Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 315
Filtrar
Mais filtros










Intervalo de ano de publicação
1.
Pathol Res Pract ; 215(6): 152383, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30890279

RESUMO

AIM: Arginase-1 (Arg-1) metabolizes l-arginine to l-ornithine and urea. It has been documented to have a role in various malignancies. However, the relationship between Arg-1 expression and clinicopathological characteristics of colorectal cancer (CRC) patients remains to be elucidated. The present study aimed to analyze the expression and prognostic value of Arg-1 in patients with CRC. MATERIAL AND METHODS: The mRNA and protein expressions of Arg-1 in fresh colorectal cancer tissue specimens and the corresponding noncancerous tissue specimens were examined by RT-qPCR (n = 24) and western blot analysis (n = 17). Arg-1 expression levels were determined in paraffin-embedded CRC tissue specimens (n = 236) by immunohistochemistry. The associations of Arg-1 expression and clinicopathological features and clinical prognosis in 236 CRC patients were analyzed. RESULTS: The expression levels of Arg-1 were significantly higher in the CRC tissues compared with the matched noncancerous tissues, and elevated Arg-1 expression was remarkably associated with stage III-IV tumors (P = 0.007), lymph node metastasis (P = 0.019) and a plasma albumin concentration <35 g/l (P = 0.022). Kaplan-Meier analysis indicated that Arg-1 overexpression was associated with adverse prognoses for overall survival (OS) (P < 0.001) and disease-free survival (DFS) (P < 0.001) in all cases. Further analysis revealed that the patients with high Arg-1 expression had significantly shorter OS and DFS at the advanced stages (III + IV) (P = 0.032 for OS, and P = 0.012 for DFS) but not at the early stages (I + II) (P = 0.194 for OS, and P = 0.065 for DFS). Multivariate analysis revealed that Arg-1 overexpression was an independent prognostic factor for OS (P = 0.002) and DFS (P < 0.001) in patients with CRC. CONCLUSION: The data indicated that Arg-1 overexpression in CRC may be a marker that can discriminate subgroups of patients with a poor prognosis.


Assuntos
Arginase/biossíntese , Biomarcadores Tumorais/análise , Neoplasias Colorretais/patologia , Adulto , Idoso , Arginase/análise , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Regulação para Cima
2.
J Am Heart Assoc ; 7(18): e009579, 2018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30371203

RESUMO

Background Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase ( eNOS ). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin-dependent kinase II / eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence-activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin-dependent kinase II . These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II -null mice resulted in reduced p32 protein level. In the aortas of wild-type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO -dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE-/-) mice fed a high-cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin-dependent kinase II -dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca2+-dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.


Assuntos
Arginase/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Endotélio Vascular/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Arginase/biossíntese , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Endotélio Vascular/patologia , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/patologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação , RNA/genética , Transdução de Sinais
3.
Biomed Res Int ; 2018: 2109865, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30320132

RESUMO

ARG1, which encodes Arginase1, is expressed in the liver cytoplasm and plays a major role in the hepatic urea cycle. The past research works shed light on the fact that ARG1 participates in anti-inflammation, tumor immunity, and immunosuppression-related diseases. Nevertheless, the concrete role and clinical significance of ARG1 in the progression of hepatocellular carcinoma (HCC) remain unclear. Herein, we aimed at examining the expression and clinicopathological significance of ARG1 in HCC, together with determining the effect of ARG1 on the progression and metastasis of HCC. In the current study, evaluation of the expression of ARG1 and clinicopathological significance of ARG1 was carried out in the human HCC tissues microarray, and the ARG1 overexpression vector and shRNA-ARG1 plasmids were constructed for the assessment of the concrete effect of ARG1 on cellular behaviors of Huh7 cells. As our data revealed, ARG1 was significantly downregulated in HCC, and the higher expression of ARG1 was positively correlated with more aggressive tumor growth, size, ALT, and GGT level. Significantly, we found that the high expression of ARG1 was correlated with poor DFS of HCC patients. Besides, in vitro study revealed that overexpression of ARG1 could enhance arginase activity, cell viability, migration, and invasion of Huh7 cells, and loss-of-function of ARG1 by shRNA interference could inhibit these cellular behaviors. Additionally, overexpression of ARG1 led to a significant increase in the expression of Vimentin, N-cadherin, and ß-catenin both at protein and mRNA levels, which promotes the EMT process. On the other hand, these proteins' expression was significantly downregulated in ARG1 silenced Huh7 cells. Besides, the level of E-cadherin protein was upregulated in ARG1 knocked down cells. In conclusion, ARG1 might play a pivotal role as an oncogene in the progression of HCC through promoting the EMT process.


Assuntos
Arginase/biossíntese , Carcinoma Hepatocelular/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/enzimologia , Proteínas de Neoplasias/biossíntese , Adulto , Idoso , Arginase/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular , Sobrevivência Celular , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Proteínas de Neoplasias/genética
4.
J Neuroimmunol ; 323: 94-104, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30196840

RESUMO

Activation states of immune cells (among them, the so-called pro- or anti-inflammatory states) influence the pathogenesis of multiple sclerosis (MS). The neuropeptide calcitonin gene-related peptide (CGRP) can exert a pro- or anti-inflammatory role in a context-dependent manner. In mice CGRP was found to attenuate the development of experimental autoimmune encephalomyelitis (EAE, a common MS animal model). We analyzed CGRP effects on the expression of cytokines and markers of activation states, as well as its intracellular cascade, following intrathecal administration during EAE immunization. Real Time quantitative-PCR (RT-PCR) showed that IL-1beta and IL-6 (associated to a pro-inflammatory state in EAE), but also Ym1 (also known as Chil3), Arg1 and CD163 (associated to an anti-inflammatory state in EAE) were decreased in the encephalon (devoid of cerebellum). In the cerebellum itself, IL-1beta and Ym1 were decreased. TNF-alpha (associated to a pro-inflammatory state in EAE), but also IL-10 (associated to another type of anti-inflammatory state) and BDNF were unchanged in these two regions. No changes were detected in the spinal cord. Additional tendencies toward a change (as revealed by RT-PCR) were again decreases: IL-10 in the encephalon and Arg1 in the spinal cord. CGRP decreased percentage of Ym1+/CD68+ immunoreactive cells and cell density of infiltrates in the cervical spinal cord pia mater. Instead, Ym1 in the underlying parenchyma and at thoracic and lumbar levels, as well as Arg1, were unchanged. In cultured microglia the neuropeptide decreased Ym1, but not Arg1, immunoreactivity. Inducible NOS (iNOS) was unchanged in spinal cord microglia and astrocytes. The neuropeptide increased the activation of ERK1/2 in the astrocytes of the spinal cord and in culture, but did not influence the activation of ERK1/2 or p38 in the spinal cord microglia. Finally, in areas adjacent to infiltration sites CGRP-treated microglia showed a larger ramification radius. In conclusion, CGRP-induced EAE amelioration was associated to a concomitant, context-dependent decrease in the expression of markers belonging to both pro- or anti-inflammatory activation states of immune cells. It can be hypothesized that CGRP-induced EAE attenuation is obtained through a novel mechanism that promotes down-regulation of immune cell activation that facilitates the establishment of a beneficial environment in EAE provided possibly also by other factors.


Assuntos
Arginase/antagonistas & inibidores , Peptídeo Relacionado com Gene de Calcitonina/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Interleucina-1beta/antagonistas & inibidores , Interleucina-6/antagonistas & inibidores , Lectinas/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Arginase/biossíntese , Arginase/genética , Biomarcadores/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Células Cultivadas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Expressão Gênica , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Interleucina-6/biossíntese , Interleucina-6/genética , Lectinas/biossíntese , Lectinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética , beta-N-Acetil-Hexosaminidases/biossíntese , beta-N-Acetil-Hexosaminidases/genética
5.
J Am Coll Cardiol ; 72(7): 769-780, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30092954

RESUMO

BACKGROUND: Cardiovascular complications are major clinical problems in type 2 diabetes mellitus (T2DM). The authors previously demonstrated a crucial role of red blood cells (RBCs) in control of cardiac function through arginase-dependent regulation of nitric oxide export from RBCs. There is alteration of RBC function, as well as an increase in arginase activity, in T2DM. OBJECTIVES: The authors hypothesized that RBCs from patients with T2DM induce endothelial dysfunction by up-regulation of arginase. METHODS: RBCs were isolated from patients with T2DM and age-matched healthy subjects and were incubated with rat aortas or human internal mammary arteries from nondiabetic patients for vascular reactivity and biochemical studies. RESULTS: Arginase activity and arginase I protein expression were elevated in RBCs from patients with T2DM (T2DM RBCs) through an effect induced by reactive oxygen species (ROS). Co-incubation of arterial segments with T2DM RBCs, but not RBCs from age-matched healthy subjects, significantly impaired endothelial function but not smooth muscle cell function in both healthy rat aortas and human internal mammary arteries. Endothelial dysfunction induced by T2DM RBCs was prevented by inhibition of arginase and ROS both at the RBC and vascular levels. T2DM RBCs induced increased vascular arginase I expression and activity through an ROS-dependent mechanism. CONCLUSIONS: This study demonstrates a novel mechanism behind endothelial dysfunction in T2DM that is induced by RBC arginase I and ROS. Targeting arginase I in RBCs may serve as a novel therapeutic tool for the treatment of endothelial dysfunction in T2DM.


Assuntos
Arginase/biossíntese , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/enzimologia , Endotélio Vascular/enzimologia , Eritrócitos/enzimologia , Idoso , Animais , Arginase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/farmacologia
6.
Pathol Res Pract ; 214(8): 1179-1184, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29970307

RESUMO

Arginase 1 (Arg1) is involved in dampening the response of antitumor T lymphocytes. Arg1 expression has been reported in a variety of cancer cell lines and tumor-associated myeloid-derived cells. However, its examination in situ in tumor microenvironment is poorly investigated. We examined the Arg1-positive cells in tumor microenvironment of gastric carcinomas (GCs), colorectal carcinomas (CRCs) and prostate carcinomas (PCs), and analyzed their clinicopathological significance. Immunohistochemical staining for Arg1 was done in 60 GCs, 38 gastric adenomas, 40 CRCs, 10 colonic adenomas, 36 PCs, and 15 benign prostatic hyperplasia (BPH). Arg1 expression was predominantly localized in tumor microenvironment and the stroma of nonneoplastic tissues. Cells with Arg1 expression were mostly leukocytes, morphologically resembling polymorphonuclear neutrophils, and showed CD15 expression. Arg1 expression was focally expressed in cancer cells of 6 PCs, but not in those of GCs and CRCs. Arg1-positive cells were significantly more infiltrated in tumors than adenomas and nonneoplastic tissues, such as BPH, intestinal metaplasia and adjacent tissues. There were no significant findings between them and clinicopathological parameters, except for the relationship to gender and tumor differentiation in CRCs. These findings suggest that Arg1-positive cells in tumor microenvironment is involved in the occurrence of GCs, CRCs, and PCs. More expansive studies are necessary to better elucidate their clinicopathological significance in carcinomas.


Assuntos
Arginase/biossíntese , Carcinoma/patologia , Neoplasias Colorretais/patologia , Neoplasias da Próstata/patologia , Neoplasias Gástricas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Carcinoma/enzimologia , Neoplasias Colorretais/enzimologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/patologia , Neoplasias da Próstata/enzimologia , Neoplasias Gástricas/enzimologia , Microambiente Tumoral
7.
J Neuropathol Exp Neurol ; 77(7): 598-607, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29850876

RESUMO

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterized by aggregation of α-synuclein in oligodendrocytes to form glial cytoplasmic inclusions. According to the distribution of neurodegeneration, MSA is subtyped as striatonigral degeneration (SND), olivopontocerebellar atrophy (OPCA), or as combination of these 2 (mixed MSA). In the current study, we aimed to investigate regional microglial populations and gene expression in the 3 different MSA subtypes. Microscopy with microglial marker Iba-1 combined with either proinflammatory marker CD68 or anti-inflammatory marker Arginase-1 was analyzed in control, SND, and OPCA cases (n = 5) using paraffin embedded sections. Western immunoblotting and cytokine array were used to determine protein expression in MSA and control brain regions. Gene expression was investigated using the NanoString nCounter Human Inflammation panel v2 mRNA Expression Assay. Analysis of neuropathological subtypes of MSA demonstrated a significant increase in microglia in the substantia nigra of OPCA cases. There was no difference in the microglial activation state in any region. Cytokine expression in MSA was comparable with controls. Decreased expression of CX3CL1 precursor protein and significantly greater CX3CR1 protein was found in MSA. NanoString analysis revealed the >2-fold greater expression of ARG1, MASP1, NOX4, PTGDR2, and C6 in MSA.


Assuntos
Encéfalo/patologia , Inflamação/genética , Inflamação/patologia , Atrofia de Múltiplos Sistemas/genética , Atrofia de Múltiplos Sistemas/patologia , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/biossíntese , Antígenos de Diferenciação Mielomonocítica/genética , Arginase/biossíntese , Arginase/genética , Proteínas de Ligação ao Cálcio , Quimiocinas/análise , Quimiocinas/biossíntese , Corpo Estriado/patologia , Citocinas/análise , Citocinas/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Proteínas dos Microfilamentos , Microglia/patologia , Atrofias Olivopontocerebelares/patologia , Substância Negra/patologia
8.
PLoS One ; 13(5): e0196921, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29771935

RESUMO

Macrophages play crucial roles in innate immune response and in the priming of adaptive immunity, and are characterized by their phenotypic heterogeneity and plasticity. Reprogramming intracellular metabolism in response to microenvironmental signals is required for M1/M2 macrophage polarization and function. Here we assessed the influence of iron on the polarization of the immune response in vivo and in vitro. Iron-enriched diet increased M2 marker Arg1 and Ym1 expression in liver and peritoneal macrophages, while iron deficiency decreased Arg1 expression. Under LPS-induced inflammatory conditions, low iron diet exacerbated the proinflammatory response, while the IL-12/IL-10 balance decreased with iron-rich diet, thus polarizing toward type 2 response. Indeed, in vitro macrophage iron loading reduced the basal percentage of cells expressing M1 co-stimulatory CD86 and MHC-II molecules. Further, iron loading of macrophages prevented the pro-inflammatory response induced by LPS through reduction of NF-κB p65 nuclear translocation with decreased iNOS, IL-1ß, IL-6, IL-12 and TNFα expression. The increase of intracellular iron also reduced LPS-induced hepcidin gene expression and abolished ferroportin down-regulation in macrophages, in line with macrophage polarization. Thus, iron modulates the inflammatory response outcome, as elevated iron levels increased M2 phenotype and negatively regulated M1 proinflammatory LPS-induced response.


Assuntos
Polaridade Celular , Ferro/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Arginase/biossíntese , Citocinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Ferro/farmacologia , Lectinas/biossíntese , Lipopolissacarídeos/toxicidade , Fígado/metabolismo , Fígado/patologia , Macrófagos Peritoneais/patologia , Camundongos , Óxido Nítrico Sintase Tipo II/biossíntese , beta-N-Acetil-Hexosaminidases/biossíntese
9.
Respir Res ; 19(1): 98, 2018 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-29792217

RESUMO

BACKGROUND: Asthma is a chronic respiratory condition, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. The hypothesis that the substantially increased expression of arginase 1 in activated macrophages limits the availability of L-arginine for nitric oxide synthesis, and thus increases AHR in lungs of mice with experimentally induced allergic asthma was recently refuted by several studies. In the present study, we tested the hypothesis that, instead, a low circulating concentration of arginine aggravates AHR in the same murine asthma model. Female FVB F/A2 tg/tg transgenic mice, which overexpress rat arginase 1 in their enterocytes, exhibit a ~ 50% decrease of their plasma L-arginine concentration. METHODS: Adult female F/A2 tg/tg mice and their wild-type littermates (F/A2 wt/wt ) were sensitized and challenged with ovalbumin (OVA/OVA). Lung function was assessed with the flexiVent™ system. Adaptive changes in the expression of arginine-metabolizing or -transporting enzymes, chemokines and cytokines, and lung histology were quantified with qPCR, ELISA, and immunohistochemistry, respectively. RESULTS: Reduction of circulating L-arginine concentration significantly increased AHR in OVA/OVA-treated mice and, to a lesser extent, even in PBS/OVA-treated mice. The pulmonary inflammatory response in OVA/OVA-treated F/A2 tg/tg and F/A2 wt/wt mice was comparable. OVA/OVA-treated F/A2 tg/tg mice differed from similarly treated female mice, in which arginase 1 expression in lung macrophages was eliminated, by a complete absence of an adaptive increase in the expression of arginine-metabolizing or -transporting enzymes. CONCLUSION: A reduction of the circulating L-arginine concentration rather than the macrophage-mediated increase of arginine catabolism worsens AHR.


Assuntos
Arginina/sangue , Asma/sangue , Pulmão/metabolismo , Hipersensibilidade Respiratória/sangue , Animais , Arginase/biossíntese , Arginina/deficiência , Asma/patologia , Hiper-Reatividade Brônquica/sangue , Hiper-Reatividade Brônquica/patologia , Feminino , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Hipersensibilidade Respiratória/patologia
10.
RNA Biol ; 15(6): 756-762, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29578372

RESUMO

mRNA based therapies hold great promise for the treatment of genetic diseases. However, this therapeutic approach suffers from multiple challenges including the short half-life of exogenously administered mRNA and subsequent protein production. Modulation of untranslated regions (UTR) represents one approach to enhance both mRNA stability and translation efficiency. The current studies describe and validate screening methods using a diverse set of 5'UTR and 3'UTR combinations for improved expression of the Arginase 1 (ARG1) protein, a potential therapeutic mRNA target. Data revealed a number of critical aspects which need to be considered when developing a screening approach for engineering mRNA improvements. First, plasmid-based screening methods do not correlate with protein expression driven by exogenously expressed mRNA. Second, improved ARG1 protein production was driven by increased translation and not improved mRNA stability. Finally, the 5' UTR appears to be the key driver in protein expression for exogenously delivered mRNA. From the testing of the combinatorial library, the 5'UTR for complement factor 3 (C3) and cytochrome p4502E1 (CYP2E1) showed the largest and most consistent increase in protein expression relative to a reference UTR. Collectively, these data provide important information for the development and optimization of therapeutic mRNAs.


Assuntos
Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Arginase , Complemento C3/genética , Citocromo P-450 CYP2E1/genética , Biossíntese de Proteínas/genética , Arginase/biossíntese , Arginase/genética
11.
Front Immunol ; 9: 50, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29422898

RESUMO

Although it has been demonstrated that cAMP pathway affect both adaptive and innate cell functions, the role of this pathway in the regulation of T-cell-mediated central nervous system (CNS) autoimmune inflammation, such as in experimental autoimmune encephalomyelitis (EAE), remains unclear. It is also unclear how cAMP pathway affects the function of CD4 T cells in vivo at the site of inflammation. We found that adenylyl cyclase activator Forskolin besides inhibition of functions autoimmune CD4 T cells also upregulated microRNA (miR)-124 in the CNS during EAE, which is associated with M2 phenotype of microglia/macrophages. Our study further established that in addition to direct influence of cAMP pathway on CD4 T cells, stimulation of this pathway promoted macrophage polarization toward M2 leading to indirect inhibition of function of T cells in the CNS. We demonstrated that Forskolin together with IL-4 or with Forskolin together with IL-4 and IFNγ effectively stimulated M2 phenotype of macrophages indicating high potency of this pathway in reprogramming of macrophage polarization in Th2- and even in Th1/Th2-mixed inflammatory conditions such as EAE. Mechanistically, Forskolin and/or IL-4 activated ERK pathway in macrophages resulting in the upregulation of M2-associated molecules miR-124, arginase (Arg)1, and Mannose receptor C-type 1 (Mrc1), which was reversed by ERK inhibitors. Administration of Forskolin after the onset of EAE substantially upregulated M2 markers Arg1, Mrc1, Fizz1, and Ym1 and inhibited M1 markers nitric oxide synthetase 2 and CD86 in the CNS during EAE resulting in decrease in macrophage/microglia activation, lymphocyte and CD4 T cell infiltration, and the recovery from the disease. Forskolin inhibited proliferation and IFNγ production by CD4 T cells in the CNS but had rather weak direct effect on proliferation of autoimmune T cells in the periphery and in vitro, suggesting prevalence of indirect effect of Forskolin on differentiation and functions of autoimmune CD4 T cells in vivo. Thus, our data indicate that Forskolin has potency to skew balance toward M2 affecting ERK pathway in macrophages and indirectly inhibit pathogenic CD4 T cells in the CNS leading to the suppression of autoimmune inflammation. These data may have also implications for future therapeutic approaches to inhibit autoimmune Th1 cells at the site of tissue inflammation.


Assuntos
Autoimunidade/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Macrófagos/classificação , Macrófagos/imunologia , Animais , Arginase/biossíntese , Autoimunidade/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Interferon gama/metabolismo , Interleucina-4/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/biossíntese , MicroRNAs/genética , Microglia/citologia , Microglia/imunologia , Receptores de Superfície Celular/biossíntese
12.
Front Immunol ; 9: 1, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29403488

RESUMO

The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF-/-) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF-/- mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.


Assuntos
Interleucina-6/imunologia , Fígado/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Fator de Necrose Tumoral alfa/genética , Animais , Arginase/biossíntese , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Receptor gp130 de Citocina/metabolismo , Inflamação/imunologia , Interleucina-4/metabolismo , Interleucina-6/biossíntese , Lectinas Tipo C/biossíntese , Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Fígado/parasitologia , Fígado/patologia , Ativação de Macrófagos/genética , Macrófagos/citologia , Lectinas de Ligação a Manose/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Carga Parasitária , Receptor de Fator Estimulador de Colônias de Macrófagos/biossíntese , Receptores de Superfície Celular/biossíntese , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT6/metabolismo
13.
Clin Exp Pharmacol Physiol ; 45(6): 556-562, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29266319

RESUMO

The L-arginine/NO pathway is an important regulator of pulmonary hypertension, the leading cause of mortality in patients with the chronic lung disease of prematurity, bronchopulmonary dysplasia. L-arginine can be metabolized by NO synthase (NOS) to form L-citrulline and NO, a potent vasodilator. Alternatively, L-arginine can be metabolized by arginase to form urea and L-ornithine, a precursor to collagen and proline formation important in vascular remodelling. In the current study, we hypothesized that C3H/HeN mice exposed to prolonged hyperoxia would have increased arginase expression and pulmonary vascular wall cell proliferation. C3H/HeN mice were exposed to 14 days of 85% O2 or room air and lung homogenates analyzed by western blot for protein levels of arginase I, arginase II, endothelial NOS (eNOS), ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), and α-smooth muscle actin (α-SMA). Hyperoxia did not change arginase I or eNOS protein levels. However, arginase II protein levels were 15-fold greater after hyperoxia exposure than in lungs exposed to room air. Greater protein levels of ODC and OAT were found in lungs following hyperoxic exposure than in room air animals. α-SMA protein levels were found to be 7-fold greater in the hyperoxia exposed lungs than in room air lungs. In the hyperoxia exposed lungs there was evidence of greater pulmonary vascular wall cell proliferation by α-SMA immunohistochemistry than in room air lungs. Taken together, these data are consistent with a more proliferative vascular phenotype, and may explain the propensity of patients with bronchopulmonary dysplasia to develop pulmonary hypertension.


Assuntos
Actinas/metabolismo , Arginase/biossíntese , Displasia Broncopulmonar/complicações , Displasia Broncopulmonar/metabolismo , Hiperóxia/complicações , Animais , Displasia Broncopulmonar/patologia , Proliferação de Células , Modelos Animais de Doenças , Indução Enzimática , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Ornitina Descarboxilase/metabolismo , Ornitina-Oxo-Ácido Transaminase/metabolismo
14.
Exp Neurol ; 301(Pt B): 120-132, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28843543

RESUMO

We studied the expression of pro- and anti-inflammatory molecules in microglia and infiltrating monocyte-derived macrophages after permanent Middle Cerebral Artery Occlusion (pMCAO). LysM-EGFP knock-in mice were used to distinguish between these two cell types, as peripheral myeloid cells are LysM-EGFP+, while microglia are not. This was confirmed with P2ry12 (a microglial specific marker), Iba-1 and EGFP immunostaining. The peak of LysM-EGFP+ myeloid cell infiltration was 72h post-ischemia, and were distributed evenly in the lesion core, surrounded by a dense region of microglia. Flow cytometry showed that a higher percentage of microglia expressed TNF-α at 3 (24.3% vs 1.4%) and 7 (18.8% vs 3.4%) days post-pMCAO as compared to infiltrating macrophages. Microglia and macrophages were purified by fluorescence activated cell sorting 72h post-ischemia to assess the mRNA expression of inflammatory markers. Macrophages upregulated expression of mRNA for arginase-1 (Arg-1) by 1000-fold, and IL-1ß by 90-fold as compared to microglia. At the protein level, a significantly number of macrophages expressed Arg-1, while few if any microglia expressed Arg-1. However, IL-1ß protein was not detected in macrophages by flow cytometry or immunofluorescence labeling of tissue sections. It was, however, detected in astrocytes along the lesion border. A PCR-array screen of 84 inflammatory genes revealed that pro-inflammatory chemokines and cytokines were predominantly upregulated in macrophages but down-regulated in microglia in the ischemic brain. Our results show clear differences in the inflammatory expression profiles between microglia and macrophages 72h post-ischemia which may shape repair and pro-regenerative mechanisms after stroke.


Assuntos
Isquemia Encefálica/patologia , Inflamação/patologia , Macrófagos/patologia , Microglia/patologia , Animais , Arginase/biossíntese , Arginase/genética , Quimiocinas/metabolismo , Doença Crônica , Citocinas/biossíntese , Técnicas de Introdução de Genes , Redes Reguladoras de Genes , Proteínas de Fluorescência Verde/genética , Infarto da Artéria Cerebral Média/patologia , Interleucina-1beta/biossíntese , Interleucina-1beta/genética , Masculino , Camundongos , Fator de Necrose Tumoral alfa/metabolismo
15.
Shock ; 50(2): 233-239, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-28953574

RESUMO

BACKGROUND: Sepsis is a major cause of acute kidney injury (AKI), with high rates of morbidity and mortality. M2 macrophages have been shown to play important roles in the secretion of anti-inflammatory and tissue repair mediators. In this study, we investigate the role of M2 macrophages in sepsis-induced AKI by depleting these cells in vivo through the systemic administration of liposomal clodronate (LC). METHODS: Male Sprague-Dawley rats were subjected to cecal ligation and puncture (CLP) or sham surgery. Biochemical and histological renal damage was assessed. Macrophage infiltration and M2 macrophage depletion were assessed by immunohistochemistry. RT-PCR was used to investigate the expression of the inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1), and found in inflammatory zone 1 (FIZZ1) mRNAs. Western blots were performed to assay the tissue levels of interleukin-10 (IL-10) and tumor necrosis factor alpha (TNF-α). RESULTS: M2 macrophages were obviously detected 72 h after sepsis-induced AKI. Kidney injury was more severe, renal function was decreased, and blood creatinine and blood urea nitrogen (BUN) levels were higher after M2 macrophage depletion. M2 macrophage depletion significantly inhibited the proliferation of tubular cells. M2 macrophage depletion also downregulated IL-10 expression and increased TNF-α secretion during sepsis-induced AKI. CONCLUSIONS: M2 macrophages attenuate sepsis-induced AKI, presumably by upregulating IL-10 expression and suppressing TNF-α secretion.


Assuntos
Lesão Renal Aguda/metabolismo , Macrófagos/metabolismo , Sepse/metabolismo , Lesão Renal Aguda/etiologia , Lesão Renal Aguda/patologia , Animais , Arginase/biossíntese , Regulação da Expressão Gênica , Interleucina-10/biossíntese , Macrófagos/patologia , Masculino , Óxido Nítrico Sintase Tipo II/biossíntese , Ratos , Ratos Sprague-Dawley , Sepse/complicações , Sepse/patologia , Fator de Necrose Tumoral alfa/biossíntese
16.
Pathog Dis ; 75(8)2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29045624

RESUMO

Leishmanioses are neglected diseases and the parasite Leishmania survives and proliferates within mononuclear phagocytes, particularly macrophages. In vitro studies of the immunology and cell biology of leishmaniosis are performed in murine peritoneum and bone marrow macrophages and immortalized cell lines despite the normal and injured tissue-specific heterogeneity of macrophages. In this work, we established an ex vivo methodology to culture lesional cells from BALB/c mice infected with Leishmania amazonensis. The cells were successfully isolated from footpad skin lesions and those exhibiting macrophage morphology were maintained in long-term culture (12 days), while the small number of lymphocytes, polymorphonuclear and unidentified cells died after 1 day of culture. The frequency of infected cells decreased over 2 days. Most lesional cells cultivated ex vivo were myeloid CD11b+ CD14+ F4/80+ CD68+ cells. Low levels of IFN-γ and IL-4, IL-10 production and low arginase and phagocytic activities were detected in ex vivo lesional cell cultures. The ex vivo model developed in this study open perspectives for studying the biology of leishmanial lesions in cellular subpopulations and at the single-cell level.


Assuntos
Antígenos de Superfície/imunologia , Leishmania/imunologia , Leishmaniose Cutânea/imunologia , Macrófagos Peritoneais/imunologia , Pele/citologia , Animais , Arginase/biossíntese , Técnicas de Cultura de Células , Células Cultivadas , Modelos Animais de Doenças , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Leishmaniose Cutânea/microbiologia , Linfócitos/microbiologia , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/microbiologia , Fagocitose/imunologia , Pele/patologia
17.
Tumour Biol ; 39(7): 1010428317712512, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28718378

RESUMO

Glioblastoma is the most common and malignant tumour that occurs primarily in nervous system and has a high morbidity. Research on glioblastoma has recently focused on human cytomegalovirus, belonging to the beta subfamily of Herpesviridae that plays crucial roles in cancer development and progression. This study aimed to investigate the role of human cytomegalovirus-associated microRNA-613 in glioblastoma. In this study, we demonstrate that microRNA-613 expression was frequently reduced in human cytomegalovirus-positive glioblastoma specimens/cells compared with human cytomegalovirus-negative glioblastoma tissue/cells, and a significant correlation was observed between the reduction in microRNA-613 expression and the presence of unfavourable variables, including tumour size (p = 0.0118), World Health Organization stage (p = 0.0169), the overall survival (p = 0.0107) and disease-free (p = 0.0159) survival of patients. Overexpression of microRNA-613 in the glioblastoma cell lines U87 and U251 retarded cell growth and induced cell apoptosis. Upregulation of microRNA-613 inhibited glioblastoma cell clone formation, invasion and migration. Furthermore, we demonstrated that arginase-2 was directly regulated by microRNA-613 and played an essential role in mediating the biological effects of microRNA-613 in glioblastoma. Re-expression of arginase-2 markedly reversed the inhibitory properties of microRNA-613 in glioblastoma cells. Taken together, our data provide compelling evidence that human cytomegalovirus reduced the level of microRNA-613 which functions as an anti-onco-miRNA in glioblastoma, primarily by downregulating the expression of arginase-2.


Assuntos
Arginase/biossíntese , Citomegalovirus/genética , Glioblastoma/genética , MicroRNAs/genética , Adulto , Idoso , Apoptose/genética , Arginase/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Citomegalovirus/patogenicidade , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Glioblastoma/virologia , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Invasividade Neoplásica/genética
18.
Nat Prod Commun ; 12(1): 111-114, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30549841

RESUMO

The anti-fatigue effect was investigated of the probiotic supplement, OM-X®, on forced swimming capacity in mice. Mice were administered either vehicle (distilled water; DW) or OM-X® (85 mg/kg body weight) by gavage for 4 weeks. Forced swimming tests were conducted weekly using the Ishihara-modified Matsumoto swimming pool. The endurance swimming time of the final forced swimming exercise in mice fed with OM-X® group showed an approximately 2-fold increase compared with the vehicle control group. Biomedical parameters, including blood lactate, blood superoxide dismutase (SOD) activity, serum triacylglycerol (TG), hepatic total lipids (TL), TG and phospholipid (PL) were significantly lower in mice fed-with OM-X® than those in the vehicle control group. Furthermore, the mRNA expression levels of carbamoyl phosphate synthetase 1 (Cpsl) and arginase I (Argl), in the urea cycle, were increased by OM-X® feeding. Thus, our findings suggest promotion of lipid metabolism and up-regulation of the urea cycle, at least in part, for the anti-fatigue effect mediated by OM-X®.


Assuntos
Resistência Física/efeitos dos fármacos , Extratos Vegetais/farmacologia , Natação , Animais , Arginase/biossíntese , Peso Corporal/efeitos dos fármacos , Suplementos Nutricionais , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Fermentação , Glicogênio/metabolismo , Ácido Láctico/sangue , Lipídeos/sangue , Masculino , Camundongos , Fadiga Muscular/efeitos dos fármacos , Superóxido Dismutase/sangue , Ureia/metabolismo
19.
Cancer Cell ; 30(3): 377-390, 2016 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-27622331

RESUMO

Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.


Assuntos
Células Dendríticas/imunologia , Imunoterapia Adotiva/métodos , Neoplasias/imunologia , Neoplasias/terapia , Óxido Nítrico Sintase Tipo II/imunologia , Linfócitos T Citotóxicos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Arginase/biossíntese , Arginase/imunologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/biossíntese , Microambiente Tumoral , Fator de Necrose Tumoral alfa/biossíntese
20.
Oncotarget ; 7(30): 47221-47231, 2016 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-27363017

RESUMO

BACKGROUND: Both arginase (ARG2) and human cytomegalovirus (HCMV) have been implicated in tumorigenesis. However, the role of ARG2 in the pathogenesis of glioblastoma (GBM) and the HCMV effects on ARG2 are unknown. We hypothesize that HCMV may contribute to tumorigenesis by increasing ARG2 expression. RESULTS: ARG2 promotes tumorigenesis by increasing cellular proliferation, migration, invasion and vasculogenic mimicry in GBM cells, at least in part due to overexpression of MMP2/9. The nor-NOHA significantly reduced migration and tube formation of ARG2-overexpressing cells. HCMV immediate-early proteins (IE1/2) or its downstream pathways upregulated the expression of ARG2 in U-251 MG cells. Immunostaining of GBM tissue sections confirmed the overexpression of ARG2, consistent with data from subsets of Gene Expression Omnibus. Moreover, higher levels of ARG2 expression tended to be associated with poorer survival in GBM patient by analyzing data from TCGA. METHODS: The role of ARG2 in tumorigenesis was examined by proliferation-, migration-, invasion-, wound healing- and tube formation assays using an ARG2-overexpressing cell line and ARG inhibitor, N (omega)-hydroxy-nor-L-arginine (nor-NOHA) and siRNA against ARG2 coupled with functional assays measuring MMP2/9 activity, VEGF levels and nitric oxide synthase activity. Association between HCMV and ARG2 were examined in vitro with 3 different GBM cell lines, and ex vivo with immunostaining on GBM tissue sections. The viral mechanism mediating ARG2 induction was examined by siRNA approach. Correlation between ARG2 expression and patient survival was extrapolated from bioinformatics analysis on data from The Cancer Genome Atlas (TCGA). CONCLUSIONS: ARG2 promotes tumorigenesis, and HCMV may contribute to GBM pathogenesis by upregulating ARG2.


Assuntos
Arginase/biossíntese , Citomegalovirus/fisiologia , Glioblastoma/virologia , Arginase/genética , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Citomegalovirus/genética , Citomegalovirus/metabolismo , Infecções por Citomegalovirus/enzimologia , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Progressão da Doença , Glioblastoma/irrigação sanguínea , Glioblastoma/enzimologia , Glioblastoma/patologia , Humanos , Imuno-Histoquímica , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Neovascularização Patológica/virologia , Transfecção , Regulação para Cima
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA