RESUMO
Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.
Assuntos
Proliferação de Células/fisiologia , Células Dendríticas/efeitos dos fármacos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Linfócitos/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Antígenos de Superfície/biossíntese , Apoptose , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Humanos , Tolerância Imunológica/fisiologia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Leucócitos Mononucleares/fisiologia , Monócitos/citologia , Monócitos/ultraestrutura , Necrose , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologiaRESUMO
Adherent umbilical cord blood stromal cells (AUCBSCs) are multipotent cells with differentiation capacities. Therefore, these cells have been investigated for their potential in cell-based therapies. Quantum Dots (QDs) are an alternative to organic dyes and fluorescent proteins because of their long-term photostability. In this study we determined the effects of the cell passage on AUCBSCs morphology, phenotype, and differentiation potential. QDs labeled AUCBSCs in the fourth cell passage were differentiated in the three mesodermal lineages and were evaluated using cytochemical methods and transmission electron microscopy (TEM). Gene and protein expression of the AUCBSCs immunophenotypic markers were also evaluated in the labeled cells by real-time quantitative PCR and flow cytometry. In this study we were able to define the best cellular passage to work with AUCBSCs and we also demonstrated that the use of fluorescent QDs can be an efficient nano-biotechnological tool in differentiation studies because labeled cells do not have their characteristics compromised.
Assuntos
Adesão Celular , Diferenciação Celular , Proliferação de Células , Sangue Fetal/citologia , Nanotecnologia , Células Estromais/citologia , Contagem de Células , Linhagem da Célula , Células Cultivadas , Sangue Fetal/metabolismo , Citometria de Fluxo , Humanos , Imunofenotipagem , Células Estromais/metabolismo , Células Estromais/ultraestruturaRESUMO
Despite recent advances, patients with malignant brain tumors still have a poor prognosis. Glioblastoma (WHO grade 4 astrocytoma), the most malignant brain tumor, represents 50% of all astrocytomas, with a median survival rate of <1 year. It is, therefore, extremely important to search for new diagnostic and therapeutic approaches for patients with glioblastoma. This study describes the application of superparamagnetic nanoparticles of iron oxide, as well as monoclonal antibodies, of immunophenotypic significance, conjoined to quantum dots for the ultrastructural assessment of glioblastoma cells. For this proposal, an immunophenotypic study by flow cytometry was carried out, followed by transmission electron microscopy analysis. The process of tumor cell labeling using nanoparticles can successfully contribute to the identification of tumorigenic cells and consequently for better understanding of glioblastoma genesis and recurrence. In addition, this method may help further studies in tumor imaging, diagnosis, and prognostic markers detection.
Assuntos
Glioblastoma/diagnóstico , Glioblastoma/ultraestrutura , Nanopartículas , Coloração e Rotulagem/métodos , Antígeno AC133 , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Biomarcadores/análise , Biomarcadores/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Vesículas Citoplasmáticas/metabolismo , Endocitose/imunologia , Endoglina , Citometria de Fluxo , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/metabolismo , Glioblastoma/metabolismo , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Imunofenotipagem , Integrina beta1/imunologia , Integrina beta1/metabolismo , Nanopartículas de Magnetita/química , Microscopia Eletrônica de Transmissão , Nanomedicina/métodos , Nanopartículas/química , Peptídeos/imunologia , Peptídeos/metabolismo , Pontos Quânticos , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Células Tumorais CultivadasRESUMO
Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.
Assuntos
Humanos , Proliferação de Células/fisiologia , Células Dendríticas/efeitos dos fármacos , /metabolismo , Linfócitos/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Apoptose , Antígenos de Superfície/biossíntese , Técnicas de Cultura de Células , Células Cultivadas , Diferenciação Celular/fisiologia , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Tolerância Imunológica/fisiologia , /genética , Leucócitos Mononucleares/fisiologia , Monócitos/citologia , Monócitos/ultraestrutura , Necrose , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologiaRESUMO
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by chronic joint inflammation and continuous immune cell infiltration in the synovium. These changes are linked to inflammatory cytokine release, leading to eventual destruction of cartilage and bone. During the last decade new therapeutic modalities have improved the prognosis, with the introduction of novel biological response modifiers including anti-TNFalpha CTLA4Ig and, more recently, anti-IL6. In the present study we looked at the immunological effects of these three forms of therapy. Serum, obtained from patients with RA was analyzed for TNFalpha, IL6, IL10, IFNgamma, and VEGF, and in parallel, circulating plasmacytoid and myeloid dendritic cells (DC) were enumerated before and after three infusions of the respective biological treatments. After treatment with anti-IL6, we found a significant reduction of IL6 and TNFalpha levels and the percentage of both DC subsets decreased. Although the results did not reach statistical significance for anti-TNFalpha treatment, similar trends were observed. Meanwhile, CTLA4Ig therapy led to the reduction IFNgamma levels only. None of the treatments modified significantly VEGF or IL10 levels. These findings may explain why patients with RA improve more rapidly on IL-6 therapy than with the other two modalities.