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1.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445576

RESUMO

CD40 crosslinking plays an important role in regulating cell migration, adhesion and proliferation in renal cell carcinoma (RCC). CD40/CD40L interaction on RCC cells activates different intracellular pathways but the molecular mechanisms leading to cell scattering are not yet clearly defined. Aim of our study was to investigate the main intracellular pathways activated by CD40 ligation and their specific involvement in RCC cell migration. CD40 ligation increased the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH (2)-terminal kinase (JNK) and p38 MAPK. Furthermore, CD40 crosslinking activated different transcriptional factors on RCC cell lines: AP-1, NFkB and some members of the Nuclear Factor of Activated T cells (NFAT) family. Interestingly, the specific inhibition of NFAT factors by cyclosporine A, completely blocked RCC cell motility induced by CD40 ligation. In tumor tissue, we observed a higher expression of NFAT factors and in particular an increased activation and nuclear migration of NFATc4 on RCC tumor tissues belonging to patients that developed metastases when compared to those who did not. Moreover, CD40-CD40L interaction induced a cytoskeleton reorganization and increased the expression of integrin ß1 on RCC cell lines, and this effect was reversed by cyclosporine A and NFAT inhibition. These data suggest that CD40 ligation induces the activation of different intracellular signaling pathways, in particular the NFATs factors, that could represent a potential therapeutic target in the setting of patients with metastatic RCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/patologia , Fatores de Transcrição NFATC/metabolismo , Idoso , Apoptose , Biomarcadores Tumorais/genética , Antígenos CD40/genética , Ligante de CD40/genética , Movimento Celular , Proliferação de Células , Reagentes para Ligações Cruzadas , Feminino , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/genética , Metástase Neoplásica , Prognóstico , Células Tumorais Cultivadas
2.
Methods Mol Biol ; 2325: 29-39, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34053048

RESUMO

During the last two decades, the immunology field has been focused on the study of conventional T-cells, leading to important advances in identification of specific subsets involved in promoting and suppressing immune response in patients with cancer, autoimmune disease or transplanted patients. In these recent years, the research on unconventional subset of CD4-CD8- double-negative T-cells is growing. DNTs are a unique subset of T-cells characterized by being CD4-CD8-CD3+ and express either ß or α T-cell receptors (TCR). In this chapter, we describe the methods used to phenotypically characterize and isolate these cells in order to study their functional profile.


Assuntos
Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Técnicas de Cultura de Células/métodos , Citometria de Fluxo/métodos , Linfócitos T Citotóxicos/metabolismo , Citocinas/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Fenótipo , Coloração e Rotulagem/métodos , Linfócitos T Citotóxicos/citologia
3.
Methods Mol Biol ; 2325: 65-77, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34053051

RESUMO

Enzyme-linked immune absorbent spot (Elispot) is a quantitative method for measuring relevant parameters of T-cell activation. The sensitivity of Elispot allows the detection of low-frequency antigen-specific T-cells that secrete cytokines and effector molecules, such as granzyme B and perforin. Cytotoxic T-cell (CTL) studies have taken advantage with this high-throughput technology by providing insights of quantity and immune kinetics. Accuracy, sensitivity, reproducibility, and robustness of Elispot resulted in a wide range of applications in research as well as in diagnostic field. Actually, CTL monitoring by Elispot is a gold standard for the evaluation of antigen-specific T-cell immunity in clinical trials and vaccine candidates where the ability to detect rare antigen-specific T-cells is of relevance for immune diagnostic. The most utilized Elispot assay is the Interferon-gamma (IFN-γ) test, a marker for CD8+ CTL activation, but Elispot can be also used to distinguish different subsets of activated T-cells by using other cytokines such as T-helper (Th) 1 type cells (characterized by the production of IFN-γ, IL-2, IL-6, IL-12, IL-21 and TNF-α), Th2 (producing cytokines like IL-4, IL-5, IL-10 and IL-13), and Th17 (IL-17) cells.The reliability of Elispot generated data, by the evaluation of T-cell frequency recognizing individual antigen/peptide, is the core of this method currently applied widely to investigate specific immune responses in cancer, infections, allergies, and autoimmune diseases. The Elispot Assay is competing with other methods measuring single-cell cytokine production, e.g., intracellular cytokine by FACS or Milteny cytokine secretion assay. Other types of lymphocyte frequency and function assays include limiting dilution assay (LDA), cytotoxic T-cell assay (CTL), and tetramer staining. Compared with respect to sensitivity the Elispot Assay is outranking other methods to define frequency of antigen-specific lymphocytes. The method described herein would like to offer helpful and clear protocols for researchers that apply Elispot. IFN-γ and Perforin Elispot assays will be described.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Citocinas/metabolismo , ELISPOT/métodos , Humanos , Interferon gama/metabolismo , Perforina/metabolismo
4.
Aging (Albany NY) ; 12(8): 7585-7602, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345771

RESUMO

Pentraxin-3 (PTX3) belongs to the pentraxine family, innate immune regulators involved in angiogenesis, proliferation and immune escape in cancer. Here, we evaluated PTX3 tissue expression and serum levels as biomarkers of clear cell renal cell carcinoma (ccRCC) and analyzed the possible role of complement system activation on tumor site. A 10-year retrospective cohort study including patients undergoing nephrectomy for ccRCC was also performed. PTX3 expression was elevated in both neoplastic renal cell lines and tissues, while it was absent in both normal renal proximal tubular cells (HK2) and normal renal tissues. Analysis of complement system activation on tumor tissues showed the co-expression of PTX3 with C1q, C3aR, C5R1 and CD59, but not with C5b-9 terminal complex. RCC patients showed higher serum PTX3 levels as compared to non-neoplastic patients (p<0.0001). Higher PTX3 serum levels were observed in patients with higher Fuhrman grade (p<0.01), lymph node (p<0.0001), and visceral metastases (p<0.001). Patients with higher PTX3 levels also showed significantly lower survival rates (p=0.002). Our results suggest that expression of PTX3 can affect the immunoflogosis in the ccRCC microenvironment, by activating the classical pathway of CS (C1q) and releasing pro-angiogenic factors (C3a, C5a). The up-regulation of CD59 also inhibits the complement-mediated cellular lysis.


Assuntos
Proteína C-Reativa/genética , Carcinoma de Células Renais/genética , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Componente Amiloide P Sérico/genética , Proteínas de Fase Aguda , Biomarcadores Tumorais/metabolismo , Proteína C-Reativa/biossíntese , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Prognóstico , Estudos Retrospectivos , Componente Amiloide P Sérico/biossíntese , Microambiente Tumoral
5.
Nephrol Dial Transplant ; 33(1): 65-75, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28992140

RESUMO

Background: Inflammation and immune system alterations contribute to bone damage in many pathologies by inducing the differentiation of osteoclasts (OCs), the bone resorbing cells. This link is largely unexplored in chronic kidney disease (CKD) and haemodialysis (HD) patients, in which reduced renal function is accompanied by an increased inflammatory state and skeletal abnormality. Methods: We used ex vivo culture experiments to investigate the osteoclastogenic potential of peripheral blood mononuclear cells (PBMCs) of CKD and HD patients, focusing on immune cell subsets and inflammatory cytokines such as LIGHT and receptor activator of nuclear factor κB ligand (RANKL). Results: We observed spontaneous osteoclastogenesis with a significant increase in OC formation and bone resorbing activity in late-stage CKD and HD patients when compared with early-stage CKD patients and healthy donors, likely due to an increased expression of RANKL and LIGHT (homologous to Lymphotoxins exhibiting Inducible expression and competing with herpes simplex virus Glycoprotein D for herpes virus entry mediator [HVEM], a receptor expressed by T lymphocytes) in PBMCs. Specific inhibition of these cytokines in PBMCs isolated from CKD stages 3b-5 and HD patients induced the reduction of OC formation in vitro. The phenotypic characterization of peripheral blood cells revealed a significant increase of OC precursors (CD14+CD11b+CD51/61+) and CD14+CD16+ monocytes in advanced CKD and HD patients compared with the control group. Conclusions: Our results suggest that circulating inflammatory monocytes from advanced CKD or HD patients trans differentiate into OCs in vitro and play a relevant role in mineral bone disorders and that LIGHT and RANKL represent new potential therapeutic targets in these settings.


Assuntos
Reabsorção Óssea/etiologia , Diferenciação Celular/imunologia , Inflamação/complicações , Leucócitos Mononucleares/patologia , Osteoclastos/patologia , Insuficiência Renal Crônica/etiologia , Reabsorção Óssea/patologia , Estudos de Casos e Controles , Células Cultivadas , Feminino , Humanos , Inflamação/fisiopatologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Osteoclastos/imunologia , Ligante RANK/metabolismo , Insuficiência Renal Crônica/patologia
6.
Oncotarget ; 8(25): 40412-40424, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28418894

RESUMO

Clear cell Renal Cell Carcinoma (ccRCC) causes over 13,000 deaths each year, and about 20,000 new cases/year in Europe. In most cases, the causes are unknown and, most importantly, there are no reliable biomarkers for the diagnosis and prognosis of this disease. The search for sensitive biomarkers for early diagnosis and prognosis of clear cell Renal Cell Carcinoma (ccRCC) is currently a fast growing field. We carried out proteomics analysis of 93 urinary samples of healthy subjects (HS) and patients affected by ccRCC, prostate cancer (PCa) and chronic kidney disease (CKD), that was able to successfully distinguish each group.The most significant candidate biomarker was identified by mass spectrometry as Raf Kinase Inhibitor Protein (RKIP), a key regulator of cell signaling, already described in several cancer types as a metastasis suppressor. By combining ELISA, immunoblotting and tissue microarray, we demonstrated that, in ccRCC, urinary excretion of RKIP and its phosphorylated form (p-RKIP) reflected the tissue expression of these putative biomarkers. Baseline urinary RKIP, evaluated in an independent cohort of 56 ccRCC patients and 28 HS, successfully distinguished both groups and, most importantly, a cut-off value of 10 ng/mg/g Pr/uCr enabled a highly accurate prediction of Cancer-specific survival and Progression-free survival. Furthermore, p-RKIP was totally undetectable in both tissue and urine samples of ccRCC, showing a great potential for diagnostics purposes.Our data indicate that urinary RKIP encompasses both the unphosphorylated and the phosphorylated form and that their combined evaluation can help in the diagnosis and prognosis of ccRCC.


Assuntos
Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/urina , Neoplasias Renais/diagnóstico , Neoplasias Renais/urina , Proteína de Ligação a Fosfatidiletanolamina/urina , Insuficiência Renal Crônica/urina , Adulto , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/urina , Carcinoma de Células Renais/patologia , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Europa (Continente) , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/patologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Insuficiência Renal Crônica/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise Serial de Tecidos
7.
Urol Oncol ; 35(7): 461.e15-461.e27, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28359744

RESUMO

OBJECTIVE: To investigate the expression of the kynurenine (KYN) pathway components and the prognostic role of the KYN-to-tryptophan ratio (KTR) in a cohort of patients with clear cell renal cell carcinoma (ccRCC). MATERIALS AND METHODS: The expression of KYN pathway components was investigated by tissue microarray-based immunohistochemistry, indirect immunofluorescence, and confocal microscopy analysis in 100 ccRCC cases and 30 normal renal samples. The role of this pathway in sustaining cancer cell proliferation, migration, and chemoresistance was evaluated. In addition, tryptophan and KYN concentrations and their ratio were measured in serum of 195 patients with ccRCC using a sandwich enzyme-linked immunosorbent assay. The role of KTR as a prognostic factor for ccRCC cancer-specific survival (CSS) and progression-free survival (PFS) was assessed. RESULTS: Tissue microarray-based immunohistochemistry and indirect immunofluorescence staining showed an increased signal for KYN pathway components in ccRCC. Kaplan-Meier curves showed significant differences in CSS and PFS among groups of patients with high vs. low KTR. In particular, patients with high KTR values had a 5-year survival rate of 76.9% as compared with 92.3% for subjects with low levels (P  < 0.0001). Similar findings were observed for PFS (72.8% vs. 96.8% at 5y). At multivariate analysis, KTR was an independent adverse prognostic factor for CSS (hazard ratio  = 1.24, P  =  0.001), and PFS (hazard ratio  =  1.14, P  =  0.001). CONCLUSIONS: The involvement of the KYN pathway enzymes and catabolites in ccRCC occurs via both immune and nonimmune mechanisms. Our data suggest that KTR could serve as a marker of ccRCC aggressiveness and as a prognostic factor for CSS and PFS.


Assuntos
Carcinoma de Células Renais/imunologia , Cinurenina/metabolismo , Idoso , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
8.
Nephrol Dial Transplant ; 32(9): 1540-1549, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27915246

RESUMO

Background: The aim of this study was to investigate neutrophil activation and its role in long pentraxin-3 (PTX3) release and oxidative stress generation during haemodialysis (HD) and to correlate neutrophil PTX3 and oxidant expression with endothelial dysfunction. Methods: Forty-seven uraemic patients on stable HD, 12 healthy subjects and 15 patients with congestive heart failure (New York Heart Association classes III and IV) were enrolled. Neutrophil PTX3 protein expression was evaluated by confocal microscopy. l -selectin expression, intracellular PTX3 localization and reactive oxygen species (ROS) generation in human neutrophils were measured by flow cytometry. NADPH-dependent superoxide generation was investigated by chemiluminescence. PTX3 plasma concentrations were measured by ELISA. Endothelial dysfunction was studied by flow-mediated dilation (FMD). Results: The low baseline levels of FMD significantly improved after HD, but worsened by 24 h. A significant up-regulation of PTX3 protein expression, localized within secondary granules, was detected in neutrophils isolated at 30 and 240 min of HD, along with an increase in l -selectin expression. The up-regulation in intracellular PTX3 in neutrophils was associated with a significant increase in PTX3 plasma concentrations at 240 min. HD increased ROS production and NADPH oxidase activity in neutrophils. In a univariate analysis, pre-treatment with FMD was inversely correlated with PTX3 expression and ROS generation in neutrophils. In a multivariate analysis, both circulating pre-HD PTX3 and intracellular ROS generation by neutrophils were independent predictors of abnormal FMD. Conclusions: Neutrophil overexpression of PTX3 is associated with ROS overproduction and endothelial dysfunction and may represent an emerging marker of vascular damage progression in HD patients.


Assuntos
Biomarcadores/sangue , Proteína C-Reativa/metabolismo , Endotélio Vascular/patologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Diálise Renal , Componente Amiloide P Sérico/metabolismo , Doenças Vasculares/patologia , Endotélio Vascular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativação de Neutrófilo , Estresse Oxidativo , Regulação para Cima , Doenças Vasculares/sangue
9.
FASEB J ; 31(1): 308-319, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27881486

RESUMO

The purpose of our study was to evaluate how hyperglycemia (HG) influences Lys63 protein ubiquitination and its involvement in tubular damage and fibrosis in diabetic nephropathy (DN). Gene and protein expression of UBE2v1, a ubiquitin-conjugating E2-enzyme variant that mediates Lys63-linked ubiquitination, and Lys63-ubiquitinated proteins increased in HK2 tubular cells under HG. Matrix-assisted laser desorption/ionization-time of flight/tandem mass spectrometry identified 30 Lys63-ubiquitinated proteins, mainly involved in cellular organization, such as ß-actin, whose Lys63 ubiquitination increased under HG, leading to cytoskeleton disorganization. This effect was reversed by the inhibitor of the Ubc13/UBE2v1 complex NSC697923. Western blot analysis confirmed that UBE2v1 silencing in HK2 under HG, restored Lys63-ß-actin ubiquitination levels to the basal condition. Immunohistochemistry on patients with type 2 diabetic (T2D) revealed an increase in UBE2v1- and Lys63-ubiquitinated proteins, particularly in kidneys of patients with DN compared with control kidneys and other nondiabetic renal diseases, such as membranous nephropathy. Increased Lys63 ubiquitination both in vivo in patients with DN and in vitro, correlated with α-SMA expression, whereas UBE2v1 silencing reduced HG-induced α-SMA protein levels, returning them to basal expression. In conclusion, UBE2v1- and Lys63-ubiquitinated proteins increase in vitro under HG, as well as in vivo in T2D, is augmented in patients with DN, and may affect cytoskeleton organization and influence epithelial-to-mesenchymal transition. This process may drive the progression of tubular damage and interstitial fibrosis in patients with DN.-Pontrelli, P., Conserva, F., Papale, M., Oranger, A., Barozzino, M., Vocino, G., Rochetti, M. T., Gigante, M., Castellano, G., Rossini, M., Simone, S., Laviola, L., Giorgino, F., Grandaliano, G., Di Paolo, S., Gesualdo, L. Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.


Assuntos
Nefropatias Diabéticas/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Sequência de Aminoácidos , Biomarcadores , Linhagem Celular , Células Epiteliais/metabolismo , Inativação Gênica , Humanos , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/genética , Proteínas Ubiquitinadas
10.
Int J Oncol ; 49(2): 457-70, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27278998

RESUMO

Renal cell carcinoma (RCC) is the most common kidney cancer, and accounts for ~3% of all adult malignancies. RCC has proven refractory to conventional treatment modalities but appears to be the only histological form that shows any consistent response to immunotherapeutic approaches. The development of a clinically effective vaccine remains a major strategic target for devising active specific immunotherapy in RCC. We aimed to identify a highly immunogenic antigenic format for immunotherapeutic approaches, so as to boost immune responses in RCC patients. We established and cloned an immunogenic cell line, RCC85#21 named Elthem, which was derived from a non-aggressive and non-metastatic clear cell carcinoma. The cell line characterization was performed by genomics (real-time PCR, genome instability), proteomics (two dimensional electrophoresis, mass spectro-metry) and immunological analysis (mixed lymphocytes tumor cell cultures). Real-time PCR confirmed the RCC85#21 cell expression of tumor antigens and cytokine genes. No difference in microsatellite instability (MSI) in RCC85#21 cell line was found as compared to control, loss of heterozygosity was observed in the RCC85#21 clone, but not in the renal cancer cell lines from which it was generated. The image analysis of RCC85#21 by two-dimensional gels showed 700±26 spots and 119 spots were identified by mass spectrometry analysis. RCC85#21 promoted a significant RCC-specific T cells activation by exhibiting a cytotoxic phenotype after mixed lymphocyte and tumor cell cultures. CD8+ T cells isolated from RCC patients displayed an elevated reactivity against RCC85#21 and efficiently lysed the RCC85#21 clone. The RCC85#21 immunogenic cell line will be suitable for immune stimulation. The identification of novel tumor associated antigens will allow the evaluation of the immune response in vitro and, subsequently, in vivo paving the way for new immunotherapeutic strategies in the RCC setting.


Assuntos
Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Antígenos de Neoplasias/imunologia , Carcinoma de Células Renais/genética , Eletroforese em Gel Bidimensional/métodos , Humanos , Imunofenotipagem , Células K562 , Neoplasias Renais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Biologia de Sistemas/métodos
11.
J Transl Med ; 14: 84, 2016 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-27063186

RESUMO

BACKGROUND: Mammalian microRNAs (miR) regulate the expression of genes relevant for the development of adaptive and innate immunity against cancer. Since T cell dysfunction has previously been reported in patients with renal cell carcinoma (RCC; clear cell type), we aimed to analyze these immune cells for genetic and protein differences when compared to normal donor T cells freshly after isolation and 35 days after in vitro stimulation (IVS) with HLA-matched RCC tumor cells. METHODS: We investigated gene expression profiles of tumor-reactive CD8(+) T cells obtained from RCC patient and compared with their HLA-matched healthy sibling donors using a microarray approach. In addition, miRNAs analysis was performed in a validation cohort of peripheral blood CD8(+) T cells from 25 RCC patients compared to 15 healthy volunteers. RESULTS: We observed that CD8(+) T cells from RCC patients expressed reduced levels of anti-apoptotic and proliferation-associated gene products when compared with normal donor T cells both pre- and post-IVS. In particular, JAK3 and MCL-1 were down-regulated in patient CD8(+) T cells versus their normal counterparts, likely due to defective suppressor activity of miR-29b and miR-198 in RCC CD8(+) T cells. Indeed, specific inhibition of miR-29b or miR-198 in peripheral blood mononuclear cells (PBMCs) isolated from RCC patients, resulted in the up-regulation of JAK3 and MCL-1 proteins and significant improvement of cell survival in vitro. CONCLUSIONS: Our results suggest that miR-29b and miR-198 dysregulation in RCC patient CD8(+) T cells is associated with dysfunctional immunity and foreshadow the development of miR-targeted therapeutics to correct such T cell defects in vivo.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Regulação para Baixo/genética , Janus Quinase 3/metabolismo , MicroRNAs/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Adulto , Idoso , Apoptose/genética , Separação Celular , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Janus Quinase 3/genética , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Leucócitos Mononucleares/metabolismo , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fenótipo , Doadores de Tecidos , Transfecção , Transplante Homólogo , Células Tumorais Cultivadas , Regulação para Cima/genética
12.
Medicine (Baltimore) ; 94(45): e1917, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26559258

RESUMO

Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies, and clear cell RCC (ccRCC), that has a high metastatic index and high relapse rate, is the most common histological subtype. The identification of new biomarkers in ccRCC is fundamental for stratifying patients into prognostic risk groups and to guide therapy. The renoprotective antiaging gene, αKlotho, has recently been found to work as a tumor suppressor in different human cancers. Here, we evaluated αKlotho expression in tissue and serum of ccRCC patients and correlated it with disease progression. Tissue αKlotho expression was studied by quantitative RT-PCR and immunohistochemistry. In addition, soluble serum αKlotho levels were preoperatively measured in 160 patients who underwent nephrectomy for RCC with ELISA. Estimates of cancer-specific (CSS) and progression-free survival (PFS) were calculated according to the Kaplan-Meier method. Multivariate analysis was performed to identify the most significant variables for predicting CSS and PFS. αKlotho protein levels were significantly decreased in RCC tissues compared with normal tissues (P < 0.01) and the more advanced the disease, the more evident the down-regulation. This trend was also observed in serum samples. Statistically significant differences resulted between serum αKlotho levels and tumor size (P = 0.003), Fuhrman grade (P = 0.007), and clinical stage (P = 0.0004). CSS and PFS were significantly shorter in patients with lower levels of αKlotho (P < 0.0001 and P = 0.0004, respectively). At multivariate analysis low serum levels of αKlotho were independent adverse prognostic factors for CSS (HR = 2.11; P = 0.03) and PFS (HR = 2.18; P = 0.03).These results indicate that a decreased αKlotho expression is correlated with RCC progression, and suggest a key role of declining αKlotho in the onset of cancer metastasis.


Assuntos
Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Glucuronidase/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/mortalidade , Masculino , Pessoa de Meia-Idade , Nefrectomia , Sensibilidade e Especificidade
13.
Medicine (Baltimore) ; 94(46): e2117, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26579829

RESUMO

Glucose-6-phosphate isomerase (GPI), also known as phosphoglucose isomerase, was initially identified as the second glycolytic enzyme that catalyzes the interconversion of glucose-6-phosphate to fructose-6-phosphate. Later studies demonstrated that GPI was the same as the autocrine motility factor (AMF), and that it mediates its biological effects through the interaction with its surface receptor (AMFR/gp78). In this study, we assessed the role of GPI/AMF as a prognostic factor for clear cell renal cell carcinoma (ccRCC) cancer-specific (CSS) and progression-free survival (PFS). In addition, we evaluated the expression and localization of GPI/AMF and AMFR, using tissue microarray-based immunohistochemistry (TMA-IHC), indirect immunofluorescence (IF), and confocal microscopy analysis.Primary renal tumor and nonneoplastic tissues were collected from 180 patients who underwent nephrectomy for ccRCC. TMA-IHC and IF staining showed an increased signal for both GPI and AMFR in cancer cells, and their colocalization on plasma membrane. Kaplan-Meier curves showed significant differences in CSS and PFS among groups of patients with high versus low GPI expression. In particular, patients with high tissue levels of GPI had a 5-year survival rate of 58.8%, as compared to 92.1% for subjects with low levels (P < 0.0001). Similar findings were observed for PFS (56.8% vs 93.3% at 5 years). At multivariate analysis, GPI was an independent adverse prognostic factor for CSS (HR = 1.26; P = 0.001), and PFS (HR = 1.16; P = 0.01).In conclusion, our data suggest that GPI could serve as a marker of ccRCC aggressiveness and a prognostic factor for CSS and PFS.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/mortalidade , Regulação Neoplásica da Expressão Gênica , Glucose-6-Fosfato Isomerase/metabolismo , Neoplasias Renais/mortalidade , Rim/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Citocinas/genética , Citocinas/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Glucose-6-Fosfato Isomerase/genética , Humanos , Imuno-Histoquímica/métodos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Análise de Sobrevida , Regulação para Cima
14.
Bone ; 81: 228-236, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26208797

RESUMO

Subjects with hypergonadotropic hypogonadism due to Turner's syndrome show low cortical mineral density, osteoporosis and risk of fractures. It is not clear if this bone fragility derives from chromosomal abnormalities or is the result of inadequate bone formation due to estrogen deficiency. The aim of this study was to investigate the cellular mechanisms underlying bone fragility in subjects with Turner's syndrome before induction of puberty and after hormonal replacement therapy (HRT). For this purpose, we have evaluated the osteoclastogenic potential of non-fractioned and T-cell depleted cultures of peripheral blood mononuclear cells (PBMCs) belonging to girls with Turner's syndrome who had not been treated with HRT yet, girls and young women who were on HRT and age-matched controls. Untreated subjects showed high FSH serum levels, whereas the other subjects displayed normal FSH serum levels. T-cell immunophenotype was analyzed through flow cytometry. Biochemical and DXA analyses were performed. Spontaneous osteoclastogenesis in non-fractioned and T-cell depleted cultures of PBMC belonging to girls with high FSH levels was more evident than in cultures of subjects with normal FSH levels. In the former, osteoclastogenesis was sustained by monocytes expressing high levels of c-fms, TNF-α and RANK, and T-cells producing high RANKL and TNF-α; in the latter it was supported by T-cells expressing high RANKL levels. CD4(+)CD25(high) T-cells were reduced in all subjects, whereas CD3(+)/CD16(+)/CD56(+) NKT-cells were increased in those with high FSH levels. High RANKL and CTX levels were detected in the sera. Bone impairment was already detectable by DXA in subjects aged under 10, although it became more evident with aging. In conclusion, our results demonstrated that bone fragility in subjects with Turner's syndrome is associated to enhanced osteoclastogenesis. This process seems to be due to high FSH serum levels before HRT, whereas it is caused by high RANKL during HRT.


Assuntos
Densidade Óssea/fisiologia , Calcificação Fisiológica/fisiologia , Osteoclastos/metabolismo , Osteogênese/fisiologia , Síndrome de Turner/sangue , Adolescente , Adulto , Densidade Óssea/efeitos dos fármacos , Calcificação Fisiológica/efeitos dos fármacos , Células Cultivadas , Criança , Pré-Escolar , Feminino , Hormônio Foliculoestimulante/sangue , Terapia de Reposição Hormonal/métodos , Humanos , Lactente , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Maturidade Sexual/efeitos dos fármacos , Maturidade Sexual/fisiologia , Síndrome de Turner/tratamento farmacológico , Adulto Jovem
15.
Nephrol Dial Transplant ; 30(9): 1480-7, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26056176

RESUMO

BACKGROUND: Coagulation and complement activation represent key events in ischaemia-reperfusion-induced renal injury leading to delayed graft function (DGF). It is still unclear whether the coagulation cascade may also influence the acquired immunity. The aim of the present study was to investigate the expression of protease-activated receptor 1 (PAR-1), the main thrombin receptor, by graft-infiltrating dendritic cells (DCs), and to evaluate whether thrombin may influence DCs complement production and T-cell response. METHODS: PAR-1, BDCA1, CD11c, BDCA4, fibrin, C3c and C3d protein expression were evaluated by confocal microscopy. Cultured DCs were obtained incubating monocytes (Ms) with IL-4 and GM-CSF. DC maturation was obtained with IFN-g+sCD40L or with a cytokine cocktail (IL-1b, TNF-a, PGE2, IL-6). PAR1 protein expression on cultured DC was evaluated by flow-cytometry. Complement receptors, C3, IL12/IL17p40 and IL10 gene expression was evaluated by qPCR. T cell phenotype was evaluated by ELISPOT. IFN-g protein presence was evaluated by ELISA. RESULTS: PAR-1 was expressed by infiltrating myeloid DCs in pre-transplant and in DGF biopsies. In DGF grafts, myeloid DCs localized within fibrin and C3d deposits and expressed C3c. In vitro, PAR-1 protein expression was increased in monocyte-derived immature DCs and in cytokine-induced mature DCs compared to monocytes. PAR-1 activation caused a time-dependent increase in C3 and complement receptors expression. Moreover, thrombin stimulation, while reducing interleukin-10 mRNA abundance, induced interleukin-12/IL-17 p40 gene expression, and promoted C3a ability to increase interleukin-12/IL17 mRNA abundance. These changes in the DCs' cytokine pattern influenced their ability to induce interferon-g production by T cells, suggesting the activation of a T helper-1 bias. CONCLUSION: Our data suggest that PAR-1 is expressed by DCs in DGF grafts and its activation may induce complement production and a Th1 bias. This observation suggests a potential pathogenic link between DGF and acquired allo-response leading to graft damage.


Assuntos
Citocinas/metabolismo , Função Retardada do Enxerto/imunologia , Células Dendríticas/imunologia , Transplante de Rim/efeitos adversos , Túbulos Renais Proximais/imunologia , Linfócitos T/imunologia , Trombina/farmacologia , Células Cultivadas , Citocinas/genética , Função Retardada do Enxerto/tratamento farmacológico , Função Retardada do Enxerto/patologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Hemostáticos/farmacologia , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia
16.
J Pathol ; 237(1): 72-84, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25925804

RESUMO

Chronic antibody-mediated rejection (CAMR) represents the main cause of kidney graft loss. To uncover the molecular mechanisms underlying this condition, we characterized the molecular signature of peripheral blood mononuclear cells (PBMCs) and, separately, of CD4(+) T lymphocytes isolated from CAMR patients, compared to kidney transplant recipients with normal graft function and histology. We enrolled 29 patients with biopsy-proven CAMR, 29 stable transplant recipients (controls), and 8 transplant recipients with clinical and histological evidence of interstitial fibrosis/tubular atrophy. Messenger RNA and microRNA profiling of PBMCs and CD4(+) T lymphocytes was performed using Agilent microarrays in eight randomly selected patients per group from CAMR and control subjects. Results were evaluated statistically and by functional pathway analysis (Ingenuity Pathway Analysis) and validated in the remaining subjects. In PBMCs, 45 genes were differentially expressed between the two groups, most of which were up-regulated in CAMR and were involved in type I interferon signalling. In the same patients, 16 microRNAs were down-regulated in CAMR subjects compared to controls: four were predicted modulators of six mRNAs identified in the transcriptional analysis. In silico functional analysis supported the involvement of type I interferon signalling. To further confirm this result, we investigated the transcriptomic profiles of CD4(+) T lymphocytes in an independent group of patients, observing that the activation of type I interferon signalling was a specific hallmark of CAMR. In addition, in CAMR patients, we detected a reduction of circulating BDCA2(+) dendritic cells, the natural type I interferon-producing cells, and their recruitment into the graft along with increased expression of MXA, a type I interferon-induced protein, at the tubulointerstitial and vascular level. Finally, interferon alpha mRNA expression was significantly increased in CAMR compared to control biopsies. We conclude that type I interferon signalling may represent the molecular signature of CAMR.


Assuntos
Rejeição de Enxerto/imunologia , Interferon Tipo I/imunologia , Transplante de Rim/efeitos adversos , Rim/imunologia , Rim/cirurgia , Leucócitos Mononucleares/imunologia , Adulto , Idoso , Biomarcadores/metabolismo , Biópsia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Doença Crônica , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Estudos de Associação Genética , Rejeição de Enxerto/sangue , Rejeição de Enxerto/genética , Humanos , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Rim/metabolismo , Rim/patologia , Leucócitos Mononucleares/metabolismo , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcrição Genética , Transcriptoma , Resultado do Tratamento
17.
Arthritis Res Ther ; 17: 72, 2015 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-25889472

RESUMO

INTRODUCTION: Type I interferons are pivotal in the activation of autoimmune response in systemic lupus erythematous. However, the pathogenic role of interferon-alpha in patients affected by lupus nephritis remains uncertain. The aim of our study was to investigate the presence of a specific interferon signature in lupus nephritis and the effects of interferon-alpha at renal level. METHODS: We performed immunohistochemical analysis for MXA-protein and in situ hybridization to detect interferon-alpha signature and production in human lupus nephritis. Through microarray studies, we analyzed the gene expression profile of renal tubular epithelial cells, stimulated with interferon-alpha. We validated microarray results through real-time polymerase chain reaction, flow cytometry on renal tubular epithelial cells, and through immunohistochemical analysis and confocal microscopy on renal biopsies. RESULTS: Type I interferons signature was characterized by MXA-specific staining in renal tubular epithelial cells; in addition, in situ hybridization showed that renal tubular epithelial cells were the major producers of interferon-alpha, indicating a potential autocrine effect. Whole-genome expression profile showed interferon-alpha induced up-regulation of genes involved in innate immunity, protein ubiquitination and switching to immunoproteasome. In accordance with the in vitro data, class IV lupus nephritis showed up-regulation of the immunoproteasome subunit LMP7 in tubular epithelial cells associated with type I interferon signature. CONCLUSIONS: Our data indicate that type I interferons might have a pathogenic role in lupus nephritis characterized by an autocrine effect of interferon-alpha on renal tubular epithelial cells. Therefore we hypothesize that inhibition of type I interferons might represent a therapeutic target to prevent tubulo-interstitial damage in patients with lupus nephritis.


Assuntos
Regulação da Expressão Gênica , Interferon Tipo I/biossíntese , Interferon-alfa/biossíntese , Túbulos Renais/metabolismo , Nefrite Lúpica/genética , Complexo de Endopeptidases do Proteassoma/genética , RNA/genética , Biópsia , Western Blotting , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Túbulos Renais/patologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Complexo Principal de Histocompatibilidade , Microscopia Confocal , Complexo de Endopeptidases do Proteassoma/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos
18.
Oncotarget ; 5(17): 7446-57, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25277184

RESUMO

In some tumours, despite a wild-type p53 gene, the p53 pathway is inactivated by alterations in its regulators or by unknown mechanisms, leading to resistance to cytotoxic therapies. Understanding the mechanisms of functional inactivation of wild-type p53 in these tumours may help to define prospective targets for treating cancer by restoring p53 activity. Recently, we identified TRIM8 as a new p53 modulator, which stabilizes p53 impairing its association with MDM2 and inducing the reduction of cell proliferation. In this paper we demonstrated that TRIM8 deficit dramatically impairs p53-mediated cellular responses to chemotherapeutic drugs and that TRIM8 is down regulated in patients affected by clear cell Renal Cell Carcinoma (ccRCC), an aggressive drug-resistant cancer showing wild-type p53. These results suggest that down regulation of TRIM8 might be an alternative way to suppress p53 activity in RCC. Interestingly, we show that TRIM8 expression recovery in RCC cell lines renders these cells sensitive to chemotherapeutic treatments following p53 pathway re-activation. These findings provide the first mechanistic link between TRIM8 and the drug resistance of ccRCC and suggest more generally that TRIM8 could be used as enhancer of the chemotherapy efficacy in cancers where p53 is wild-type and its pathway is defective.


Assuntos
Carcinoma de Células Renais/patologia , Proteínas de Transporte/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Renais/patologia , Proteínas do Tecido Nervoso/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Carcinoma de Células Renais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Imunoprecipitação , Neoplasias Renais/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteína Supressora de Tumor p53/metabolismo
19.
Crit Care ; 18(5): 520, 2014 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-25261195

RESUMO

INTRODUCTION: The pathophysiology of endotoxemia-induced acute kidney injury (AKI) is characterized by an intense activation of the host immune system and renal resident cells by lipopolysaccharide (LPS) and derived proinflammatory products. However, the occurrence of renal fibrosis in this setting has been poorly investigated. The aim of the present study was to investigate the possible association between endothelial dysfunction and acute development of tissue fibrosis in a swine model of LPS-induced AKI. Moreover, we studied the possible effects of coupled plasma filtration adsorption (CPFA) in this setting. METHODS: After 9 hours from LPS infusion and 6 hours of CPFA treatment, histologic and biochemical changes were analyzed in pigs. Apoptosis and endothelial dysfunction were assessed on renal biopsies. The levels of LPS-binding protein (LBP) were quantified with enzyme-linked immunosorbent assay (ELISA). Endothelial cells (ECs) were stimulated in vitro with LPS and cultured in the presence of swine sera and were analyzed with FACS and real-time RT-PCR. RESULTS: In a swine model of LPS-induced AKI, we observed that acute tubulointerstitial fibrosis occurred within 9 hours from LPS injection. Acute fibrosis was associated with dysfunctional alpha-smooth muscle actin (α-SMA)+ ECs characterized by active proliferation (Ki-67+) without apoptosis (caspase-3-). LPS led to EC dysfunction in vitro with significant vimentin and N-cadherin expression and increased collagen I mRNA synthesis. Therapeutic intervention by citrate-based CPFA significantly prevented acute fibrosis in endotoxemic animals, by preserving the EC phenotype in both peritubular capillaries and renal arteries. We found that the removal of LBP from plasma was crucial to eliminate the effects of LPS on EC dysfunction, by blocking LPS-induced collagen I production. CONCLUSIONS: Our data indicate that EC dysfunction might be pivotal in the acute development of tubulointerstitial fibrosis in LPS-induced AKI. Selective removal of the LPS adaptor protein LBP might represent a future therapeutic option to prevent EC dysfunction and tissue fibrosis in endotoxemia-induced AKI.


Assuntos
Injúria Renal Aguda/patologia , Células Endoteliais/fisiologia , Rim/patologia , Injúria Renal Aguda/induzido quimicamente , Proteínas de Fase Aguda , Animais , Proteínas de Transporte , Caspase 3/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Fibrose , Rim/irrigação sanguínea , Rim/fisiopatologia , Glicoproteínas de Membrana , Suínos
20.
Methods Mol Biol ; 1186: 1-11, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25149299

RESUMO

Adoptive transfer of tumor-reactive cytotoxic T cells (CTLs) is a promising therapeutic approach for the treatment of solid tumors. To develop these strategies, it is necessary to isolate specific leukocyte subpopulations from peripheral blood or tumor tissue (referred to as tumor-infiltrating lymphocytes (TIL)) that will be reinfused into the patient after expansion in vitro. The ideal cell isolation approach should be performed rapidly, thereby maximizing the recovery and viability of the purified cells. Here, we describe the negative or the positive separation procedures to isolate CD8(+) T cells from whole blood, from peripheral blood mononuclear cells (PBMCs), or from cancer tissue. Purified CD8(+) cells will be used for different downstream applications such as protein and gene expression profile analysis in order to assess their intrinsic cytotoxic ability.


Assuntos
Carcinoma de Células Renais/imunologia , Neoplasias Renais/imunologia , Linfócitos do Interstício Tumoral/citologia , Formação de Roseta/métodos , Linfócitos T Citotóxicos/citologia , Células Apresentadoras de Antígenos/imunologia , Diferenciação Celular/imunologia , Centrifugação com Gradiente de Concentração , Granzimas/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Leucócitos Mononucleares/citologia , Microesferas , Perforina/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia
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