RESUMEN
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.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Fibronectinas/inmunología , Proteínas Recombinantes de Fusión/inmunología , Receptor Toll-Like 4/inmunología , Animales , Células Presentadoras de Antígenos/metabolismo , Línea Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Fibronectinas/química , Fibronectinas/genética , Hepacivirus/inmunología , Humanos , Inmunoterapia Activa , Ligandos , Ratones , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Estreptavidina/química , Linfocitos T/inmunología , Linfocitos T/patología , Receptor Toll-Like 4/metabolismoRESUMEN
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.
Asunto(s)
Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Neovascularización Coroidal/tratamiento farmacológico , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Progresión de la Enfermedad , Angiografía con Fluoresceína , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Fragmentos de Péptidos/administración & dosificación , Péptidos/administración & dosificación , Fosforilación/efectos de los fármacos , Ratas , Receptores de Factores de Crecimiento Transformadores beta/administración & dosificación , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Riñón/efectos de los fármacos , NADPH Oxidasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/uso terapéutico , Proteoglicanos/química , Receptores de Factores de Crecimiento Transformadores beta/química , Receptores de Factores de Crecimiento Transformadores beta/uso terapéutico , Animales , Fibrosis/metabolismo , Fibrosis/prevención & control , Riñón/metabolismo , Riñón/patología , Masculino , Ratas , Ratas Endogámicas SHR , Transducción de Señal/efectos de los fármacosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Hipertensión/enzimología , NADPH Oxidasas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Animales , Presión Sanguínea/efectos de los fármacos , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fibroblastos/efectos de los fármacos , Corazón/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Miocardio/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Ratas , Ratas Endogámicas WKY , Receptores de Factores de Crecimiento Transformadores beta/uso terapéutico , Factor de Crecimiento Transformador beta1/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
PURPOSE: To assess the effect of transforming growth factor (TGF)-ß inhibitor peptides (P17 and P144) on the development of laser-induced choroidal neovascularization (LI-CNV) in a rat model. METHODS: Sixty-one Long-Evans rats underwent diode LI-CNV model. Forty-eight hours later, treatment was administered. The intravenous control group (IV-control) and intravenous P17 group (IV-17) received five doses (0.2 mg every 72 hours) of vehicle and P17, respectively. Four groups received intravitreal injections of P17 low-dose (LD-17; 1 mg/mL) and high-dose (HD-17; 20 mg/mL) and P144 low-dose (LD-144; 1 mg/mL) and high-dose (HD-144; 3 mg/mL), and fellow eyes received vehicle. CNV evolution was assessed weekly by fluorescein angiography (FA). After death, VEGF, TGF-ß and PDGF protein levels were measured by ELISA in RPE and retina homogenates. Data were analyzed with commercially available statistical analysis software. RESULTS: The mean CNV area, measured in pixels, was significantly lower at the second and fourth weeks in IV-17 (P < 0.05) and from the second week in HD-17 (P < 0.05), whereas LD-144 and HD-144 showed significant differences at every time point (P < 0.05). LD-17 showed significantly lower protein levels of TGF-ß in retina and PDGF in RPE (P < 0.05), whereas HD-17 showed lower levels of VEGF (RPE and retina; P < 0.05), TGF-ß (RPE and retina; P < 0.05), and PDGF (RPE; P < 0.05). HD-144 showed lower VEGF levels in the retina (P < 0.05). CONCLUSIONS: TGF-ß inhibition with these peptides represents a promising new therapeutic line for CNV targeting a different pathway than current therapies. More studies are needed to assess this effect on early CNV, alone or in combination with anti-VEGF.
Asunto(s)
Neovascularización Coroidal/tratamiento farmacológico , Modelos Animales de Enfermedad , Fragmentos de Péptidos/farmacología , Péptidos/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Animales , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Ensayo de Inmunoadsorción Enzimática , Angiografía con Fluoresceína , Inyecciones Intravenosas , Inyecciones Intravítreas , Coagulación con Láser , Láseres de Semiconductores , Masculino , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Ratas , Ratas Long-Evans , Receptores de Factores de Crecimiento Transformadores beta , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
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.
Asunto(s)
Transdiferenciación Celular/efectos de los fármacos , Diálisis Peritoneal/efectos adversos , Fibrosis Peritoneal/etiología , Peritoneo/patología , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Biomarcadores/metabolismo , Células Cultivadas , Soluciones para Diálisis/efectos adversos , Femenino , Inyecciones Intraperitoneales , Queratinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/metabolismo , Miofibroblastos/patología , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/uso terapéutico , Péptidos/farmacología , Péptidos/uso terapéutico , Fibrosis Peritoneal/metabolismo , Fibrosis Peritoneal/patología , Fibrosis Peritoneal/prevención & control , Fenotipo , Receptores de Factores de Crecimiento Transformadores beta/uso terapéutico , Proteína de Unión al Calcio S100A4 , Proteínas S100/metabolismo , Factor de Crecimiento Transformador beta1/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Adenoviridae/fisiología , Células Dendríticas/virología , Hepacivirus/inmunología , Hepatitis C/inmunología , Linfocitos T Colaboradores-Inductores/fisiología , Virión/fisiología , Adenoviridae/genética , Animales , Ligando de CD40/genética , Ligando de CD40/fisiología , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/patología , Modelos Animales de Enfermedad , Hepatitis C/patología , Hepatitis C/prevención & control , Humanos , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Colaboradores-Inductores/patología , Transducción Genética , Vacunas contra Hepatitis Viral/inmunología , Vacunas contra Hepatitis Viral/uso terapéutico , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismoRESUMEN
Low antigen expression and an absence of coimmunostimulatory signals may be partly responsible for the low immunogenicity of many tumors. It may be possible to overcome this situation by defining a combination of adjuvants and antigens that can activate a high-avidity antitumor response. Using the poorly immunogenic B16-OVA melanoma cells as tumor model, we tested different combinations of adjuvants and antigens to treat established tumors. In the absence of exogenous antigens, repeated administration of the TLR7 ligand Imiquimod together with anti-CD40 agonistic antibodies activated only innate immunity, which was insufficient to reject intradermal tumors. Administering this adjuvant combination together with OVA as a tumor antigen induced T-cell responses that delayed tumor growth. However, administering a combination of anti-CD40 plus TLR3 and TLR7 ligands, together with antigen targeting to dendritic cells through TLR4, was sufficient to induce tumor rejection in 50% of mice. This response was associated with a greater activation of innate immunity and induction of high-avidity polyfunctional CD8(+) T-cell responses, which each contributed to tumor rejection. This therapy activated T-cell responses not only against OVA, which conferred protection against a rechallenge with B16-OVA cells, but also activated T-cell responses against other melanoma-associated antigens. Our findings support the concept that multiple adjuvant combination and antigen targeting may be a useful immunotherapeutic strategy against poorly immunogenic tumors.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Antígenos de Neoplasias/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Aminoquinolinas/farmacología , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Afinidad de Anticuerpos , Antígenos de Neoplasias/farmacología , Antígenos CD4/inmunología , Femenino , Imiquimod , Inmunidad Innata/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Poli I-C/farmacología , Timoma/genética , Timoma/inmunología , TransfecciónRESUMEN
Melanoma progression is associated with the expression of different growth factors, cytokines, and chemokines. Because TGFß1 is a pleiotropic cytokine involved not only in physiologic processes but also in cancer development, we analyzed in A375 human melanoma cells, the effect of TGFß1 on monocyte chemoattractant protein-1 (MCP-1) and interleukin-10 (IL-10) expression, two known factors responsible for melanoma progression. TGFß1 increased the expression of MCP-1 and IL-10 in A375 cells, an effect mediated by the cross-talk between Smad, PI3K (phosphoinositide 3-kinase)/AKT, and BRAF-MAPK (mitogen activated protein kinase) signaling pathways. Supernatants from TGFß1-treated A375 cells enhanced MCP-1-dependent migration of monocytes, which, in turn, expressed high levels of TGF,ß1, bFGF, and VEGF mRNA. Moreover, these supernatants also inhibited functional properties of dendritic cells through IL-10-dependent mechanisms. When using in vitro, the TGFß1-blocking peptide P144, TGFß1-dependent Smad3 phosphorylation, and expression of MCP-1 and IL-10 were inhibited. In vivo, treatment of A375 tumor-bearing athymic mice with P144 significantly reduced tumor growth, associated with a lower macrophage infiltrate and decreased intratumor MCP-1 and VEGF levels, as well as angiogenesis. Finally, in C57BL/6 mice with B16-OVA melanoma tumors, when administered with immunotherapy, P144 decreased tumor growth and intratumor IL-10 levels, linked to enhanced activation of dendritic cells and natural killer cells, as well as anti-OVA T-cell responses. These results show new effects of TGFß1 on melanoma cells, which promote tumor progression and immunosuppression, strongly reinforcing the relevance of this cytokine as a molecular target in melanoma.
Asunto(s)
Quimiocina CCL2/biosíntesis , Interleucina-10/biosíntesis , Melanoma/inmunología , Factor de Crecimiento Transformador beta1/farmacología , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Interleucina-10/inmunología , Sistema de Señalización de MAP Quinasas , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Datos de Secuencia Molecular , Monocitos/efectos de los fármacos , Monocitos/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína smad3/metabolismoRESUMEN
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.
Asunto(s)
Células Dendríticas/inmunología , Hepacivirus/inmunología , Interleucina-10/antagonistas & inhibidores , Interleucina-12/biosíntesis , Secuencia de Aminoácidos , Animales , Ligando de CD40/farmacología , Línea Celular , Células Dendríticas/metabolismo , Antígenos de la Hepatitis C/farmacología , Humanos , Interferón-alfa/biosíntesis , Interleucina-10/inmunología , Ratones , Biblioteca de Péptidos , Factor de Transcripción STAT3/metabolismo , Receptor Toll-Like 9/fisiología , Proteínas del Núcleo Viral/farmacologíaRESUMEN
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.
Asunto(s)
Fibroblastos/efectos de los fármacos , Péptidos/farmacología , Fibrosis Pulmonar/prevención & control , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Actinas/genética , Actinas/metabolismo , Animales , Bleomicina , Western Blotting , Línea Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/genética , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Músculo Liso/química , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
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.
Asunto(s)
Factores de Transcripción Forkhead/antagonistas & inhibidores , Péptidos/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Vacunas/inmunología , Animales , Femenino , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Microscopía Confocal , Biblioteca de Péptidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/metabolismo , TransfecciónRESUMEN
Staphylococcus epidermidis releases a complex of at least four peptides, termed phenol-soluble modulins (PSM), which stimulate macrophages to produce proinflammatory cytokines via activation of TLR2 signalling pathway. We demonstrated that covalent linkage of PSM peptides to an antigen facilitate its capture by dendritic cells and, in combination with different TLR ligands, can favour the in vivo induction of strong and persistent antigen-specific immune responses. Treatment of mice grafted with HPV16-E7-expressing tumor cells (TC-1) with poly(I:C) and a peptide containing αMod linked to the H-2D(b)-restricted cytotoxic T-cell epitope E7(49-57) from HPV16-E7 protein allowed complete tumor regression in 100% of the animals. Surprisingly, this immunomodulatory property of modulin-derived peptides was TLR2 independent and partially dependent upon the EGF-receptor signalling pathway. Our results suggest that alpha or gamma modulin peptides may serve as a suitable antigen carrier for the development of anti-tumoral or anti-viral vaccines.
Asunto(s)
Antígenos de Neoplasias/inmunología , Toxinas Bacterianas/inmunología , Neoplasias Experimentales/tratamiento farmacológico , Proteínas E7 de Papillomavirus/inmunología , Animales , Presentación de Antígeno , Antígenos de Neoplasias/administración & dosificación , Toxinas Bacterianas/administración & dosificación , Células Dendríticas/inmunología , Epítopos de Linfocito T/inmunología , Receptores ErbB/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias Experimentales/prevención & control , Proteínas E7 de Papillomavirus/administración & dosificación , Poli I-C/inmunología , Staphylococcus epidermidis , Receptor Toll-Like 2/inmunologíaRESUMEN
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.
Asunto(s)
Fibronectinas/uso terapéutico , Hepacivirus/inmunología , Hepatitis C/prevención & control , Proteínas Recombinantes/uso terapéutico , Proteínas Virales de Fusión/uso terapéutico , Proteínas no Estructurales Virales/uso terapéutico , Vacunas Virales/uso terapéutico , Animales , Antivirales/uso terapéutico , Línea Celular , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Quimioterapia Combinada , Escherichia coli/metabolismo , Femenino , Fibronectinas/biosíntesis , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatitis C/inmunología , Hepatitis C/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Poli I-C/uso terapéutico , Estructura Terciaria de Proteína , ARN Viral/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Virales de Fusión/biosíntesis , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/genética , Vacunas Virales/biosíntesisRESUMEN
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.
Asunto(s)
Inmunoterapia , Melanoma Experimental/terapia , Fragmentos de Péptidos/farmacología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antígenos CD40/inmunología , Células Dendríticas/inmunología , Femenino , Citometría de Flujo , Proteínas de Homeodominio/fisiología , Humanos , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Poli I-C/inmunología , Poli I-C/uso terapéutico , Receptores de Factores de Crecimiento Transformadores beta , Tasa de Supervivencia , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Células Tumorales CultivadasRESUMEN
AIM: We investigated whether P144, a synthetic peptide from transforming growth factor-beta(1) (TGF-beta(1)) type III receptor betaglycan, exhibits cardiac antifibrotic properties. METHODS AND RESULTS: The study was carried out in one group of 10-week-old normotensive Wistar-Kyoto rats treated with vehicle (V-WKY), one group of 10-week-old spontaneously hypertensive rats treated with vehicle (V-SHR), and one group of 10-week-old SHR treated with P144 (P144-SHR) for 12 weeks. Two more groups of 10-week-old untreated WKY and SHR were used to assess baseline values of the parameters tested. In addition, the effects of P144 on rat cardiac fibroblasts stimulated with TGF-beta(1) were also studied. Compared with V-WKY, V-SHR exhibited significant increases in the myocardial expression of phosphorylated Smad2, 38 and 42 kDa connective tissue growth factor (CTGF) isoforms, procollagen alpha1 (I) mRNA, and collagen type I protein, as well as in the expression of lysyl oxidase (LOX) mRNA and protein, collagen cross-linking and deposition. P144 administration was associated with significant reduction in all these parameters in P144-SHR. TGF-beta(1)-stimulated fibroblasts exhibited significant increases in phosphorylated Smad2, 38 and 42 kDa CTGF proteins, and procollagen alpha(1) (I) mRNA compared with control fibroblasts. No significant differences were found in these parameters between fibroblasts incubated with TGF-beta(1) and P144 and control fibroblasts. CONCLUSION: These results show that P144 inhibits TGF-beta(1)-dependent signalling pathway and collagen type I synthesis in cardiac fibroblasts. These effects may be involved in the ability of this peptide to prevent myocardial fibrosis in SHR.
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Cardiomiopatías/prevención & control , Fármacos Cardiovasculares/farmacología , Hipertensión/tratamiento farmacológico , Miocardio/metabolismo , Fragmentos de Péptidos/farmacología , Proteoglicanos/efectos de los fármacos , Receptores de Factores de Crecimiento Transformadores beta/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Presión Sanguínea/efectos de los fármacos , Cardiomiopatías/etiología , Cardiomiopatías/patología , Fármacos Cardiovasculares/administración & dosificación , Línea Celular , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis , Hipertensión/complicaciones , Hipertensión/patología , Inyecciones Intraperitoneales , Masculino , Miocardio/patología , Fragmentos de Péptidos/administración & dosificación , Proteína-Lisina 6-Oxidasa/metabolismo , Proteoglicanos/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de Factores de Crecimiento Transformadores beta/administración & dosificación , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal/genética , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
The role of transforming growth factor beta (TGFbeta) in tumor promotion and in angiogenesis is context-dependent. While TGFbeta prevents tumor growth and angiogenesis in early phases of tumor development, evidence is accumulating about its pro-angiogenic and tumor promotion activities in late-stages of tumor progression. Here we have studied, in an experimental context previously reported to disclose the pro-angiogenic effects of TGFbeta, the blocking activity of TGFbeta antagonist peptides. In agreement with previous results, we have observed that TGFbeta exerts a powerful pro-angiogenic activity on human normal dermal microvascular endothelial cells (MVEC), by promoting invasion and capillary morphogenesis in Matrigel. No apoptotic activity of TGFbeta was observed. By RT-PCR we have shown that TGFbeta up-regulates expression not only of plasminogen activator inhibitor type-1 (PAI-1), but also of the urokinase-type plasminogen activator receptor (uPAR), whose inhibition by specific antibodies blunted the TGFbeta angiogenic response in vitro. The SMAD2/3 and FAK signaling pathways were activated by TGFbeta in MVEC, as an early and late response, respectively. The use of two different TGFbeta1 antagonist peptides, derived from TGFbeta type III receptor sequence and 15-mer phage display technology, inhibited the signaling and pro-angiogenic response in vitro, as well as uPAR and PAI-1 up-regulation of MVEC following TGFbeta challenge. The anti-angiogenic properties of both inhibitors were evident also in the in vivo TGFbeta Matrigel Sponge Assay. These results may be relevant to develop a potentially fruitful strategy for the therapy of late-stage-associated tumor angiogenesis.
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Neovascularización Patológica/prevención & control , Péptidos/farmacología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Secuencia de Bases , Western Blotting , Capilares/crecimiento & desarrollo , Proliferación Celular/efectos de los fármacos , Cartilla de ADN , Humanos , Inmunohistoquímica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/fisiologíaRESUMEN
Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-beta1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-beta1 and TGF-beta2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-beta may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.
Asunto(s)
Regulación hacia Abajo/inmunología , Activación de Linfocitos/inmunología , Péptidos/farmacología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Factor de Crecimiento Transformador beta1/inmunología , Animales , Vacunas contra el Cáncer/inmunología , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Factores de Transcripción Forkhead/inmunología , Humanos , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Neoplasias/inmunología , Neoplasias/patología , Isoformas de Proteínas/inmunología , Linfocitos T Reguladores/citología , Factor de Crecimiento Transformador beta2/antagonistas & inhibidores , Factor de Crecimiento Transformador beta2/inmunologíaRESUMEN
Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, we developed a murine lung cancer model. Comparative transcriptomic analysis identified genes encoding signaling molecules (such as TCF4 and PRKD3) and cell anchorage-related proteins (MCAM and SUSD5), some of which were basally modulated by transforming growth factor-beta (TGF-beta) in tumor cells and in conditions mimicking tumor-stromal interactions. Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-beta blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGF-beta-dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. Our findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization.