RESUMO
The off-label use of imiquimod (IQ) for hemangioma treatment has shown clinical benefits. We have previously reported a selective direct IQ-cytotoxic effect on transformed (H5V) vs. normal (1G11) endothelial cells (EC). In the present study, we investigated the mechanism underlying this selective cytotoxicity in terms of TLR7/8 receptor expression, NF-κB signalling and time-dependent modifications of oxidative stress parameters (ROS: reactive oxygen species, catalase and superoxide dismutase activities, GSH/GSSG and lipid peroxidation). TLR7/8 level was extremely low in both cell lines, and IQ did not upregulate TLR7/8 expression or activate NF-κB signalling. IQ significantly induced ROS in H5V after 2 h and strongly affected antioxidant defenses. After 12 h, enzyme activities were restored to baseline levels but a robust drop in GSH/GSSG persisted together with increased lipid peroxidation levels and a marked mitochondrial dysfunction. Although in normal IQ-treated EC some oxidative stress parameters were affected after 4 h, mitochondrial health and GSH/GSSG ratio remained notably unaffected after 12 h. Therefore, the early alterations (0-2 h) in transformed EC breached redox homeostasis as strongly as to enhance their susceptibility to IQ. This interesting facet of IQ as redox disruptor could broaden its therapeutic potential for other skin malignancies, alone or in adjuvant schemes.
Assuntos
Glutationa , NF-kappa B , Antioxidantes/metabolismo , Células Endoteliais/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Dissulfeto de Glutationa/farmacologia , Homeostase , Imiquimode/farmacologia , NF-kappa B/metabolismo , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Receptor 7 Toll-LikeRESUMO
Infantile hemangiomas are the most common benign tumors of infancy, characterized by unregulated angiogenesis and endothelial cells with high mitotic rate. Although spontaneous regression occurs, sometimes treatment is required and alternatives to corticosteroids should be considered to reduce side effects. Imiquimod is an imidazoquinoline, approved for some skin pathologies other than hemangioma. It is proposed that the effectiveness of imiquimod comes from the activation of immune cells at tumor microenvironment. However, the possibility to selectively kill different cell types and to directly impede angiogenesis has been scarcely explored in vitro for endothelial cells. In this work we showed a dramatic cytotoxicity on hemangioma cell, with a significant lower IC50 value in hemangioma compared to normal endothelial cells and melanoma (employed as a non-endothelial tumor cell line). Nuclear morphometric and flow-cytometry assays revealed imiquimod-induced apoptosis on hemangioma and melanoma cells but a small percentage of senescence on normal endothelial cells. At sub-lethal conditions, cell migration, a key step in angiogenesis turned out to be inhibited in a tumor-selective manner along with actin cytoskeleton disorganization on hemangioma cells. Altogether, these findings pointed out the selective cytotoxic effects of imiquimod on transformed endothelial cells, evidencing the potential for imiquimod to be a therapeutic alternative to reduce extensive superficial hemangioma lesions.
Assuntos
Aminoquinolinas/farmacologia , Antineoplásicos/farmacologia , Hemangioma/patologia , Neoplasias Cutâneas/patologia , Aminoquinolinas/uso terapêutico , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células Endoteliais/efeitos dos fármacos , Hemangioma/tratamento farmacológico , Humanos , Imiquimode , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Neoplasias Cutâneas/tratamento farmacológico , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/ultraestruturaRESUMO
Dendritic cells (DCs) play a pivotal role in the orchestration of immune responses, and are thus key targets in cancer vaccine design. Since the 2010 FDA approval of the first cancer DC-based vaccine (Sipuleucel-T), there has been a surge of interest in exploiting these cells as a therapeutic option for the treatment of tumors of diverse origin. In spite of the encouraging results obtained in the clinic, many elements of DC-based vaccination strategies need to be optimized. In this context, the use of experimental cancer models can help direct efforts toward an effective vaccine design. This paper reviews recent findings in murine models regarding the antitumoral mechanisms of DC-based vaccination, covering issues related to antigen sources, the use of adjuvants and maturing agents, and the role of DC subsets and their interaction in the initiation of antitumoral immune responses. The summary of such diverse aspects will highlight advantages and drawbacks in the use of murine models, and contribute to the design of successful DC-based translational approaches for cancer treatment.
RESUMO
Immune evasion strategies are important for the onset and the maintenance of viral infections. Many viruses have evolved mechanisms to counteract or suppress the host immune response. We have previously characterized two syncytial (syn) variants of Herpes simplex 1 (HSV-1) strain F, syn14-1 and syn17-2, obtained by selective pressure with a natural carrageenan. These variants showed a differential pathology in vaginal and respiratory mucosa infection in comparison with parental strain. In this paper, we evaluated the modulation of immune response in respiratory mucosa by these HSV-1 variants. We observed altered levels of Tumor Necrosis Factor-α and Interleukin-6 in lungs of animals infected with the syn14-1 and syn17-2 variants compared with the parental strain. Also, we detected differences in the recruitment of immune cells to the lung in syn variants infected mice. Both variants exhibit one point mutation in the sequence of the gene of glycoprotein D detected in the ectodomain of syn14-1 and the cytoplasmic tail of syn17-2. Results obtained in the present study contribute to the characterization of HSV-1 syn variants and the participation of the cellular inflammatory response in viral pathogenesis.
Assuntos
Citocinas/metabolismo , Herpesvirus Humano 1/imunologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/patologia , Animais , Feminino , Herpesvirus Humano 1/genética , Camundongos , Camundongos Endogâmicos BALB C , Mucosa/imunologia , Mucosa/patologia , Mutação Puntual , Infecções Respiratórias/virologia , Proteínas do Envelope Viral/genéticaRESUMO
De novo ectopic lymphoid tissue formation is known to occur in certain disease and inflammatory settings. After an effective vaccination with dendritic cells (DC) charged with melanoma apoptotic/necrotic cells (Apo/Nec), a subcutaneous tertiary lymphoid structure was organized, where retained vaccine cells interacted with recruited inflammatory and T cells. In this work we report for the first time the recruitment of two morphologically different CD207(+) cells to vaccination site. The time-course behavior of CD207(+) cells was reciprocal between vaccination site and draining lymph nodes (DLNs). After 6-10 days, CD207(+) cells localized at the paracortical region of DLNs, in close contact with T cell population. DLNs were enriched in a peculiar MHCII(+) CD11c((-)) CD207(+) population, whose role remains to be determined. Whether CD207(+) cells migration to the vaccination site can be associated with a differential anti-tumoral response remains as an open and exciting question.
Assuntos
Antígenos de Superfície/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Linfonodos/imunologia , Tecido Linfoide/fisiologia , Lectinas de Ligação a Manose/imunologia , Animais , Movimento Celular , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC-) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC- fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC- fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccine's ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo.
Assuntos
Apresentação de Antígeno/imunologia , Células da Medula Óssea/imunologia , Antígeno CD11c/imunologia , Vacinas Anticâncer/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Animais , Apresentação de Antígeno/genética , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Células da Medula Óssea/metabolismo , Antígeno CD11c/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
Decorin and biglycan proteoglycans play important roles in the organization of the extracellular matrix, and in the regulation of cell adhesion and migration. Given morphological and functional endothelial heterogeneity, information is needed regarding whether endothelial cells (ECs) from different vascular beds possess different profiles of proteoglycan constituents of the basement membranes. Here, we report that endothelia from different murine organs and EC lines derived thereof produce and secrete different patterns of proteoglycans. A faint colocalization between decorin and PECAM/CD31 was found on tissue sections from mouse heart, lung and kidney by immunofluorescence. Three EC lines derived from these organs produced decorin (100-kDa) and its core protein (45-kDa). Extracellular decorin recognition in culture supernatant was only possible after chondroitin lyase digestion suggesting that the core protein of secreted proteoglycan is more encrypted by glycosaminoglycans than the intracellular one. Heart and lung ECs were able to produce and release decorin. Kidney ECs synthesized the proteoglycan and its core protein but no secretion was detected in culture supernatants. Although biglycan production was recorded in all EC lines, secretion was almost undetectable, consistent with immunofluorescence results. In addition, no biglycan secretion was detected after EC growth supplement treatment, indicating that biglycan is synthesized, secreted and quickly degraded extracellularly by metalloproteinase-2. Low molecular-mass dermatan sulfate was the predominant glycosaminoglycan identified bound to the core protein. ECs from different vascular beds, with differences in morphology, physiology and cell biology show differences in the proteoglycan profile, extending their heterogeneity to potential differences in cell migration capacities.
Assuntos
Biglicano/metabolismo , Decorina/metabolismo , Células Endoteliais/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Dermatan Sulfato/metabolismo , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Rim/citologia , Rim/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/citologia , Miocárdio/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
Antigen presentation by dendritic cells (DC) is of key importance for the initiation of the primary immune response. Mice vaccinated with DC charged with apoptotic/necrotic B16 cells (DC-Apo/Nec) are protected against B16 challenge. The aim of this study was to assess vaccine cell migration in our system and to find out if there is an immunological response taking place at the vaccination site. The formation of a pseudocapsule, peripheral node addresin expression in small venules, and the recruitment of a wide variety of cellular populations, including macrophages, polymorphonuclear lymphocytes, and CD8+ and CD4+ T lymphocytes found in association with DC, evidenced the formation of tertiary lymphoid tissue in the vaccination site in our experimental system.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Tecido Linfoide/fisiologia , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Vacinação/métodos , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Histocitoquímica , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tela Subcutânea/imunologia , Análise de SobrevidaRESUMO
BACKGROUND: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression. METHODS: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 x 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC. RESULTS: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 +/- 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed. CONCLUSION: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis. TRIAL REGISTRATION: Clinicaltrials.gov (NHI) NCT00515983.
Assuntos
Vacinas Anticâncer/toxicidade , Células Dendríticas/imunologia , Células Dendríticas/transplante , Interleucina-10/genética , Melanoma/imunologia , Melanoma/patologia , Adolescente , Adulto , Apoptose , Progressão da Doença , Feminino , Genótipo , Humanos , Hipersensibilidade Tardia , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Necrose , Estadiamento de Neoplasias , Seleção de Pacientes , Fagocitose , Polimorfismo Genético , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Transplante AutólogoRESUMO
Chemokines such as monocyte chemoattractant protein (MCP)-1 are key agonists that attract macrophages to tumors. In melanoma, it has been previously shown that variable levels of MCP-1/CCL2 appear to correlate with infiltrating macrophages and tumor fate, with low to intermediate levels of the chemokine contributing to melanoma development. To work under such conditions, a poorly tumorigenic human melanoma cell line was transfected with an expression vector encoding MCP-1. We found that M2 macrophages are associated to MCP-1+ tumors, triggering a profuse vascular network. To target the protumoral macrophages recruitment and reverting tumor growth promotion, clodronate-laden liposomes (Clod-Lip) or bindarit were administered to melanoma-bearing mice. Macrophage depletion after Clod-Lip treatment induced development of smaller tumors than in untreated mice. Immunohistochemical analysis with an anti-CD31 antibody revealed scarce vascular structures mainly characterized by narrow vascular lights. Pharmacological inhibition of MCP-1 with bindarit also reduced tumor growth and macrophage recruitment, rendering necrotic tumor masses. We suggest that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.