RESUMO
UNLABELLED: The high levels of interleukin 10 (IL-10) present in chronic hepatitis C virus (HCV) infection have been suggested as responsible for the poor antiviral cellular immune responses found in these patients. To overcome the immunosuppressive effect of IL-10 on antigen-presenting cells such as dendritic cells (DCs), we developed peptide inhibitors of IL-10 to restore DC functions and concomitantly induce efficient antiviral immune responses. Two IL-10-binding peptides (p9 and p13) were selected using a phage-displayed library and their capacity to inhibit IL-10 was assessed in a bioassay and in STAT-3 (signal transducer and activator of transcription 3) phosphorylation experiments in vitro. In cultures of human leukocytes where HCV core protein induces the production of IL-10, p13 restored the ability of plasmacytoid DC to produce interferon alpha (IFN-α) after Toll-like receptor 9 (TLR9) stimulation. Similarly, when myeloid DCs were stimulated with CD40L in the presence of HCV core, p9 enhanced IL-12 production by inhibiting HCV core-induced as well as CD40L-induced IL-10. Moreover, in vitro, p13 potentiated the effect of maturation stimuli on human and murine DC, increasing their IL-12 production and stimulatory activity, which resulted in enhanced proliferation and IFN-γ production by responding T-cells. Finally, immunization with p13-treated murine DC induced stronger anti-HCV T-cell responses not only in wildtype mice but also in HCV transgenic mice and in mice transiently expressing HCV core in the liver. CONCLUSION: These results suggest that IL-10 inhibiting peptides may have important applications to enhance anti-HCV immune responses by restoring the immunostimulatory capabilities of DC.
Assuntos
Células Dendríticas/imunologia , Hepacivirus/imunologia , Interleucina-10/antagonistas & inibidores , Interleucina-12/biossíntese , Sequência de Aminoácidos , Animais , Ligante de CD40/farmacologia , Linhagem Celular , Células Dendríticas/metabolismo , Antígenos da Hepatite C/farmacologia , Humanos , Interferon-alfa/biossíntese , Interleucina-10/imunologia , Camundongos , Biblioteca de Peptídeos , Fator de Transcrição STAT3/metabolismo , Receptor Toll-Like 9/fisiologia , Proteínas do Core Viral/farmacologiaRESUMO
UNLABELLED: Injection of dendritic cells (DCs) presenting viral proteins constitutes a promising approach to stimulate T cell immunity against hepatitis C virus (HCV). Here we describe a strategy to enhance antigen loading and immunostimulatory functions of DCs useful in the preparation of therapeutic vaccines. Incubation of murine DCs with CFm40L, an adapter molecule containing the coxsackie-adenovirus receptor fused to the ecto-domain of murine CD40L-induced DC maturation, produced high amounts of interleukin-12 and up-regulation of molecules associated with T helper 1 responses. Accordingly, targeting of an adenovirus encoding HCV NS3 protein (AdNS3) to DCs with CFm40L strongly enhanced NS3 presentation in vitro, activating interferon-γ-producing T cells. Moreover, immunization of mice with these DCs promoted strong CD4 and CD8 T cell responses against HCV NS3. CFh40L, a similar adapter molecule containing human CD40L, enhanced transduction and maturation of human monocyte-derived DCs. Comparison of DCs transduced with AdNS3 and CFh40L from patients with chronic HCV infection and healthy donors revealed similar maturation levels. More importantly, DCs from the patients induced NS3-specific responses when transduced with AdNS3 and CFh40L but not with AdNS3 alone. CONCLUSION: DCs transduced with AdNS3 and the adapter molecule CFm/h40L exhibit enhanced immunostimulatory functions, induce robust anti-HCV NS3 immunity in animals, and can induce antiviral immune responses in subjects with chronic HCV infection. This strategy may serve as therapeutic vaccination for patients with chronic hepatitis C.
Assuntos
Adenoviridae/fisiologia , Células Dendríticas/virologia , Hepacivirus/imunologia , Hepatite C/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Vírion/fisiologia , Adenoviridae/genética , Animais , Ligante de CD40/genética , Ligante de CD40/fisiologia , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Modelos Animais de Doenças , Hepatite C/patologia , Hepatite C/prevenção & controle , Humanos , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Transdução Genética , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/uso terapêutico , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismoRESUMO
Immunosuppressive activity of regulatory T cells (Treg) may contribute to the progression of cancer or infectious diseases by preventing the induction of specific immune responses. Using a phage-displayed random peptide library, we identified a 15-mer synthetic peptide, P60, able to bind to forkhead/winged helix transcription factor 3 (FOXP3), a factor required for development and function of Treg. P60 enters the cells, inhibits FOXP3 nuclear translocation, and reduces its ability to suppress the transcription factors NF-κB and NFAT. In vitro, P60 inhibited murine and human-derived Treg and improved effector T cell stimulation. P60 administration to newborn mice induced a lymphoproliferative autoimmune syndrome resembling the reported pathology in scurfy mice lacking functional Foxp3. However, P60 did not cause toxic effects in adult mice and, when given to BALB/c mice immunized with the cytotoxic T cell epitope AH1 from CT26 tumor cells, it induced protection against tumor implantation. Similarly, P60 improved the antiviral efficacy of a recombinant adenovirus expressing NS3 protein from hepatitis C virus. Functional inhibition of Treg by the FOXP3-inhibitory peptide P60 constitutes a strategy to enhance antitumor and antiviral immunotherapies.
Assuntos
Fatores de Transcrição Forkhead/antagonistas & inibidores , Peptídeos/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Vacinas/imunologia , Animais , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Microscopia Confocal , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/metabolismo , TransfecçãoRESUMO
During peritoneal dialysis (PD), mesothelial cells undergo mesothelial-to-mesenchymal transition (MMT), a process associated with peritoneal-membrane dysfunction. Because TGF-ß1 can induce MMT, we evaluated the efficacy of TGF-ß1-blocking peptides in modulating MMT and ameliorating peritoneal damage in a mouse model of PD. Exposure of the peritoneum to PD fluid induced fibrosis, angiogenesis, functional impairment, and the accumulation of fibroblasts. In addition to expressing fibroblast-specific protein-1 (FSP-1), some fibroblasts co-expressed cytokeratin, indicating their mesothelial origin. These intermediate-phenotype (Cyto(+)/FSP-1(+)) fibroblasts had features of myofibroblasts with fibrogenic capacity. PD fluid treatment triggered the appearance of CD31(+)/FSP-1(+) and CD45(+)/FSP-1(+) cells, suggesting that fibroblasts also originate from endothelial cells and from cells recruited from bone marrow. Administration of blocking peptides significantly ameliorated fibrosis and angiogenesis, improved peritoneal function, and reduced the number of FSP-1(+) cells, especially in the Cyto(+)/FSP-1(+) subpopulation. Conversely, overexpression of TGF-ß1 in the peritoneum by adenovirus-mediated gene transfer led to a marked accumulation of fibroblasts, most of which derived from the mesothelium. Taken together, these results demonstrate that TGF-ß1 drives the peritoneal deterioration induced by dialysis fluid and highlights a role of TGF-ß1-mediated MMT in the pathophysiology of peritoneal-membrane dysfunction.
Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/etiologia , Peritônio/patologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Soluções para Diálise/efeitos adversos , Feminino , Injeções Intraperitoneais , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Fibrose Peritoneal/metabolismo , Fibrose Peritoneal/patologia , Fibrose Peritoneal/prevenção & controle , Fenótipo , Receptores de Fatores de Crescimento Transformadores beta/uso terapêutico , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidoresRESUMO
Pulmonary fibrosis encompasses several respiratory diseases characterized by epithelial cell injury, inflammation and fibrosis. Transforming growth factor (TGF)-ß1 is one of the main profibrogenic cytokines involved in the pathogenesis of lung fibrosis. It induces fibroblast differentiation into myofibroblasts, which produce high levels of collagen and concomitantly loss of lung elasticity and reduction of the respiratory function. In the present study, we have investigated the effects of P17 (a TGF-ß inhibitor peptide) on IMR-90 lung fibroblast differentiation in vitro, as well as on the inhibition of the development of bleomycin-induced pulmonary fibrosis in mice. It was found that in IMR-90 cells, P17 inhibited TGF-ß1-induced expression of connective tissue growth factor and α-smooth muscle actin. In vivo, treatment of mice with P17 2days after bleomycin administration decreased lung fibrosis, areas of myofibroblast-like cells and lymphocyte infiltrate. P17 also reduced mRNA expression of collagen type I, fibronectin and the fibronectin splice isoform EDA in the lung, and increased the expression of IFN-γ mRNA. Finally, therapeutic treatment with P17 in mice with already established fibrosis was able to significantly attenuate the progression of lung fibrosis. These results suggest that P17 may be useful in the treatment of pulmonary fibrosis.
Assuntos
Fibroblastos/efeitos dos fármacos , Peptídeos/farmacologia , Fibrose Pulmonar/prevenção & controle , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Actinas/genética , Actinas/metabolismo , Animais , Bleomicina , Western Blotting , Linhagem Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/química , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Transforming growth factor-beta (TGF-beta) is a cytokine with potent immunosuppressive effects and is overexpressed in many tumors. Therefore, development of molecules able to inhibit TGF-beta is of paramount importance to improve the efficacy of antitumor immunotherapy. TGF-beta inhibitor peptides P144 and P17 were combined with the administration of adjuvant molecules poly(I:C) and agonistic anti-CD40 antibodies, and their effect on the growth of E.G7-OVA established tumors and on antitumor immune response was evaluated. Tumor rejection efficacy of a single administration of adjuvants was enhanced from 15 to 70 % when combined with repeated injections of TGF-beta inhibitor peptides. Simultaneous administration of adjuvants and TGF-beta inhibitor peptides was required for maximal therapeutic efficacy. Although tumor cells produced TGF-beta, it was found that the beneficial effect of peptide administration was mainly due to the inhibition of TGF-beta produced by regulatory CD4(+)CD25(+) T cells rather than by tumor cells. The enhanced antitumor effect was accompanied by a higher activity of dendritic cells, natural killer cells and tumor antigen-specific T cells, as well as by a decrease in the number of myeloid-derived suppressor cells. In conclusion, administration of peptide inhibitors of TGF-beta in therapeutic vaccination enhances the efficacy of immunotherapy by increasing antitumor immune responses. These peptide inhibitors may have important applications for current immunotherapeutic strategies.
Assuntos
Imunoterapia , Melanoma Experimental/terapia , Fragmentos de Peptídeos/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Adjuvantes Imunológicos/uso terapêutico , Animais , Antígenos CD40/imunologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Proteínas de Homeodomínio/fisiologia , Humanos , Células Matadoras Naturais/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Poli I-C/imunologia , Poli I-C/uso terapêutico , Receptores de Fatores de Crescimento Transformadores beta , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND/AIMS: Vaccination strategies able to induce strong T-cell responses might contribute to eradicate hepatitis C virus (HCV) infection. We previously demonstrated that fusion of an antigen to the extra domain A from fibronectin (EDA) targets the antigen to TLR4-expressing dendritic cells (DC) and improves its immunogenicity. Here, we studied if fusion of EDA with the non-structural HCV protein NS3 might constitute an effective immunogen against HCV. METHODS: Recombinant NS3 and the fusion protein EDA-NS3 were produced and purified from E. coli, and tested in vitro for their capacity to activate maturation of DC and to favour antigen presentation. HHD transgenic mice expressing the human HLA-A2 molecule were immunized with recombinant proteins in the absence or presence of poly(I:C) and anti-CD40 agonistic antibodies and responses elicited by vaccination were tested in vitro, and in vivo, by their capacity to downregulate intrahepatic expression of HCV-NS3 RNA. RESULTS: EDA-NS3, but not NS3 alone, upregulated the expression of maturation markers, as well as Delta-like 1 and Delta-like 4 Notch ligands in DC and induced the production of IL-12. Mice immunized with EDA-NS3 had strong and long lasting NS3-specific CD4+ and CD8+ T-cell responses and, in combination with poly(I:C) and anti-CD40, downregulated intrahepatic expression of HCV-NS3 RNA. CONCLUSIONS: Recombinant EDA-NS3 may be considered for the development of vaccines against HCV infection.
Assuntos
Fibronectinas/uso terapêutico , Hepacivirus/imunologia , Hepatite C/prevenção & controle , Proteínas Recombinantes/uso terapêutico , Proteínas Virais de Fusão/uso terapêutico , Proteínas não Estruturais Virais/uso terapêutico , Vacinas Virais/uso terapêutico , Animais , Antivirais/uso terapêutico , Linhagem Celular , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Escherichia coli/metabolismo , Feminino , Fibronectinas/biossíntese , Antígeno HLA-A2/genética , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/imunologia , Hepatite C/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Poli I-C/uso terapêutico , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Virais de Fusão/biossíntese , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genética , Vacinas Virais/biossínteseRESUMO
Chronic infection by hepatitis C virus (HCV) is characterized by the absence of efficient antiviral T-cell responses. Thus, vaccination strategies to induce strong anti-HCV T-cell responses are of paramount importance for prophylactic and therapeutic purposes. Dendritic cells (DCs) are the most potent antigen presenting cells; therefore, immunization with these cells loaded with viral antigens offers a new approach for induction of antiviral immunity. Here we show that immunization with DCs transfected with an adenovirus encoding non-structural 3 protein, from HCV (AdNS3), induced multiepitopic CD4 T helper cell 1 (Th1) and CD8 T-cell responses in different mouse strains. These responses prevented the growth of a tumorexpressing HCV proteins, in short- and long-term experiments. Moreover, immunization with AdNS3-transfected DCs did not induce anti-adenoviral antibodies, as compared to direct immunization with AdNS3, but elicited T-cell responses even in the presence of pre-existing anti-adenoviral antibodies. Finally, responses induced by this protocol down-regulated the expression of HCV RNA in the liver. In conclusion, DCs transfected with AdNS3 may prove to be an efficient anti-HCV vaccine.
Assuntos
Adenoviridae , Células Dendríticas/metabolismo , Células Dendríticas/transplante , Vetores Genéticos , Hepacivirus/imunologia , Transdução Genética , Vacinas contra Hepatite Viral/administração & dosagem , Proteínas não Estruturais Virais/genética , Adenoviridae/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Feminino , Vetores Genéticos/imunologia , Hepatite C/imunologia , Hepatite C/prevenção & controle , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologia , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/imunologiaRESUMO
Following recognition of antigens by T helper (Th) lymphocytes, T cell help is elicited to induce humoral and cellular immune responses. These antigens are presented as short peptides, T helper peptides (THP), bound to MHC class II molecules. Since both endogenous THP (from antigens of interest) or exogenous THP (not encompassed by the sequence of the antigen of interest) are able to elicit T cell help, we decided to engineer promiscuous exogenous THP capable of binding to several HLA-DR molecules, in order to cover an important proportion of the human population. Some of these exogenous THP were able to bind to all seven HLA-DR molecules tested and were immunogenic in vivo in HLA-DR4 transgenic mice. Among them, peptides p37, p62 and p45 elicited Th1 cytokine profiles in vivo, providing help for the induction of potent CTL responses. Finally, in vitro stimulation assays carried out using human cells, showed that these peptides could induce T cell responses using cells obtained from individuals with a broad spectrum of HLA-DR molecules. Thus, engineered exogenous THP may be a valuable tool for the induction of immune responses in a large proportion of human population.
Assuntos
Formação de Anticorpos/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Peptídeos/farmacologia , Engenharia de Proteínas , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Antígenos HLA-DR/imunologia , Humanos , Imunidade Celular/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Development of vaccination strategies against hepatitis C virus (HCV) is of paramount importance. With this aim, we tested the ability of dendritic cell-activating reagents polyinosinic-polycytidylic acid (poly(I:C)) and anti-CD40, as adjuvants to induce T-cell responses against HCV. Immunization of mice with these adjuvants induced dendritic cell maturation in vivo. Also, joint administration of poly(I:C) and anti-CD40 plus HCV antigens had a synergistic effect on the induction of anti-HCV T-cell responses. CD4 responses displayed a Th1 cytokine profile, and CD8 responses could be induced by immunization with a minimal CD8 epitope. Addition of a low amount of NS3 protein (as a source of Th epitopes) to the immunization mixture enhanced CD8 responses, whereas immunization with higher doses of NS3 induced both CD4 and CD8 responses. Surprisingly, immunization with NS3 protein but not with CD8 epitopes was able to induce CD8 responses and able to recognize cells expressing HCV antigens endogenously. Moreover, immunization with these adjuvants activated NK cells, which in turn helped to induce Th1 responses. Finally, this combined immunization protocol afforded long-lasting T-cell responses, suggesting that this strategy may prove to be useful in vaccination and/or treatment of HCV infection.
Assuntos
Anticorpos/imunologia , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD40/imunologia , Linfócitos T CD8-Positivos/imunologia , Hepacivirus/imunologia , Hepatite C/imunologia , Imunização , Peptídeos/imunologia , Poli I-C/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos/administração & dosagem , Especificidade de Anticorpos , Antígenos Virais/administração & dosagem , Diferenciação Celular , Linhagem Celular Tumoral , Citocinas/biossíntese , Células Dendríticas/citologia , Sinergismo Farmacológico , Epitopos de Linfócito T/imunologia , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/administração & dosagem , Peptídeos/síntese química , Poli I-C/administração & dosagem , Fatores de Tempo , Proteínas não Estruturais Virais/imunologiaRESUMO
Transforming growth factor-beta (TGF-beta) plays a crucial role in the pathogenesis of skin fibrotic diseases. Systemic TGF-beta inhibitors effectively inhibit fibrosis in different animal models; however, systemic inhibition of TGF-beta raises important safety issues because of the pleiotropic physiological effects of this factor. In this study, we have investigated whether topical application of P144 (a peptide inhibitor of TGF-beta1) ameliorates skin fibrosis in a well-characterized model of human scleroderma. C3H mice received daily subcutaneous injections of bleomycin for 4 wk, and were treated daily with either a lipogel containing P144 or control vehicle. Topical application of P144 significantly reduced skin fibrosis and soluble collagen content. Most importantly, in mice with established fibrosis, topical treatment with P144 lipogel for 2 wk significantly decreased skin fibrosis and soluble collagen content. Immunohistochemical studies in P144-treated mice revealed a remarkable suppression of connective tissue growth factor expression, fibroblast SMAD2/3 phosphorylation, and alpha-smooth muscle actin positive myofibroblast development, whereas mast cell and mononuclear cell infiltration was not modified. These data suggest that topical application of P144, a peptide inhibitor of TGF-beta1, is a feasible strategy to treat pathological skin scarring and skin fibrotic diseases for which there is no specific therapy.
Assuntos
Cicatriz/tratamento farmacológico , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/administração & dosagem , Pele/patologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Administração Tópica , Animais , Bleomicina/antagonistas & inibidores , Cicatriz/metabolismo , Emulsões , Fibrose/induzido quimicamente , Géis , Camundongos , Pele/efeitos dos fármacos , Pele/metabolismo , Fator de Crescimento Transformador beta1RESUMO
PURPOSE: The purpose of this research was to identify promiscuous T-helper cell determinants (THd) from carcinoembryonic antigen (CEA) to be used to prime T-cell help for cancer therapy. CEA was selected because this antigen is expressed in an important variety of carcinomas. EXPERIMENTAL DESIGN: Potential promiscuous THd from CEA were predicted using available computer algorithms. Predicted peptides were synthesized and tested in binding experiments to different HLA-DR molecules. Binder peptides were then used to prime T-cell responses both in vitro and in vivo. RESULTS: Twenty 15-mer peptides from CEA were predicted to bind to different HLA-DR molecules. The promiscuous character of these peptides was demonstrated in binding experiments. Fifteen of 20 peptides tested were able to bind to HLA-DR4, but only CEA (625-639) was shown to be presented after processing of recombinant CEA. CEA (625-639) was also found to be presented by HLA-DR53. Moreover, immunization of HLA-DR4 transgenic mice with CEA (625-639) in conjunction with class I epitope OVA (257-264), induced a CTL response specific of OVA (257-264). CONCLUSIONS: CEA (625-639) might be a relevant promiscuous THd peptide for cancer therapy.
Assuntos
Antígeno Carcinoembrionário/química , Antígeno Carcinoembrionário/imunologia , Peptídeos/química , Linfócitos T Auxiliares-Indutores/química , Algoritmos , Animais , Antígenos/química , Carcinoma/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Epitopos/química , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Antígenos HLA-DR/química , Cadeias HLA-DRB4 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , Proteínas Recombinantes/química , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismoRESUMO
PURPOSE: Many epitopes from tumor antigens recognized by CTLs canbe poorly immunogenic. This low immunogenicity can be improved by carrying out amino acid replacements in their sequence. We have applied this strategy to enhance the immunogenicity of the HLA-A2-restricted CTL epitope CEA691 (IMIGVLVGV) from carcinoembryonic antigen (CEA), which is expressed by a wide variety of tumors. EXPERIMENTAL DESIGN: Substituted peptides from CEA691 were synthesized and tested in HLA-A2-binding assays, and in recognition by CEA691-specific CTL. Selected peptides were used to induce CTL responses in vivo in HLA-A2Kb transgenic mice and in vitro with human cells. RESULTS: Our experiments afforded several peptides with enhanced binding and/or recognition by CTL specific of CEA691. However, when HLA-A2Kb mice were immunized with these peptides only a few induced a CTL response that cross-reacted with CEA691. Additional replacement of their NH(2)-terminal amino acid by Y (tyrosine) afforded peptides YMIGMLVGV and YMIGVLLGV with enhanced in vivo and in vitro immunogenicity than CEA691. Indeed, they activated a greater number of precursor cells that recognized CEA691-pulsed cells and tumor cells expressing CEA. CONCLUSIONS: Our results widen the possibility of treating CEA-expressing tumors with enhanced efficacy.
Assuntos
Antígeno Carcinoembrionário/imunologia , Epitopos/imunologia , Antígeno HLA-A2/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Vacinas Anticâncer/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/síntese química , Ligação Proteica , Tirosina/imunologia , VacinaçãoRESUMO
PURPOSE: The product of the carcinoembryonic antigen (CEA) gene is an attractive candidate for T-cell-based immunotherapy because it is frequently expressed in epithelial solid carcinomas. Although many CEA peptide epitopes capable of stimulating CTLs have been identified, no MHC class II-restricted T helper epitope has yet been reported. EXPERIMENTAL DESIGN: The amino acid sequence of CEA was examined for the presence of potential T helper epitopes, and candidate peptides were used to stimulate in vitro T-cell responses. RESULTS: We describe here that using an algorithm to identify promiscuous helper T-cell epitopes, a peptide of CEA occupying residue positions 653 to 667 (CEA(653-667)), was effective in inducing in vitro T helper responses in the context of the HLA-DR4, HLA-DR7, and HLA-DR 9 alleles. Most significantly, some of the peptide-reactive helper T lymphocytes were also capable of recognizing naturally processed antigen in the form of recombinant CEA protein or cell lysates from tumors that express CEA. Interestingly, the newly identified helper T-cell epitope was found to overlap with a previously described HLA-A24-restricted CTL epitope, CEA(652-660), which could facilitate the development of a therapeutic vaccine capable of eliciting both CTL and T helper responses in patients suffering from epithelial carcinomas. CONCLUSION: These results indicate that T helper lymphocytes are capable of recognizing CEA as a tumor antigen and that epitope CEA(653-667) could be used for immunotherapy against tumors expressing CEA.
Assuntos
Antígeno Carcinoembrionário/imunologia , Neoplasias do Colo/imunologia , Epitopos de Linfócito T/imunologia , Linfoma de Células T/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Algoritmos , Alelos , Apresentação de Antígeno/imunologia , Citotoxicidade Imunológica , Mapeamento de Epitopos , Epitopos de Linfócito T/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Antígenos HLA-DR/imunologia , Subtipos Sorológicos de HLA-DR , Antígeno HLA-DR4/imunologia , Antígeno HLA-DR7/imunologia , Humanos , Fragmentos de Peptídeos/imunologia , Células Tumorais CultivadasRESUMO
Hepatitis C virus (HCV) is a highly prevalent virus and one of the major agents of chronic hepatitis. Since HCV NS3 protease is essential for the processing of HCV polyprotein, this protease is a target of choice to control HCV replication. Peptide inhibitors of NS3 were developed by selective amino acid replacement of six leader sequences, corresponding to regions of HCV polyprotein that are cleaved by NS3. The large numbers of potential 14-mer and 16-mer peptide inhibitors thus obtained were tested against NS3 using the fluorescent probe RETS1 and peptide cofactor SVVIVGRIILSGRA from NS4A protein. This afforded several peptide inhibitors with an IC50 of around 2 microM. These peptides may be good leading compounds for the development of peptidomimetics to control HCV replication in the treatment of chronic hepatitis C.
Assuntos
Oligopeptídeos/química , Oligopeptídeos/farmacologia , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/química , Antivirais/farmacologia , Hepatite C/tratamento farmacológico , Humanos , Hidrólise , Concentração Inibidora 50 , Cinética , Mimetismo Molecular , Inibidores de Proteases/química , Relação Estrutura-AtividadeRESUMO
Peptide vaccines derived from CD8+ T-cell epitopes have shown variable efficacy in cancer patients. Thus, some peptide vaccines are capable of activating CD8+ T-cell responses, even in the absence of CD4+ T-cell epitopes or dendritic cell (DC)-activating adjuvants. However, the mechanisms underlying the clinical activity of these potent peptides are poorly understood. Using CT26 and ovalbumin-expressing B16 murine allograft tumor models, we found that the antitumor effect of helper cell-independent CD8 T-cell peptide vaccines is inhibited by the blockade of CD40 ligand (CD40L) in vivo. Furthermore, in vitro stimulation with antigenic peptides of cells derived from immunized mice induced the expression of CD40L on the surface of CD8+ T cells and fostered DC maturation, an effect that was partially inhibited by CD40L-blocking antibodies. Interestingly, CD40L blockade also inhibited CD8+ T-cell responses, even in the presence of fully mature DCs, suggesting a role for CD40L not only in promoting DC maturation but also in mediating CD8+ T-cell co-stimulation. Importantly, these potent peptides share features with bona fide CD4 epitopes, since they foster responses against less immunogenic CD8+ T-cell epitopes in a CD40L-dependent manner. The analysis of peptides used for the vaccination of cancer patients in clinical trials showed that these peptides also induce the expression of CD40L on the surface of CD8+ T cells. Taken together, these results suggest that CD40L expression induced by potent CD8+ T-cell epitopes can activate antitumor CD8+ T-cell responses, potentially amplifying the immunological responses to less immunogenic CD8+ T-cell epitopes and bypassing the requirement for CD4+ helper T cells in vaccination protocols.
RESUMO
The purpose of this study was to assess the effects of transforming growth factor beta (TGF-ß) inhibitor peptides (P17 & P144) on early laser-induced choroidal neovascularization (LI-CNV) lesions in rats, two weeks after laser CNV induction. Seventy-one Long Evans rats underwent diode laser application in an established LI-CNV model. Baseline fluorescein angiography (FA) was performed 14 days following laser procedure, and treatments were administered 16 days post-laser application via different administration routes. Intravenous groups included control (IV-Control), P17 (IV-17), and P144 (IV-144) groups, whereas intravitreal groups included P17 (IVT-17), P144 (IVT-144), and a mixture of both peptides (IVT-17+144) (with fellow eyes receiving vehicle alone). CNV evolution was assessed using FA performed weekly for four weeks after treatment. Following sacrifice, VEGF, TGF-ß, COX-2, IGF-1, PAI-1, IL-6, MMP-2, MMP-9, and TNF-α gene expression was assessed using RT-PCR. VEGF and p-SMAD2 protein levels were also assessed by western-blot, while MMP-2 activity was assessed with gelatin zymography. Regarding the FA analysis, the mean CNV area was lower from the 3(rd) week in IVT-17 and IVT-144 groups, and also from the 2(nd) week in IVT-17+144. Biochemical analysis revealed that gene expression was lower for VEGF and COX-2 genes in IV-17 and IV-144 groups, VEGF gene in IVT-17+144 group and MMP-2 gene in IVT-17 and IVT-144 groups. VEGF protein expression was also decreased in IV-17, IV-144, IVT-17 and IVT-144, whereas pSMAD-2 levels were lower in IV-17, IV-144 and IVT-17+144 groups. Zymogram analysis revealed decreased MMP-2 activity in IV-17, IV-144, IVT-17 and IVT-144 groups. These data suggest that the use of TGF-ß inhibitor peptides (P17 & P144) decrease the development of early CNV lesions by targeting different mediators than those typically affected using current anti-angiogenic therapies. Its potential role in the treatment of early CNV appears promising as a single therapy or adjuvant to anti-VEGF drugs.
Assuntos
Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Neovascularização de Coroide/tratamento farmacológico , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Progressão da Doença , Angiofluoresceinografia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Peptídeos/administração & dosagem , Fosforilação/efeitos dos fármacos , Ratos , Receptores de Fatores de Crescimento Transformadores beta/administração & dosagem , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
AIMS: The NADPH oxidases constitute a major source of superoxide anion (·O2(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by transforming growth factor-ß1 (TGF-ß1). We investigated whether a chronic treatment with P144, a peptide synthesized from type III TGF-ß1 receptor, inhibited NADPH oxidases in the renal cortex of spontaneously hypertensive rats (SHR). RESULTS: Here, we show that chronic administration of P144 significantly reduced the NADPH oxidase expression and activity as well as the oxidative stress observed in control vehicle-treated SHR (V-SHR). In addition, P144 was also able to reduce the significant increase in the renal fibrosis and in mRNA expression of different components of collagen metabolism, as well as in the levels of connective tissue growth factor observed in V-SHR. Finally, TGF-ß1-stimulated NRK52E exhibited a significant increase in NADPH oxidase expression and activity as well as a TGF-ß1-dependent intracellular pathway that were inhibited in the presence of P144. INNOVATION: Our experimental evidence suggests that reversing oxidative stress may be therapeutically useful in preventing fibrosis-associated renal damage. We show here that (i) the TGF-ß1-NADPH oxidases axis is crucial in the development of fibrosis in an experimental hypertensive renal disease animal model, and (ii) the use of P144 reverses TGF-ß1-dependent NADPH oxidase activity; thus, P144 may be considered a novel therapeutic tool in kidney disease associated with hypertension. CONCLUSION: We demonstrate that P144 inhibits NADPH oxidases and prevents oxidative stress in kidneys from hypertensive rats. Our data also suggest that these effects are associated with the renal antifibrotic effect of P144.
Assuntos
Rim/efeitos dos fármacos , NADPH Oxidases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/uso terapêutico , Proteoglicanas/química , Receptores de Fatores de Crescimento Transformadores beta/química , Receptores de Fatores de Crescimento Transformadores beta/uso terapêutico , Animais , Fibrose/metabolismo , Fibrose/prevenção & controle , Rim/metabolismo , Rim/patologia , Masculino , Ratos , Ratos Endogâmicos SHR , Transdução de Sinais/efeitos dos fármacosRESUMO
The development of tools for efficient targeting of antigens to antigen presenting cells is of great importance for vaccine development. We have previously shown that fusion proteins containing antigens fused to the extra domain A from fibronectin (EDA), an endogenous TLR4 ligand, which targets antigens to TLR4-expressing dendritic cells (DC), are highly immunogenic. To facilitate the procedure of joining EDA to any antigen of choice, we have prepared the fusion protein EDAvidin by linking EDA to the N terminus of streptavidin, allowing its conjugation with biotinylated antigens. We found that EDAvidin, as streptavidin, forms tetramers and binds biotin or biotinylated proteins with a Kd ~ 2.6 × 10(-14) mol/L. EDAvidin favours the uptake of biotinylated green fluorescent protein by DC. Moreover, EDAvidin retains the proinflammatory properties of EDA, inducing NF- κß by TLR4-expressing cells, as well as the production of TNF- α by the human monocyte cell line THP1 and IL-12 by DC. More importantly, immunization of mice with EDAvidin conjugated with the biotinylated nonstructural NS3 protein from hepatitis C virus induces a strong anti-NS3 T cell immune response. These results open a new way to use the EDA-based delivery tool to target any antigen of choice to DC for vaccination against infectious diseases and cancer.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Fibronectinas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fibronectinas/química , Fibronectinas/genética , Hepacivirus/imunologia , Humanos , Imunoterapia Ativa , Ligantes , Camundongos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Estreptavidina/química , Linfócitos T/imunologia , Linfócitos T/patologia , Receptor 4 Toll-Like/metabolismoRESUMO
NADPH oxidases constitute a major source of superoxide anion (·O(2)(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-ß 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-ß 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR) to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-ß 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-ß 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-ß 1.