RESUMO
Brucella Lumazine Synthase (BLS) is a highly immunogenic decameric protein which can accept the fusion of foreign proteins at its ten N-termini. These chimeras are very efficient to elicit systemic and oral immunity without adjuvants. BLS signaling via Toll-Like Receptor 4 (TLR4) regulates innate and adaptive immune responses, inducing dendritic cell maturation and CD8(+) T-cell cytotoxicity. In this work we study the effect induced by BLS in TLR4-expressing B16 melanoma. In order to evaluate the effectiveness of BLS as a preventive vaccine, C57BL/6J mice were immunized with BLS or BLS-OVA, and 35 days later were subcutaneously inoculated with B16-OVA melanoma. BLS or BLS-OVA induced a significant inhibition of tumor growth, and 50% of mice immunized with the highest dose of BLS did not develop visible tumors. This effect was not observed in TLR4-deficient mice. For treatment experiments, mice were injected with BLS or BLS-OVA 2 days after the inoculation of B16 cells. Both treatments induced significant and equal tumor growth delay and increased survival. Moreover, BLS and BLS-OVA stimulation were also effective in TLR4-deficient mice. In order to study whether BLS has a direct effect on tumor cells, B16 cells were preincubated with BLS, and after 48h, cells were inoculated. Tumors induced by BLS-stimulated cells had inhibited growth and survival was increased. In the BLS group, 40% of mice did not develop tumors. This effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Our work demonstrates that BLS immunization induces a preventive antitumor response that depends on mice TLR4. We also show that BLS generates a therapeutic effect in mice inoculated with B16 cells. Our results show that BLS acts directly in cultured tumor cells via TLR4, highly suggesting that BLS elicits its therapeutic effects acting on the TLR4 from B16 melanoma cells.
Assuntos
Brucella/enzimologia , Complexos Multienzimáticos/metabolismo , Receptor 4 Toll-Like/genética , Animais , Apoptose , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/mortalidade , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/imunologia , Ovalbumina/genética , Ovalbumina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Taxa de Sobrevida , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/metabolismo , Transplante HomólogoRESUMO
The full-length pigeon ovalbumin (OVA) gene cDNA was cloned and sequenced by reverse transcription-polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends. A 386-amino acid protein was predicted for the obtained sequence, which had 67% identity with the chicken protein. Similar to chicken OVA, the pigeon OVA gene is a non-inhibitory serine protease inhibitor. Quantitative PCR analysis revealed that pigeon OVA mRNA was highly expressed in the oviduct, and trace amounts were detected in other tissues. During the reproductive cycle, pigeon oviduct OVA mRNA expression reached its peak during the egg-laying stage, decreased with brooding, and then increased again during the squab-feeding period. Moreover, the relative OVA expression level in pigeon oviduct epithelial cells could be upregulated by a constant concentration of steroid hormones.
Assuntos
Columbidae/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ovalbumina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Columbidae/crescimento & desenvolvimento , Columbidae/fisiologia , Feminino , Hormônios Esteroides Gonadais/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Ovalbumina/metabolismo , Oviductos/citologia , Oviductos/metabolismo , Oviductos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We successfully replaced the ovalbumin gene of a magnum region in chickens with a human plasminogen activator. We constructed pL-eGFP, pL-tPAGFP and pL-2.8OVtPAGFP vectors and cultured 293FT chicken embryo fibroblasts, chicken primordial germ cells, Hela C127 cells, and oviduct epithelial cells. All vectors were expressed in the transfected cells, except pL-2.8OVtPAGFP vector, which was only expressed in oviduct epithelial cells. A lentivirus with pL-2.8OVtPAGFP was injected in fertilized eggs; 11 chicks hatched in the G0 generation, four of them carried the tPAGFP. Two cockerels from the G0 generation were crossed with four wild-type hens. Three chicks in G1 carried the tPAGFP. We concluded that by using an oviduct-specific vector for transfection, human recombinant plasminogen activator protein can be expressed in the oviducts of laying hens. This character is inherited and can be reproduced with a need for repeated transfection.
Assuntos
Animais Geneticamente Modificados/genética , Galinhas/genética , Engenharia Genética/métodos , Vetores Genéticos/química , Ativadores de Plasminogênio/genética , Transfecção/métodos , Animais , Animais Geneticamente Modificados/metabolismo , Embrião de Galinha , Galinhas/metabolismo , Cruzamentos Genéticos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Reporter , Células Germinativas/citologia , Células Germinativas/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Lentivirus/genética , Masculino , Ovalbumina/genética , Ovalbumina/metabolismo , Oviductos/metabolismo , Ativadores de Plasminogênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ZigotoRESUMO
Baculoviruses (BV) are DNA viruses that are pathogenic for insects. Although BV infect a range of mammalian cell types, they do not replicate in these cells. Indeed, the potential effects of these insect viruses on the immune responses of mammals are only just beginning to be studied. We show in this paper that a recombinant Autographa californica multiple nuclear polyhedrosis virus carrying a fragment of ovalbumin (OVA) on the VP39 capsid protein (BV-OVA) has the capacity to act as an adjuvant and vector of antigens in mice, thereby promoting specific CD4 and cytotoxic T cell responses against OVA. BV also induced in vivo maturation of dendritic cells and the production of inflammatory cytokines, thus promoting innate and adaptive immune responses. The OVA-specific response induced by BV-OVA was strong enough to reject a challenge with OVA-expressing melanoma cells (MO5 cells) and effectively prolonged survival of MO5 bearing mice. All these findings, together with the absence of pre-existing immunity to BV in humans and the lack of viral gene expression in mammalian cells, make BV a candidate for vaccination.
Assuntos
Baculoviridae/imunologia , Baculoviridae/metabolismo , Proteínas do Capsídeo/imunologia , Ovalbumina/imunologia , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/metabolismo , Animais , Baculoviridae/genética , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Células Cultivadas , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Imunoeletrônica , Ovalbumina/genética , Ovalbumina/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
Galectin (Gal) constitute a family of carbohydrate-recognizing molecules ubiquitously expressed in mammals. In the immune system, they regulate many processes such as inflammation, adhesion, and apoptosis. Here, we report the expression in the spleen of the two same Gal-8 splice variants described previously in the thymus. Gal-8 was found to induce two separate biological activities on T lymphocytes: a robust naive CD4(+) T cell proliferation in the absence of antigen and notably, a costimulatory signal that synergized the cognate OVA peptide in DO11.10 mice transgenic for TCR(OVA). The antigen-independent proliferation induced by Gal-8 displayed increased expression of pro- and anti-inflammatory cytokines, thus suggesting the polyclonal expansion of Th1 and Th2 clones. The costimulatory effect on antigen-specific T cell activation was evidenced when the Gal and the peptide were assayed at doses suboptimal to induce T cell proliferation. By mass spectra analysis, several integrins and leukocyte surface markers, including CD45 isoforms, as well as other molecules specific to macrophages, neutrophils, and platelets, were identified as putative Gal-8 counter-receptors. Gal-8 triggered pZAP70 and pERK1/2. Moreover, pretreatment with specific inhibitors of CD45 phosphatase or ERK1/2 prevented its antigen-dependent and -independent T cell-proliferative activities. This seems to be associated with the agonistic binding to CD45, which lowers the activation threshold of the TCR signaling pathway. Taken together, our findings support a distinctive role for locally produced Gal-8 as an enhancer of otherwise borderline immune responses and also suggest that Gal-8 might fuel the reactivity at inflammatory foci.
Assuntos
Proliferação de Células , Galectinas/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Ativação Linfocitária/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Galectinas/genética , Galectinas/farmacologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Células Jurkat , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/genética , Ovalbumina/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo , Linfócitos T/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Dendritic cells (DCs) are professional antigen presenting cells with the ability to induce and regulate an immune response. DCs that capture and present antigen under noninflammatory conditions maintain an immature phenotype and acquire tolerogenic properties. These DCs generate regulatory T lymphocytes that potentiate tolerogenic responses. Here we developed a method for the generation of immature murine DCs able to process and present a specific antigen in a tolerogenic context. Immature DCs were prepared from bone marrow precursors after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of vitamin D(3) and characterized by their low expression of major histocompatibility complex class (MHC) II and CD86 molecules. Purified phagosomes containing either MHC II molecules or ovalbumin were used to deliver antigens to immature DCs. More than 80% of the DCs captured the phagosomes, while maintaining a low expression of maturation markers and showing basal levels of secretion of activating cytokines such as interleukin (IL)-2 and IL-12. Treatment of the immature DCs with lipopolysaccharides (LPS) increased IL-10 secretion, in agreement with their anti-inflammatory and immune regulatory properties. Cocultures of transgenic OT-II T lymphocytes with the immature DCs carrying OVA-phagosomes succeeded in generating a subpopulation of regulatory T lymphocytes characterized by the expression of CD4, CD25, CD62L, and Foxp3. Taken together, our results suggest that vitamin D(3) generates immune tolerance through the modulation of DC phenotype and could be useful to induce tolerance to allotransplants.
Assuntos
Células Dendríticas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-2/efeitos dos fármacos , Células da Medula Óssea/imunologia , Calcitriol/farmacologia , Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/genética , Fagocitose/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
T-cell activation results from engagement of the T-cell receptor (TCR) by cognate peptide-major histocompatibility complex (pMHC) complexes on the surface of antigen-presenting cells (APC). Previous studies have provided evidence supporting the notion that the half-life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters that can influence T-cell activation. However, whether the half-life of the TCR/pMHC interaction can modulate T-cell activation in response to a pathogen challenge remains unknown. To approach this question, we generated strains of bacteria expressing variants of the ovalbumin (OVA) antigen, carrying point mutations in the SIINFEKL sequence. When bound to H-2K(b), this peptide is the cognate ligand for the OT-I TCR. Variants of the H-2K(b)/SIINFEKL bind to the OT-I TCR with distinct half-lives. Here we show that dendritic cells (DCs) infected with bacteria expressing OVA variants were incapable of activating OT-I T cells when the half-life of the TCR/H-2K(b)/OVA interaction was excessively short. Consistent with these data, T-cell activation was only observed in mice infected with bacteria expressing OVA variants that bound to OT-I with a half-life above a certain threshold. Considered together, our data suggest that the half-life of TCR/pMHC interaction can significantly modulate T-cell activation in vivo, as well as influence recognition of antigens expressed by bacteria. These observations underscore the importance of the TCR/pMHC half-life on the clearance of pathogens.
Assuntos
Antígenos de Bactérias/imunologia , Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Infecções por Salmonella/imunologia , Animais , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Meia-Vida , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Ovalbumina/genética , Ovalbumina/imunologia , Salmonella typhimurium/imunologiaRESUMO
The effect of Bacillus CalmetteGue´rin (BCG) treatment in allergic pulmonary reaction was studied in micegenetically selected accordingly to a High (H-IVA) or Low (L-IVA) antibody responsiveness. Mice wereimmunized with ovalbumin (OVA) or OVA plus BCG. Two days after nasal antigenic challenge, seric IgE andIgG1 anti-OVA, eosinophils in pulmonary tissue, inflammatory cells in bronchoalveolar lavage and the complianceand conductance of respiratory system were evaluated. H-IVA mice were found more susceptible than L-IVA, andBCG was able to inhibit simultaneously the production of IgE, the bronchopulmonary inflammation and bronchialhyperresponsiveness in these genetically selected mice.
Assuntos
Animais , Ratos/genética , Vacina BCG/genética , Vacina BCG/imunologia , Ovalbumina , Ovalbumina/análise , Ovalbumina/classificação , Ovalbumina/genética , Ovalbumina/imunologiaRESUMO
Dendritic cells (DCs) are professional APCs with the unique ability to activate naive T cells, which is required for initiation of the adaptive immune response against pathogens. Therefore, interfering with DC function would be advantageous for pathogen survival and dissemination. In this study we provide evidence suggesting that Salmonella enterica serovar typhimurium, the causative agent of typhoid disease in the mouse, interferes with DC function. Our results indicate that by avoiding lysosomal degradation, S. typhimurium impairs the ability of DCs to present bacterial Ags on MHC class I and II molecules to T cells. This process could correspond to a novel mechanism developed by this pathogen to evade adaptive immunity. In contrast, when S. typhimurium is targeted to FcgammaRs on DCs by coating bacteria with Salmonella-specific IgG, bacterial Ags are efficiently processed and presented on MHC class I and class II molecules. This enhanced Ag presentation leads to a robust activation of bacteria-specific T cells. Laser confocal microscopy experiments show that virulent S. typhimurium is rerouted to the lysosomal degradation pathway of DCs when internalized through FcgammaR. These observations are supported by electron microscopy studies demonstrating that internalized S. typhimurium shows degradation signs only when coated with IgG and captured by FcgammaRs on DCs. Therefore, our data support a potential role for bacteria-specific IgG on the augmentation of Ag processing and presentation by DCs to T cells during the immune response against intracellular bacteria.
Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Receptores de IgG/metabolismo , Salmonella typhi/imunologia , Salmonella typhi/metabolismo , Animais , Apresentação de Antígeno/genética , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Linhagem Celular , Células Dendríticas/enzimologia , Células Dendríticas/metabolismo , Proteínas do Ovo/genética , Proteínas do Ovo/imunologia , Proteínas do Ovo/metabolismo , Antígenos H-2/genética , Antígenos H-2/imunologia , Antígenos H-2/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Fragmentos de Peptídeos , Fagocitose/imunologia , Plasmídeos , Receptores de IgG/fisiologia , Salmonella typhi/genética , Salmonella typhi/patogenicidade , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Virulência/genética , Virulência/imunologiaRESUMO
Objetivo: Investigar se o tratamento com BCG é capaz de bloquear a resposta alergica pulmonar de camundongos geneticamente selecionados para uma boa (HIV-A) ou má resposta ( LIV-A) de anticorpos imunizados com ovalbumina ( OVA)...
To investigate whether BCG inhibits allergic pulmonary response in genetically selected mice for high (HIV-A) or low antibody response (LIV-A)...