RESUMO
Silencing of SOCS1 protein with shRNAi lentivirus (shR-SOCS1) led to partial reversion of the tumorigenic phenotype of B16F10-Nex2 melanoma cells. SOCS1 silencing inhibited cell migration and invasion as well as in vitro growth by cell cycle arrest at S phase with increased cell size and nuclei. Down-regulation of SOCS1 decreased the expression of epidermal growth factor receptor, Ins-Rα, and fibroblast growth factor receptors. The present work aimed at analyzing the SOCS1 cell signaling and expression of proteins relevant to tumor development. An RNA microarray analysis of B16F10-Nex2 melanoma cells with SOCS1 silenced by shRNAi-SOCS1 was undertaken in comparison with cells transduced with the empty vector. Among 609 differentially expressed genes, c-Kit, Met and EphA3 cytokine/tyrosine-kinase (TK) receptors were down regulated. A significant decrease in the expression of TK receptors, the phosphorylation of mediators of ERK1/2 and p38 pathways and STAT3 (S727) were observed. Subcutaneous immunization with shR-SOCS1-transduced viable tumor cells rendered protection against melanoma in a syngeneic model, with decreased expression of PD-L1 and of matrix metallo-proteinases (MMPs) and CD-10 in those cells. The present work shows the role of SOCS1 in murine melanoma development and the potential of SOCS1-silenced tumor cells in raising an effective anti-melanoma immune response.
Assuntos
Antígeno B7-H1/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal , Imunidade , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Melanoma Experimental/genética , Antígenos Específicos de Melanoma/metabolismo , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Substâncias Protetoras/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Fator de Transcrição AP-2/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Regulação para Cima/genéticaRESUMO
Antitumor immune responses are associated with proinflammatory cytokines, whereas tumor-developing animals generally have increased the production of immunosuppressive cytokines. Here, we show that splenocytes from C57Bl/6 mice resistant to low doses of B16F10-Nex2 melanoma cells produced twofold or higher interferon-γ (IFN-γ)/interleukin-10 (IL-10) ratios, whereas cells from tumor-bearing animals produced predominantly IL-10. IL-10-knockout (IL-10KO) mice were significantly more resistant to B16F10-Nex2 development, producing increased amounts of IL-12 and IFN-γ. To neutralize IL-10 in vivo, aiming at cancer therapy, recombinant eukaryotic plasmid expressing the soluble extracellular region of the murine IL-10 receptor α-chain was constructed (pcDNA3-sIL-10R). Plasmid-treated melanoma-challenged animals showed extended survival time, the protective response was IFN-γ dependent and enhanced by co-immunization with a plasmid expressing IL-12. Dendritic cells (DCs) from IL-10KO mice, primed with B16F10-Nex2 antigens (TAg), secreted increased amounts of T-helper 1-type cytokines and increased the expression of surface activation markers. Vaccination of C57Bl/6 mice with TAg-activated IL-10KO DCs, as well as with TAg-primed DCs from C57Bl/6 mice transfected with pcDNA3-sIL10R plasmid, significantly increased animal survival. In conclusion, an IFN-γ-dependent protective response was induced against B16F10-Nex2 cells by neutralization of IL-10 with pcDNA3-sIL10R plasmid. This effect was enhanced by association with IL-12 gene therapy (80% protection), and could be mediated by TAg-primed DCs.
Assuntos
Terapia Genética/métodos , Imunoterapia Adotiva/métodos , Interferon gama/imunologia , Interleucina-12/genética , Melanoma Experimental/terapia , Receptores de Interleucina-10/genética , Animais , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Humanos , Interleucina-12/biossíntese , Interleucina-12/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmídeos/genética , Receptores de Interleucina-10/biossíntese , Receptores de Interleucina-10/imunologia , TransfecçãoRESUMO
Propionibacterium acnes has been described as a potent adjuvant to immune responses in vitro and in vivo. Presently, we analysed the modulation of peritoneal exudate cells (PEC) by heat-killed P. acnes or its purified soluble polysaccharide (PS), both injected intraperitoneally in C57Bl/6 mice, aiming at their recruitment and cytotoxicity. Both treatments induced an increase in macrophages, immature dendritic cells, B1a lymphocytes and NK1.1(+) CD3(+) cells. The bacterium caused a remarkable increase in a NK1.1(+) CD3(+) CD4(-) CD8(-) cells subpopulation, whereas the PS component seemed responsible for the recruitment of mainly macrophage cells. To assess P. acnes and PS adjuvant effect on PEC cytotoxicity we evaluated their in vitro effect on murine B16F10 melanoma cells. The effector cells from the heat-killed bacteria and PS-treated groups lysed melanoma cells in co-cultures with PEC. Mice genetically deficient in IFN-gamma, when stimulated with P. acnes or PS, had reduced PEC cytotoxicity, and the cytotoxic effect was completely abrogated in PEC from iNOS(-/-) mice. The tumoricidal activity of PEC from P. acnes-treated mice was mediated by macrophages and NKT cells stimulated with IL-12. In PS-treated mice the cytotoxicity was mediated mainly by macrophages. Moreover, both treatments increased IL-4 and IFN-gamma production by NKT cells. In conclusion, we show that P. acnes act mainly by recruiting and activating NKT double-negative cells in PEC, which were shown to be tumoricidal in vitro when induced by IL-12. Macrophages induced by both P. acnes and PS have their antitumour effect dependent on NO production.
Assuntos
Líquido Ascítico/citologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Lipopolissacarídeos/imunologia , Macrófagos Peritoneais/imunologia , Propionibacterium acnes/imunologia , Animais , Líquido Ascítico/imunologia , Morte Celular/imunologia , Citocinas/metabolismo , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/imunologia , Feminino , Fatores Imunológicos/imunologia , Imunofenotipagem , Injeções Intraperitoneais , Lipopolissacarídeos/isolamento & purificação , Ativação de Macrófagos/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico/metabolismo , Células Tumorais CultivadasRESUMO
The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15-mer sequence from gp43, denominated P10, induced T-CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen-DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T-cell epitope expressed in a tetravalent 13-mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freund's adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti-PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10-elicited T-cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.
Assuntos
Antígenos de Fungos/imunologia , Epitopos de Linfócito T/imunologia , Proteínas Fúngicas/imunologia , Glicoproteínas/imunologia , Paracoccidioidomicose/prevenção & controle , Peptídeos/síntese química , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Feminino , Vacinas Fúngicas/imunologia , Imunização , Ativação Linfocitária/imunologia , Masculino , Camundongos , Dados de Sequência Molecular , Paracoccidioides/imunologiaRESUMO
Polyclonal and monoclonal antibodies (MAbs) have been raised against B16F10 cells collected from growing tumors in vivo or grown in culture media supplemented with normal mouse serum to avoid xenogeneic reactivity. Antibody binding to glutaraldehyde-fixed melanoma cells and Melan A melanocytes was assayed using chemiluminescent-enzyme-linked immunosorbent assay (CL-ELISA) for increased sensitivity. Most of the reactivity of antitumor polyclonal IgG (92%) was inhibitable by a carbohydrate pool consisting of melibiose, mannose, lactose, and sialic acid. Two monoclonal IgG(2a) antibodies, A4 and B11, had their reactivity to melanoma cells completely and specifically inhibited by melibiose. MAb A4 did not bind to alpha-galactosyl residues abundantly expressed in a protozoan mucin used as substrate, and its binding to the tumor cells was not affected by alpha-galactosidase treatment or addition of alpha-methyl-galactopyranoside or raffinose. Recognition of a mimotope similar to melibiose is suggested. MAb is cytotoxic in vitro in a complement-mediated reaction and effectively neutralizes melanoma cells protecting syngeneic mice against tumor development in vivo. This MAb is thus an important tool for further studies on antitumor adjuvant therapy combined with other agents associated with immuno- and chemotherapy of invasive melanoma.
Assuntos
Melanoma/metabolismo , Melibiose/química , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antineoplásicos/administração & dosagem , Antígenos de Neoplasias , Vacinas Anticâncer/química , Metabolismo dos Carboidratos , Carboidratos/farmacologia , Separação Celular , Proteínas do Sistema Complemento , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Epitopos , Citometria de Fluxo , Galactose/química , Glutaral/farmacologia , Humanos , Hibridomas , Imunoglobulina G/metabolismo , Imunoterapia , Lactose/metabolismo , Antígeno MART-1 , Manose/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Mucinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Proteínas de Neoplasias/farmacologia , Conformação Proteica , Trypanosoma cruziRESUMO
An exocellular proteinase activity has been characterized in Paracoccidioides brasiliensis culture filtrates. Chromatographic analysis showed that the activity was eluted from an anion-exchange Resource Q column at 0.08-0.1 M NaCl, and by gel filtration near ovalbumin elution, in a single peak. Purification of the proteinase, however, was hampered by the low protein yield, in contrast to the high peptidase activity. Numerous chromogenic peptidyl p-nitroanilide derivatives and internally quenched fluorescent peptides, flanked by Abz (O-aminobenzoyl) and EDDnp (ethylenediaminedinitrophenyl), were tested as substrates. Cleavage was observed with Abz-MKRLTL-EDDnp, Abz-FRLVR-EDDnp, and Abz-PLGLLGR-EDDnp at Leu-Thr, Leu-Val and Leu-Leu/Leu-Gly bonds respectively as determined by isolation of the corresponding fragments by HPLC. Leucine at P1 seemed to be restrictive for the activity of the exocellular enzyme, but threonine (P'1) and leucine (P'2) in Abz-MKRLTL-EDDnp apparently were not essential. Also, a pair of alanines could substitute for lysine (P3) and arginine (P2) in this substrate, with a decrease in the Km values. The exocellular peptidase activity of P. brasiliensis had an optimum pH of > 9.0 and was irreversibly inhibited by PMSF, mercuric acetate and p-hydroxymercuribenzoate. Inhibition of the mercuriate compounds could be partially reversed by Cys/EDTA. E-64 [trans-epoxysuccinyl-L-leucylamido-(4-guanido)butene] was a weak and reversible inhibitor, whereas EDTA and pepstatin were not inhibitory. These results suggest that P. brasiliensis exocellular enzyme belongs to the subfamily of SH-containing serine proteinases.
Assuntos
Paracoccidioides/enzimologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Hidrólise , Dados de Sequência Molecular , Peptídeos/metabolismo , Inibidores de ProteasesRESUMO
The 43,000 dalton glycoprotein of Paracoccidioides brasiliensis (gp 43) is the main exocellular antigen recognized by sera from patients with paracoccidioidomycosis in a variety of serological assays. Specific conformational peptide epitopes are recognized by the human antibodies as determined by antigen deglycosylation. Procedures for the purification of the gp43 using immunoaffinity chromatography have been described. The secretion of the gp43 as a function of the growth curve, its partial aggregation with a proteolytic enzyme, ability to bind laminin, as well as to form circulating immunocomplexes in vivo could play a role in pathogenesis. Crude antigenic preparations depleted of gp43 epitopes lost their ability to elicit positive skin tests. Accordingly, the purified gp43 molecule induced delayed hypersensitivity reactions in man and infected animals, caused a T-CD4-dependent proliferation of lymph node cells from mice immunized with it, and of peripheral blood lymphocytes from an individual sensitized to P. brasiliensis by prolonged contact with the fungus. To identify the immunodominant epitopes in both humoral and cellular reactions, the gp43 gene has been cloned, sequenced, and partly expressed. It bears peptide sequences homologous to those of beta-1,3-glucanases from Candida albicans and Saccharomyces cerevisiae but has no enzymatic activity itself. The molecular weight of the unglycosylated antigen is 42,227. A single N-linked oligosaccharide chain in the gp43 contains alpha-D-mannopyranosyl, beta-D-galactofuranosyl and N-acetylglucosaminyl units with the predominant ratio of 10:2:2, and characteristics of a high mannose type.
Assuntos
Antígenos de Fungos , Paracoccidioides/imunologia , Paracoccidioidomicose/diagnóstico , Sequência de Aminoácidos , Animais , Antígenos de Fungos/genética , Antígenos de Fungos/metabolismo , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Fava Nettos' polysaccharide antigen (FNPA), shown to detect humoral and cellular responses in paracoccidioidomycosis, was investigated. Skin tests with FNPA were negative after alkaline hydrolysis or depletion of gp43 peptide epitopes. Purified antibodies to FNPA or gp43 from paracoccidioidomycosis patients cross-reacted, showing common epitopes and FNPA-specific ones. Normal human sera, in contrast to paracoccidioidomycosis sera, were unreactive with gp43 but recognized epitopes of FNPA susceptible to alkaline hydrolysis and pronase treatment. Histoplasmosis patients sera strongly reacted with FNPA carbohydrate epitopes.
Assuntos
Antígenos de Fungos/imunologia , Epitopos/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Polissacarídeos/imunologia , Animais , Anticorpos Antifúngicos , Antígenos de Fungos/isolamento & purificação , Reações Cruzadas , Cobaias , Histoplasmose/imunologia , Humanos , Masculino , Polissacarídeos/isolamento & purificação , Testes CutâneosRESUMO
Quantitative chemiluminescent enzyme-linked immunosorbent assay (ELISA) and dot-blotting procedures were developed to evaluate the reactivity of human antibodies with crude antigens and purified molecules of parasites and fungi, mainly Trypanosoma cruzi and Paracoccidioides brasiliensis. Reproducible, highly sensitive, and strictly dose-responding results were obtained, with the specificity depending on the kind of antigen used. Mixed antigens (epimastigote membrane and HIV-1 heptapeptide) applied in dots could be independently recognized by specific sera. Purified antigens (T. cruzi F2/3 and P. brasiliensis gp43) at very small concentrations gave specific reactions with patients' sera diluted > or = 1:1,000 and were very poorly reactive or unreactive with natural antibodies using the chemiluminescent immunoassays. P. brasiliensis crude antigen Fava Netto polysaccharide antigen (FNPA) contained peptide epitopes recognized by natural antibodies and carbohydrate epitopes reactive with sera from histoplasmosis patients. It is very important that sensitive chemiluminescence immunoassays be used with purified antigenic molecules to ensure specificity for the diagnosis and follow-up of parasitic and fungal infections.
Assuntos
Anticorpos Antifúngicos/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Fungos/imunologia , Antígenos de Protozoários/imunologia , Paracoccidioides/imunologia , Trypanosoma cruzi/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Humanos , Medições LuminescentesRESUMO
Several methods have been used for the preservation of fungi, all of them presenting advantages and disadvantages. The choice of the methods depends upon the laboratory availabilities, time of preservation, genetic stability of the cultures and other factors. In this work the results obtained through the utilization of Castellani's method (preservation in distilled water) for the maintenance of 174 strains belonging to the "Micoteca do Instituto de Medicina Tropical de São Paulo" are presented. These strains were analyzed after 6, 12, 18 and 24 months, with regard to the percentage of viability taking into consideration the rates of growth and contamination. The smallest percentage of viability occurred in the group of the actinomycetes (50 to 100%) and the largest one in the group of the yeasts (near 100%). According to other authors, the Castellani's method, besides being simple and economically feasible for small size laboratories, yields good results.