability observed in CIPA patients.


Assuntos
Autofagia , Hipo-Hidrose/enzimologia , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/enzimologia , Insensibilidade Congênita à Dor/enzimologia , Agregação Patológica de Proteínas/enzimologia , Deficiências na Proteostase/enzimologia , Receptor trkA/metabolismo , Adolescente , Substituição de Aminoácidos , Animais , Córtex Cerebral/enzimologia , Feminino , Células HeLa , Humanos , Hipo-Hidrose/genética , Masculino , Camundongos , Camundongos Mutantes , Doenças Neurodegenerativas/genética , Nociceptores/enzimologia , Insensibilidade Congênita à Dor/genética , Agregação Patológica de Proteínas/genética , Deficiências na Proteostase/genética , Receptor trkA/genética
7.
Oncotarget ; 7(41): 66835-66850, 2016 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-27572316

RESUMO

Tumor microenvironment has been described to play a key role in tumor growth, progression, and metastasis. Macrophages are a major cellular constituent of the tumor stroma, and particularly tumor associated macrophages (TAMs or M2-like macrophages) exert important immunosuppressive activity and a pro-tumoral role within the tumor microenvironment. Alternative-reading frame (ARF) gene is widely inactivated in human cancer. We have previously demonstrated that ARF deficiency severely impairs inflammatory response establishing a new role for ARF in the regulation of innate immunity. On the basis of these observations, we hypothesized that ARF may also regulates tumor growth through recruitment and modulation of the macrophage phenotype in the tumor microenvironment. Xenograft assays of B16F10 melanoma cells into ARF-deficient mice resulted in increased tumor growth compared to those implanted in WT control mice. Tumors from ARF-deficient mice exhibited significantly increased number of TAMs as well as microvascular density. Transwell assays showed crosstalk between tumor cells and macrophages. On the one hand, ARF-deficient macrophages modulate migratory ability of the tumor cells. And on the other, tumor cells promote the skewing of ARF-/- macrophages toward a M2-type polarization. In conclusion, these results demonstrate that ARF deficiency facilitates the infiltration of macrophages into the tumor mass and favors their polarization towards a M2 phenotype, thus promoting tumor angiogenesis and tumor growth. This work provides novel information about the critical role of ARF in the modulation of tumor microenvironment.


Assuntos
Macrófagos/imunologia , Melanoma Experimental/imunologia , Carga Tumoral/imunologia , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p14ARF/imunologia , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Humanos , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/classificação , Macrófagos/metabolismo , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/etiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Carga Tumoral/genética , Microambiente Tumoral/genética , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p14ARF/metabolismo
8.
Biochem J ; 473(14): 2061-71, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27154204

RESUMO

Endothelial activation contributes to lung inflammatory disorders by inducing leucocyte recruitment to pulmonary parenchyma. Consequently, vascular-targeted therapies constitute promising strategies for the treatment of inflammatory pathologies. In the present study, we evaluated the effect of 8,9-dehydrohispanolone-15,16-lactol diterpene (DT) on lung endothelium during inflammation. Lung endothelial cells pre-treated with DT and activated with lipopolysaccharide (LPS) or tumour necrosis factor-α (TNF-α) exhibited reduced expression of the pro-inflammatory cytokines Cxcl10, Ccl5 and Cxcl1, whereas the anti-inflammatory molecules IL1r2 and IL-10 were induced. Consistent with this result, DT pre-treatment inhibited nuclear factor κB (NF-κB) nuclear translocation, by interfering with IκBα phosphorylation, and consequently NF-κB transcriptional activity in endothelium activated by LPS or TNF-α. Furthermore, DT, probably through p38 signalling, induced transcriptional activation of genes containing activator protein 1 (AP-1)-binding elements. Inhibition of p38 prevented IL1r2 mRNA expression in endothelium incubated with DT alone or in combination with LPS or TNF-α. Accordingly, conditioned medium (CM) from these cells failed to stimulate leucocytes as measured by a reduction in adhesive ability of the leucocyte cell line J774 to fibronectin (FN). Additionally, DT reduced the expression of the endothelial adhesion molecules E-selectin, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) after activation. Similarly, expression of VCAM-1 and ICAM-1 molecules on the lung endothelial layer of C57/BL6 mice pre-treated with DT and challenged with LPS were unchanged. Finally, inhibition of vascular adhesion molecule expression by DT decreased the interaction of J774 cells with lung endothelial cells in an inflammatory environment. Our findings establish DT as a novel endothelial inhibitor for the treatment of inflammatory-related diseases triggered by Gram-negative bacteria or by the associated cytokine TNF-α.


Assuntos
Diterpenos/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inflamação/prevenção & controle , Lipopolissacarídeos/farmacologia , Animais , Linhagem Celular , Quimiocina CCL5/metabolismo , Quimiocina CXCL1/metabolismo , Quimiocina CXCL10/metabolismo , Células Endoteliais/imunologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
9.
Free Radic Biol Med ; 93: 41-51, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26828021

RESUMO

UNLABELLED: Peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) is a regulator of mitochondrial metabolism and reactive oxygen species (ROS) that is known to play a relevant role in angiogenesis. AIMS: This study aims to investigate the role of ROS on the regulation by PGC-1α of angiogenesis. METHODS AND RESULTS: We found that endothelial cells (ECs) from mice deleted for PGC-1α display attenuated adhesion to the extracellular matrix, together with slower and reversible spreading. Structural analysis demonstrates unstable formation of focal adhesions, defective cytoskeleton reorganization in response to cellular matrix adhesion, cell migration and cell-cell adhesion. Confluent cultures showed also a reduction of membrane bound VE-cadherin, suggesting defective inter-cellular junction formation. Functional consequences included impaired directional migration, and enhanced tip phenotype in aortic explants sprouting assays. At the molecular level, PGC-1α-deleted ECs exhibit a constitutive activation of the vascular endothelial growth factor-A (VEGF-A) signaling pathway and a defective response to VEGF-A. All these alterations are partially reversed by administration of the antioxidant EUK-189. The contribution of mitochondrial ROS and NOX activation was confirmed using a mitochondrial targeted antioxidant (MitoTEMPO) and a NOX inhibitor (VAS-2870). These results indicate that elevated production of ROS in the absence of PGC-1α is a key factor in the alteration of the VEGF-A signaling pathway and the capacity of endothelial cells to form stable interactions with other endothelial cells and with the extracellular matrix. Our findings show that PGC-1α control of ROS homeostasis plays an important role in the control of endothelial response to VEGF-A.


Assuntos
Neovascularização Patológica/genética , Estresse Oxidativo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/biossíntese , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Antígenos CD/metabolismo , Antioxidantes/metabolismo , Caderinas/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Compostos Organometálicos/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Salicilatos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Blood Cells Mol Dis ; 55(3): 220-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26227851

RESUMO

We studied whether chemokines may have a role in relapses in childhood acute lymphoblastic leukemia (ALL). We compared the levels of chemokine receptors in marrow samples from 82 children with ALL at diagnosis versus 15 at relapses, and quantified the levels of chemokines in central system fluid (CSF) samples. The functional role of specific chemokines was studied in vitro and in vivo. The expression of some chemokine receptors was upregulated upon leukemic relapse, both in B- and in T-ALL, and in cases of medullary and extramedullary involvement. CXCL10 induced chemotaxis in leukemic cell lines and in primary leukemic cells, depending upon the levels of CXCR3 expression. CXCL10 specifically diminished chemotherapy-induced apoptosis on ALL cells expressing CXCR3, partially inhibiting caspase activation and maintaining the levels of the antiapoptotic protein Bcl-2. Finally, immunodeficient mice engrafted with CXCR3-expressing human leukemic cells showed decreased infiltration of marrow, spleen, and CNS after receiving a CXCR3-antagonist molecule. CXCR3 signaling in ALL may have a dual function: chemotactic for the localisation of leukemic blasts in specific niches, and it may also confer resistance to chemotherapy, enhancing the chances for relapses.


Assuntos
Antineoplásicos/farmacologia , Quimiocinas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Antineoplásicos/uso terapêutico , Quimiotaxia de Leucócito , Criança , Resistencia a Medicamentos Antineoplásicos , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Recidiva
11.
Rev. int. androl. (Internet) ; 13(2): 74-78, abr.-jun. 2015. ilus
Artigo em Espanhol | IBECS | ID: ibc-141743

RESUMO

El absceso prostático es un proceso poco común que se ha asociado a sondajes permanentes, instrumentación genitourinaria, prostatitis crónicas, diabetes mellitus, síndrome de inmunodeficiencia adquirida, hemodiálisis u otras condiciones que comprometan la inmunidad. Actualmente Escherichia coli y otros agentes gramnegativos son los principales responsables en el desarrollo de los abscesos prostáticos. La ecografía transrectal es considerada una prueba sensible para el diagnóstico, así como para adoptar una actitud terapéutica. El tratamiento estándar del absceso prostático, en la mayoría de los casos, consiste en una amplia cobertura antibiótica y drenaje del absceso mediante diferentes técnicas y vías de acceso. Presentamos el caso de un paciente de 74 años con un absceso prostático producido por Enterococcus faecalis, el cual fue tratado con antibioterapia y drenaje percutáneo transperineal ecodirigido (AU)


Prostatic abscess is a rare process that has been associated with permanent catheterization, genitourinary instrumentation, chronic prostatitis, diabetes mellitus, acquired immunodeficiency, hemodialysis or other conditions that compromise immunity syndrome. Currently, Escherichia coli and other Gram negative agents are primarily responsible for the development of prostatic abscess. Transrectal ultrasound is considered a sensitive test for the diagnosis as well as the therapeutic approach. Standard treatment of prostatic abscess, in most cases, consisting of a wide spectrum antibiotics and abscess drainage using different techniques and paths. We report the case of a patient of 74 years with a prostatic abscess caused by Enterococcus faecalis which was treated with antibiotics and ultrasound-guided transperineal percutaneous drainage (AU)


Assuntos
Idoso , Humanos , Masculino , Abscesso Abdominal/cirurgia , Doenças Prostáticas/cirurgia , Sucção/métodos , Ultrassom Focalizado Transretal de Alta Intensidade/métodos
12.
Eur J Immunol ; 45(1): 273-86, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25328047

RESUMO

Alternative activation of macrophages plays an important role in a range of physiological and pathological processes. This alternative phenotype, also known as M2 macrophages, is induced by type 2 cytokines such as IL-4. The binding of IL-4 to its receptor leads to activation of two major signaling pathways: STAT-6 and PI3K. However, recent studies have described that p38 MAPK might play a role in IL-4-dependent signaling in some cells, although its role in macrophages is still controversial. In this study, we investigated whether p38 MAPK plays a role in the polarization of macrophages in mice. Our results reveal that IL-4 induces phosphorylation of p38 MAPK in thioglycollate-elicited murine peritoneal macrophages, in addition to STAT-6 and PI3K activation. Furthermore, p38 MAPK inactivation, by gene silencing or pharmacological inhibition, suppressed IL-4-induced typical M2 markers, indicating the involvement of p38 MAPK in the signaling of IL-4 leading to M2-macrophage polarization. Moreover, p38 MAPK inhibition blocked phosphorylation of STAT-6 and Akt, suggesting that p38 MAPK is upstream of these signaling pathways. Finally, we show that in an in vivo model of chitin-induced M2 polarization, p38 MAPK inhibition also diminished activation of M2 markers. Taken together, our data establish a new role for p38 MAPK during IL-4-induced alternative activation of macrophages.


Assuntos
Interleucina-4/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Receptores de Interleucina-4/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Animais , Quitina/farmacologia , Regulação da Expressão Gênica , Interleucina-4/genética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosforilação , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Interleucina-4/genética , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/imunologia , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia
15.
Fish Shellfish Immunol ; 38(1): 255-64, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24698994

RESUMO

In the current work, we have established and characterized a novel cell line from rainbow trout (Oncorhynchus mykiss). The cell line, designated as RTH (rainbow trout heart), was obtained by immortalizing heart cells with recombinant retroviruses that transduced polyoma middle T antigen. This is the first time such a strategy is used to obtain an immortalized fish cell line. The cells showed an endothelial-like morphology and characteristics, constitutively transcribing collagen, selectin and VCAM (vascular cell adhesion molecule), as well as different chemokines and chemokine receptors, but not cytokeratin. As already described for heart endothelial cells, RTH cells actively phagocytized latex beads. Furthermore, RTH cells showed a high susceptibility to viral hemorrhagic septicemia virus (VHSV). VHSV modulated the transcription of Mx, major histocompatibility complex II (MHC-II), VCAM and many of the chemokine and chemokine receptors expressed in these cells. Therefore, RTH cells constitute an excellent model to study the immune regulation of endothelial cells in fish and their role in leukocyte extravasation.


Assuntos
Miócitos Cardíacos/citologia , Novirhabdovirus/fisiologia , Oncorhynchus mykiss , Animais , Linhagem Celular , Regulação da Expressão Gênica , Miócitos Cardíacos/fisiologia , Retroviridae
16.
Free Radic Biol Med ; 75 Suppl 1: S5, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26461397

RESUMO

Peroxisome proliferator activated receptor g co-activator 1alpha (PGC-1α) is a regulator of oxidative metabolism and reactive oxygen species (ROS) homeostasis that has been show to play a relevant role in angiogenesis. PGC-1α KO mice show reduced vascular density in the retinas and KO primary vascular endothelial cells (ECs) migrate faster than the wild type, an effect that can be rescued by antioxidants, suggesting that excessive ROS levels might be relevant in PGC-1 α role in angiogenesis. This study aims to investigate the role of ROS homeostasis on the regulation by PGC-1 α of angiogenesis. We found that endothelial cells (ECs) from mice deleted for PGC-1 α display attenuated adhesion to the extracellular matrix, together with slower spreading, reduced formation of cellular junctions, a disorganized cytoskeleton and random motility, and a enhanced tip phenotype. Aditionally, PGC-1 α -deleted ECs exhibit an altered response to vascular endothelial growth factor-A (VEGF-A). In vivo, deletion of PGC-1 α results in addition to reduced retinal vascular density, sparse pericyte coverage. Exposure of PGC-1 α deleted mice to hyperoxia during retinal vascular development exacerbates these vascular abnormalities and mice show extensive retinal hemorrhaging, with highly unstructured areas and very poor perfusion, compared with wild-type mice. Structural analysis demonstrates a reduction of endothelial VE-cadherin, suggesting defective inter-cellular junctions. Interestingly, this hyperoxia-induced phenotype is partially reversed by antioxidant administration, indicating that elevated production of mitochondrial reactive oxygen species (ROS) in the absence of PGC-1 α is functionally important. Finally, in vitro studies show that antioxidant treatment improves VEGF-A signaling, suggesting that toxic effect of ROS may be caused by the alteration of the VEGF-A signaling pathway. In summary, our findings indicate that PGC-1 α control of ROS homeostasis plays an important role in the control of de novo angiogenesis, and is required for vascular stability.

17.
J Immunol ; 192(3): 1257-66, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24353268

RESUMO

Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyer's patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7(+) cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed <5% CCR7(+) cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7(+) cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD(+) cells in the gills expressed CCR7. Intriguingly, the IgD(+)CCR7(+) population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7(+) cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD(+)memIgM(-) B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD(+)IgM(-) subsets in mammals are discussed.


Assuntos
Subpopulações de Linfócitos B/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Brânquias/metabolismo , Imunoglobulina D/análise , Oncorhynchus mykiss/metabolismo , Receptores CCR7/biossíntese , Animais , Especificidade de Anticorpos , Feminino , Brânquias/citologia , Brânquias/crescimento & desenvolvimento , Rim Cefálico/citologia , Rim Cefálico/crescimento & desenvolvimento , Rim Cefálico/metabolismo , Septicemia Hemorrágica Viral/imunologia , Imunoglobulina M/análise , Tecido Linfoide/citologia , Tecido Linfoide/crescimento & desenvolvimento , Tecido Linfoide/metabolismo , Novirhabdovirus/fisiologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/crescimento & desenvolvimento , Oncorhynchus mykiss/imunologia , Especificidade de Órgãos , Receptores CCR7/genética , Receptores CCR7/imunologia
18.
PLoS One ; 8(12): e82737, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24324826

RESUMO

TWO major classes of b lymphocytes have been described to date in rainbow trout: IgM(+) and IgT(+) cells. IgM(+) cells are mainly localized in the spleen, peripheral blood and kidney but are also found in other tissues. However, differences among IgM(+) cell populations attending to its location are poorly defined in fish. Thus, the aim of this work was to characterize the expression of different immune molecules such as chemokine receptors, Toll-like receptors (TLRs) and transcription factors on sorted IgM(+) lymphocytes from different rainbow trout tissues. IgM(+) populations from blood, spleen, kidney, gills, intestine and liver were isolated by cell sorting and the constitutive levels of transcription of these genes evaluated by real-time PCR. To further characterize B cells, we identified an MS4A sequence. In humans, the MS4A family includes several genes with immune functions, such as the B cell marker CD20 or FcRß. Subsequently, we have also evaluated the mRNA levels of this MS4A gene in the different IgM(+) populations. The relevant differences in transcriptional patterns observed for each of these IgM(+) populations analyzed, point to the presence of functionally different tissue-specific B cell populations in rainbow trout. The data shown provides a pattern of genes transcribed in IgM(+) B cells not previously revealed in teleost fish. Furthermore, the constitutive expression of all the TLR genes analyzed in IgM(+) cells suggests an important role for these cells in innate immunity.


Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Heterogeneidade Genética , Imunoglobulina M/imunologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Transcrição Genética , Sequência de Aminoácidos , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Imunoglobulina M/metabolismo , Imunofenotipagem , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Oncorhynchus mykiss/metabolismo , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Filogenia , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
19.
Pediatr Neurol ; 49(6): 451-457.e1, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24080276

RESUMO

BACKGROUND: Rasmussen encephalitis is a devastating pediatric syndrome of unknown etiology that is characterized by progressive loss of neurological function and intractable focal epilepsy. Cytotoxic T lymphocytes have an active role in the pathogenic process of Rasmussen encephalitis. We studied the implication of CXCL10-CXCR3, a chemotactic axis involved in the pathogenesis of several cases of immune encephalitis. METHODS: We analyzed surgical specimens of children with Rasmussen encephalitis, and performed functional in vitro assays to test the implications of the pathological findings. RESULTS: We found that cytotoxic T lymphocytes infiltrating the damaged areas of primary biopsies expressed CXCR3, whereas neurons and astrocytes in the same areas expressed CXCL10. The in vitro assays demonstrated we found that astrocytes upregulated the expression of CXCL10 messenger RNA and the release of CXCL10 to the supernatants on stimulation with polyinosinic-polycyticylic acid, a synthetic double-stranded RNA that mimics infections with either RNA or DNA viruses. Activated T lymphocytes responded to the production of CXCL10 by astrocytes by increasing their migration in a transwell assay. Finally, the chemotaxis induced by the stimulated astrocytes was completely abrogated in the presence of a small molecule antagonist of CXCR3. CONCLUSIONS: Our results suggest that the CXCR3-CXCL10 axis has a role in recruiting pathogenic T lymphocytes into the brains of patients with Rasmussen encephalitis. This chemotactic mechanism may be targeted pharmacologically.


Assuntos
Encéfalo/patologia , Quimiocina CXCL10/metabolismo , Encefalite/metabolismo , Receptores CXCR3/metabolismo , Adolescente , Amidas/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Encefalite/patologia , Feminino , Humanos , Indutores de Interferon/farmacologia , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Poli I-C/farmacologia , Compostos de Amônio Quaternário/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Linfócitos T/patologia
20.
Rev. int. androl. (Internet) ; 11(2): 75-78, abr.-jun. 2013. ilus
Artigo em Espanhol | IBECS | ID: ibc-114899

RESUMO

El absceso testicular es una enfermedad poco frecuente, y la mayoría de los casos son una complicación de casos avanzados o no tratados de orquiepididimitis. Los síntomas clínicos y la exploración física no siempre permiten un diagnóstico de certeza, ya que el dolor y la inflamación limitan la palpación correcta del contenido escrotal. De ahí que la ecografía escrotal sea la técnica de imagen de primera elección para determinar la naturaleza de una masa escrotal y también para detectar la presencia de abscesos escrotales. Aunque el tratamiento inicial es médico, en ocasiones hay que recurrir a la orquiectomía. El objetivo de este trabajo es proponer el abordaje inguinal para el tratamiento quirúrgico de los abscesos testiculares, el cual permite mantener aislado el proceso infeccioso y evitar la «violación del escroto» en caso de tratarse de un tumor. Presentamos 2 casos de absceso testicular tratados de manera satisfactoria mediante orquiectomía inguinal (AU)


Testicular abscess is a rare condition that often arises due to complications of untreated or advanced orchiepididymitis. Clinical symptoms and physical exam do not always provide an accurate diagnosis because the pain and swelling often limit adequate palpation of the scrotal contents. Therefore, scrotal ultrasound is the imaging technique of choice to determine the nature of the scrotal mass and also to detect presence of scrotal abscesses. Although initial treatment is medical, it is sometimes necessary to resort to orchiectomy. This purpose of this paper is to propose inguinal approach for the surgical treatment of testicular abscesses. This approach makes it possible to maintain the infectious process isolated and thus avoid ‘‘violation of the scrotum’’ in the case of a tumor.We report two cases of testicular abscess successfully treated by inguinal orchiectomy (AU)


Assuntos
Humanos , Masculino , Pessoa de Meia-Idade , Idoso , Doenças Testiculares/cirurgia , Doenças Testiculares , Abscesso/complicações , Abscesso/diagnóstico , Abscesso/cirurgia , Orquiectomia/instrumentação , Orquiectomia/métodos , Orquiectomia , Orquite/patologia , Orquite/cirurgia , Orquite , Gentamicinas/uso terapêutico , Testículo/patologia , Testículo , Escroto/cirurgia , Escroto
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA