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1.
Biomarkers ; 20(5): 328-37, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26329530

RESUMO

CONTEXT: Urinary biomarkers are promising as simple alternatives to cystoscopy for the diagnosis of de novo and recurrent bladder cancer. OBJECTIVE: To identify a highly sensitive and specific biomarker candidate set with potential clinical utility in bladder cancer. MATERIALS AND METHODS: Urinary biomarker concentrations were determined by ELISA. The performance of individual markers and marker combinations was assessed using ROC analysis. RESULTS: A five-biomarker panel (IL8, MMP9, VEGFA, PTGS2 and EN2) was defined from the candidate set. DISCUSSION AND CONCLUSION: This panel showed a better overall performance than the best individual marker. Further validation studies are needed to evaluate its clinical utility in bladder cancer.


Assuntos
Biomarcadores Tumorais/urina , Neoplasias da Bexiga Urinária/diagnóstico , Humanos , Modelos Biológicos , Neoplasias da Bexiga Urinária/urina
2.
Gastroenterology ; 143(6): 1650-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22960659

RESUMO

BACKGROUND & AIMS: Mutations in components of the Wnt signaling pathway, including ß-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. METHODS: Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of ß-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. RESULTS: Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G(2)/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of ß-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of ß-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. CONCLUSIONS: Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of ß-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC.


Assuntos
Proteína Axina/genética , Proteína Axina/fisiologia , Carcinoma Hepatocelular/fisiopatologia , Deleção de Genes , Neoplasias Hepáticas/fisiopatologia , Alelos , Animais , Carcinoma Hepatocelular/patologia , Ciclo Celular/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Hepatócitos/patologia , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Mutantes , Proteínas Wnt/fisiologia , beta Catenina/fisiologia
3.
Clin Dev Immunol ; 2013: 320168, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23401696

RESUMO

Human infections involving yeast of the genus Candida often occur in the presence of bacteria, and, as such, it is important to understand how these bacteria influence innate host immunity towards Candida. Dectin-1 is a cell receptor of macrophages for Candida albicans recognition. The aim of this study was to examine dectin-1 expression by monocytes after stimulation with bacterial lipopolysaccharide (LPS), followed by heat-killed C. albicans (HKC). Freshly isolated human peripheral blood monocytes (PBMCs) and human monocytes cell line (THP-1) cells expressed low levels of dectin-1. Stimulation with LPS and GM-CSF/IL-4 was found to increase dectin-1 expression in both CD14(+) human PBMC and THP-1 cells. Enhanced dectin-1 expression resulted in increased phagocytosis of Candida. When THP-1 cells were challenged only with HKC, detectable levels of IL-23 were not evident. However, challenge by LPS followed by varying concentrations of HKC resulted in increased IL-23 expression by THP-1 cells in HKC dose-dependent manner. Increased expression of IL-17 by PBMC also occurred after stimulation with Candida and LPS. In conclusion, bacterial LPS induces an enhanced immune response to Candida by immune cells, and this occurs through increasing dectin-1 expression.


Assuntos
Antígenos de Bactérias/imunologia , Candida albicans/imunologia , Candidíase/imunologia , Lipopolissacarídeos/imunologia , Monócitos/imunologia , Candidíase/microbiologia , Linhagem Celular , Humanos , Imunidade , Imunomodulação , Interleucina-17/imunologia , Interleucina-23/imunologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Fagocitose/imunologia , Regulação para Cima
4.
Methods Mol Biol ; 1899: 15-23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649762

RESUMO

Bone marrow resident hematopoietic stem cells (HSCs) are responsible for the lifetime generation of the wide profusion of blood and immune cell types found in the body. In addition, therapeutically, in the context of bone marrow transplantation, HSCs have been successfully deployed to restore normal blood-forming capacity in patients being treated with high-dose chemotherapy for hematologic malignancies. The known ability of bone marrow transplantation to either restore or reset the immune system and to engender immune tolerance has suggested that HSCs may be applied therapeutically for a wider range of clinical conditions, including immunological/autoimmune disorders and allogeneic organ transplantation. Herein, we describe a flow-cytometry-based method to isolate mouse HSCs for continued experimental investigation into such therapeutic uses.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Animais , Transplante de Medula Óssea , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos
5.
Antioxid Redox Signal ; 22(7): 555-71, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25336178

RESUMO

AIMS: Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. RESULTS: WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a ß-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/ß-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. INNOVATION: NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. CONCLUSION: These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism.


Assuntos
Antioxidantes/metabolismo , Proteína Axina/genética , Proteína Axina/metabolismo , Hepatócitos/metabolismo , Proteína Wnt3A/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Fígado/citologia , Fígado/metabolismo , Camundongos , Camundongos Transgênicos
6.
PLoS One ; 8(5): e63967, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23724011

RESUMO

Macrophages are heterogeneous cell populations that are present in all tissues. Macrophages can be divided into classically activated inflammatory macrophages (M1) and alternatively activated anti-inflammatory macrophages (M2). It has been generally accepted that M1 macrophages are polarised in an inflammatory environment to produce pro-inflammatory cytokines, whilst M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Bacterial endotoxin (lipopolysaccharide; LPS) is a potent factor in infection, which induces M1 macrophages resulting in higher levels of iNOS, TNFα and IL-12p70 which dictate inflammatory T cell responses. M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation. Candida albicans is a commensal fungal microorganism, which has been suggested to induce immune tolerance; however, the mechanism of C. albicans-induced immune tolerance has not been investigated in detail. IL-35 is a recently identified anti-inflammatory cytokine which is a heterodimeric protein consisting of the Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 shares the protein subunit p35, with IL-12p70. IL-12p70 is the most potent cytokine to induce Th1 responses during inflammation. In this study, we demonstrate that heat-killed C. albicans (HKC) strongly suppressed LPS-induced IL-12p70 production in M2 macrophages. Candida albicans induced a high level of EBI3 expression in M2 macrophages, which served as a mechanism for IL-12p70 suppression by competitive binding of the common protein subunit (p35) of IL-35 and IL-12p70. To demonstrate that EBI3 expression had the ability to block IL-12p70 production intracellularly, a Chinese Hamster Ovary (CHO) cell line with biscistronic expression of IL-12p40 and p35 was constructed, followed by ectopic over-expression of EBI3. The over-expression of EBI3 in the IL-12p70 producing cell line effectively suppressed IL-12p70 production. IL-35 secretion was also detected in the cell line, with suppressed IL-12p70 production by immune-precipitation Western blotting. However, this secretion was not evident in M2 macrophages following stimulation by HKC. This can be explained by the constitutive expression of IL-35 receptors (gp130 and IL-12Rß2) in M2 macrophages for cytokine consumption. Our results have indicated that C. albicans can suppress host inflammatory responses in mucosal skin by suppressing LPS-induced IL-12p70 production. Lower IL-12p70 production may avoid an unnecessary Th1 response in order to retain immune tolerance, which may be one of the mechanisms by which C. albicans achieves a successful commensal lifestyle without having a detrimental effect on the host's health.


Assuntos
Candida albicans/imunologia , Regulação da Expressão Gênica , Interleucina-12/biossíntese , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Receptores de Citocinas/genética , Animais , Células da Medula Óssea , Células CHO , Linhagem Celular , Cricetulus , Expressão Gênica , Macrófagos/microbiologia , Camundongos , Antígenos de Histocompatibilidade Menor , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Citocinas/metabolismo
7.
J Biol Chem ; 280(9): 8208-20, 2005 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-15590649

RESUMO

In mice genetic ablation of expression of either melanin-concentrating hormone or the melanin-concentrating hormone-1 receptor results in alterations in energy metabolism and a lean phenotype. There is thus great interest in the function and regulation of this receptor. Using the yeast two-hybrid system we identified an interaction of the actin- and intermediate filament-binding protein periplakin with the intracellular C-terminal tail of the melanin-concentrating hormone-1 receptor. Direct association of these proteins was verified in pull-down and coimmunoprecipitation experiments. Truncations and internal deletions delineated the site of interaction to a group of 11 amino acids proximal to transmembrane helix VII, which was distinct from the binding site for the melanin-concentrating hormone-1 receptor-interacting zinc finger protein. Immunohistochemistry demonstrated coexpression of periplakin with melanin-concentrating hormone-1 receptor in specific cells of the piriform cortex, amygdala, and other structures of the adult mouse brain. Coexpression of the melanin-concentrating hormone-1 receptor with periplakin in human embryonic kidney 293 cells did not prevent agonist-mediated internalization of the receptor but did interfere with binding of (35)S-labeled guanosine 5'-3-O-(thio)triphosphate ([(35)S]GTPgammaS) to the G protein Galpha(o1) and the elevation of [Ca(2+)](i). Coexpression of the receptor with the interacting zinc finger protein did not modulate receptor internalization or G protein activation. The interaction of periplakin with receptors was selective. Coexpression of periplakin with the IP prostanoid receptor did not result in coimmunoprecipitation nor interfere with agonist-mediated binding of [(35)S]GTPgammaS to the G protein Galpha(s). Periplakin is the first protein described to modify the capacity of the melanin-concentrating hormone-1 receptor to initiate signal transduction.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/química , Receptores de Somatostatina/química , Receptores de Somatostatina/metabolismo , Actinas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biotina/química , Encéfalo/metabolismo , Cálcio/química , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , DNA/química , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Biblioteca Gênica , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Histidina/química , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Fenótipo , Fosforilação , Plaquinas , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Dedos de Zinco
8.
Anal Biochem ; 300(2): 212-20, 2002 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11779113

RESUMO

It is extremely difficult to detect guanine nucleotide exchange or hydrolysis stimulated by receptors which couple to G(s)alpha. Furthermore, G(s)alpha is largely resistant to the GTPase-activating properties of RGS proteins. Coexpression of the vasopressin V(2) receptor with a series of chimeric G protein alpha subunits in which the C-terminal 6-12 amino acids of G(i1)alpha were replaced with the equivalent sequence of G(s)alpha allowed robust vasopressin-stimulated [(35)S]GTPgammaS binding. Vasopressin did not stimulate the GTPase activity of fusion proteins between the V(2) receptor and either G(s)alpha or G(i1)alpha. However, it produced a concentration-dependent stimulation of V(max) for a V(2) receptor-G(i1)alpha/Gs6alpha fusion protein. This construct bound [(3)H]vasopressin with high affinity and this was competed by other ligands with rank order anticipated for the V(2) receptor. RGS1 enhanced vasopressin stimulation of V(2) receptor-G(i1)alpha/G(s)6alpha in a concentration-dependent manner. RGS-GAIP was substantially less potent. Enzyme kinetic analysis demonstrated that RGS1 increased both V(max) of the GTPase activity and the observed K(m) for GTP, consistent with RGS1 accelerating the rate of GTP hydrolysis of the chimeric G protein, whereas the agonist vasopressin accelerates guanine nucleotide exchange. This approach provides a sensitive assay for V(2) receptor agonist ligands and may be amenable to many other G(s)alpha-coupled receptors.


Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Engenharia de Proteínas/métodos , Receptores de Vasopressinas/agonistas , Transdução de Sinais , Sequência de Aminoácidos , Linhagem Celular , Membrana Celular/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Cinética , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
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