RESUMEN
UNLABELLED: Accurate and easy-to-use measurement tools are required to evaluate the effect of treatments on patient activity. Comfortable device placement and fixation are important for patient compliance. The aim of this study was the evaluation of the accuracy of an accelerometer at different placements and slow velocities. METHODS: A total of 43 healthy volunteers were included for a literature-based treadmill protocol using five accelerometer positions; a subset of 18 volunteers performed an extended treadmill protocol with velocities between 0.1 and 2.6m/s and finally stair climbing. RESULTS: An alternative accelerometer position at the anterolateral aspect of the middle shank did measure steps more accurately than at the manufacturer suggested position, especially during slow velocities. Participants preferred the alternative placement at the shank. The accuracy of different accelerometer positions was excellent at velocities between 1.0 and 2.2m/s. During slow velocities below 1.0m/s steps were recorded less accurately. Accepting an error of five percent, the accelerometer recorded steps accurately from 0.5m/s at the alternative placement and from 0.8m/s at the manufacturer suggested placement. Stair climbing was not recorded accurately by any accelerometer position. CONCLUSION: For measuring step number during slow velocities, the alternative position should be favoured. Stair climbing was not recorded accurately by any tested placement.
Asunto(s)
Acelerometría/instrumentación , Locomoción/fisiología , Caminata/fisiología , Adolescente , Adulto , Prueba de Esfuerzo , Femenino , Humanos , Extremidad Inferior , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Complex biological processes often require in vivo analysis, and many important research advances have been made using mice as a model for the study of various biological systems. Cutaneous melanomas are tumors originating from skin melanocytes, which are present in hair follicles, and interfollicular epidermal and dermal layers. Until recently, mouse melanoma models were largely based on transplantation models, i.e. transplantation of either syngeneic or xenogeneic melanoma cells into wild type or genetically modified animals. More recently, however, the use of novel technologies specifically modifying the genome allows for the generation of mouse strains, which may develop spontaneous melanoma. Nevertheless, it should be kept in mind that animal models provide only an approximation of reality in humans. In this review, we will discuss a representative selection of currently available transplantation and transgenic melanoma models; despite the fact that this selection will be biased by personal experience, we are confident to demonstrate how the use of mouse melanoma models facilitates translational research in several biomedical disciplines.
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Modelos Animales de Enfermedad , Melanoma Experimental , Ratones , Animales , Humanos , Melanoma Experimental/genética , Trasplante Heterólogo , Trasplante IsogénicoRESUMEN
Survivin is overexpressed in several malignancies and in tumor-associated endothelium making it an attractive target for therapeutic cytotoxic T-cell responses. Thus, it would be important to test this notion in preclinical models. Consequently, we screened the murine survivin sequence for potential binding K(b)-restricted octamer peptide epitopes. Two epitopes, which bind strongly to K(b), were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes. The natural processing and presentation of these epitopes by tumor cells was evident by the killing of murine melanoma cells by vaccination-induced T cells. Subcutaneous challenge with syngeneic melanoma demonstrated the protective immunity of this vaccination. Notably, analysis of the vessel density in subcutaneous tumors revealed that survivin-specific vaccination significantly reduced the number of intratumoral vessels. In summary, we demonstrated the immunogenicity of two K(b)-restricted peptide epitopes derived from the murine survivin protein; moreover, survivin-specific vaccination not only resulted in a reduction of tumor cells but also the tumor supplying blood vessels. The presented preclinical model for survivin-directed vaccination may serve as a valuable tool to improve already running clinical trials in a syngeneic tumor model.
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Epítopos de Linfocito T/inmunología , Antígenos H-2/inmunología , Proteínas Asociadas a Microtúbulos/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunohistoquímica , Inmunoterapia/métodos , Proteínas Inhibidoras de la Apoptosis , Melanoma/inmunología , Melanoma/terapia , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/inmunología , Proteínas Represoras , SurvivinRESUMEN
Sorafenib, originally developed as CRAF inhibitor but soon recognized as a multikinase inhibitor, is currently widely tested for the treatment of different cancers either alone or in combination therapy. However, the clinical success, particularly in immunogenic tumors such as melanoma, was less than anticipated. Because T-cell activation is tightly regulated by a multitude of kinases, we scrutinized effects of sorafenib on immune responses. To this end, comprehensive in vitro studies revealed that the presence of sorafenib concentrations comparable with observed plasma levels in patients strongly impairs the activation of T cells. Notably, even established tumor-specific immune responses are influenced by sorafenib. Indeed, ELISPOT data of peripheral blood lymphocytes obtained from melanoma patients vaccinated against survivin show markedly diminished survivin-specific immune responses in the presence of sorafenib. Surprisingly, inhibition of T-cell activation was not associated with reduced extracellular signal-regulated kinase phosphorylation. In fact, on T-cell receptor stimulation phospho-extracellular signal-regulated kinase and phospho-mitogen-activated protein kinase kinase levels were found to be elevated in the presence of sorafenib, showing the complexity of signal transduction events following T-cell receptor stimulation. In conclusion, our data show that T-cell function is sensitive toward the multikinase inhibitor sorafenib in a mitogen-activated protein kinase-independent fashion. This observation has important implications for the use of sorafenib as therapy for immunogenic cancers.
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Bencenosulfonatos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Anticuerpos/farmacología , Antígenos de Neoplasias/metabolismo , Butadienos/farmacología , Antígenos CD28/metabolismo , Complejo CD3/metabolismo , Epítopos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Activación de Linfocitos/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Niacinamida/análogos & derivados , Nitrilos/farmacología , Compuestos de Fenilurea , Fosforilación/efectos de los fármacos , Fitohemaglutininas/farmacología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacos , Sorafenib , Linfocitos T/enzimología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
CD147 is highly expressed on many tumor cells; its role for tumor invasiveness and metastasis has been deduced from its capacity to induce MMPs, i.e., MMP-1, -2, -3, and -9. However, in the murine B16 melanoma model, MMP-2/-9 expression occurs independent of CD147. To scrutinize the impact of CD147 on metastasis formation and angiogenesis in this model, CD147 was stably knocked down in B16 cells. This silencing of CD147 expression resulted in a reduced capability of the tumor cells to metastasize to the draining lymph nodes. Notably, the CD147 knock down caused a decreased VEGF expression in vivo accompanied by reduced blood vessel formation. Thus, in the B16 melanoma model, CD147 promotes metastasis formation by induction of angiogenesis in an MMP independent manner.
Asunto(s)
Basigina/fisiología , Melanoma Experimental/irrigación sanguínea , Melanoma Experimental/secundario , Neovascularización Patológica/etiología , Animales , Línea Celular Tumoral , Ratones , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/análisis , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
Among the relatively large number of known tumor-associated antigens (TAA) which are recognized by human CD8 T-cells, Melan-A/MART-1 is one of the most-if not the most-frequently used target for anti-cancer vaccines in HLA-A2 + melanoma patients. In this study, we analyzed the killing of a large panel of melanoma cells by a high avidity, MART-1-specific T-cell clone or a MART-1-specific, polyclonal T-cell culture. Strikingly, we observed that the MART-1-specific T-cells only killed around half of the analyzed melanoma cell lines. In contrast a Bcl-2-specific T-cell clone killed all melanoma cell lines, although the T-cell avidity of this clone was significantly lower. The MART-1-specific T-cell clone expressed NKG-2D and was fully capable of releasing both perforin and Granzyme B. Notably, the resistance to killing by the MART-1-specific T-cells could be overcome by pulsing of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy.
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Antígenos de Neoplasias/análisis , Antígenos de Superficie/análisis , Epítopos de Linfocito T/análisis , Epítopos/análisis , Antígeno HLA-A2/inmunología , Epítopos Inmunodominantes/análisis , Melanoma/química , Proteínas de Neoplasias/análisis , Linfocitos T Citotóxicos/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Superficie/inmunología , Línea Celular Tumoral/química , Línea Celular Tumoral/inmunología , Pruebas Inmunológicas de Citotoxicidad , Citotoxicidad Inmunológica , Epítopos/inmunología , Epítopos de Linfocito T/inmunología , Granzimas/biosíntesis , Granzimas/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Interferón gamma/metabolismo , Melanoma/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/biosíntesis , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Proteínas de Neoplasias/inmunología , Perforina , Proteínas Citotóxicas Formadoras de Poros/biosíntesis , Proteínas Citotóxicas Formadoras de Poros/inmunología , Proteínas Proto-Oncogénicas c-bcl-2/inmunologíaRESUMEN
The proinflammatory cytokine macrophage migration inhibitory factor (MIF) stimulates tumor cell proliferation, migration, and metastasis; promotes tumor angiogenesis; suppresses p53-mediated apoptosis; and inhibits antitumor immunity by largely unknown mechanisms. We here describe an overexpression of MIF in ovarian cancer that correlates with malignancy and the presence of ascites. Functionally, we find that MIF may contribute to the immune escape of ovarian carcinoma by transcriptionally down-regulating NKG2D in vitro and in vivo which impairs NK cell cytotoxicity toward tumor cells. Together with the additional tumorigenic properties of MIF, this finding provides a rationale for novel small-molecule inhibitors of MIF to be used for the treatment of MIF-secreting cancers.
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Células Asesinas Naturales/inmunología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Neoplasias Ováricas/inmunología , Receptores Inmunológicos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Escape del Tumor , Adulto , Anciano , Anciano de 80 o más Años , Ascitis , Citotoxicidad Inmunológica , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células Asesinas Naturales/metabolismo , Factores Inhibidores de la Migración de Macrófagos/inmunología , Persona de Mediana Edad , Subfamilia K de Receptores Similares a Lectina de Células NK , Neoplasias Ováricas/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores de Células Asesinas Naturales , Transcripción Genética , Factor de Crecimiento Transformador beta/inmunología , Células Tumorales CultivadasRESUMEN
B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (microMT). Whereas BALB/c microMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c microMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c microMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c microMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.
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Linfocitos B/inmunología , Diferenciación Celular/inmunología , Leishmania major/inmunología , Leishmaniasis Cutánea/inmunología , Células Th2/inmunología , Traslado Adoptivo , Animales , Anticuerpos Antiprotozoarios/inmunología , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Susceptibilidad a Enfermedades/inmunología , Inmunidad Innata/inmunología , Ratones , Ratones Endogámicos BALB C , FenotipoRESUMEN
BACKGROUND: We previously demonstrated that targeting lymphotoxin alpha (LTalpha) to the tumor evokes its immunological destruction in a syngeneic B16 melanoma model. Since treatment was associated with the induction of peritumoral tertiary lymphoid tissue, we speculated that the induced immune response was initiated at the tumor site. METHODS AND RESULTS: In order to directly test this notion, we analyzed the efficacy of tumor targeted LTalpha in LTalpha knock-out (LTalpha(-/-)) mice which lack peripheral lymph nodes. To this end, we demonstrate that tumor-targeted LTalpha mediates the induction of specific T-cell responses even in the absence of secondary lymphoid organs. In addition, this effect is accompanied by the initiation of tertiary lymphoid tissue at the tumor site in which B and T lymphocytes are compartmentalized in defined areas and which harbor expanded numbers of tumor specific T cells as demonstrated by in situ TRP-2/K(b) tetramer staining. Mechanistically, targeted LTalpha therapy seems to induce changes at the tumor site which allows a coordinated interaction of immune competent cells triggering the induction of tertiary lymphoid tissue. CONCLUSION: Thus, our data demonstrate that targeted LTalpha promotes an accelerated immune response by enabling the priming of T cells at the tumor site.
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Tejido Linfoide/inmunología , Linfotoxina-alfa/inmunología , Linfotoxina-alfa/uso terapéutico , Melanoma Experimental/terapia , Animales , Gangliósidos/inmunología , Humanos , Inmunohistoquímica , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor , Tejido Linfoide/citología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Linfocitos T/inmunologíaRESUMEN
PURPOSE: The microphthalmia-associated transcription factor (MITF) is regarded as a key oncogene of the melanocytic lineage since it was detected by a genome-wide analysis to be strongly amplified in 15% to 20% of metastatic melanomas. MITF gene amplification was shown to be associated with a reduced survival in metastatic melanoma patients, and reduction of MITF activity was shown to sensitize melanoma cell lines to chemotherapeutics, suggesting the intratumoral MITF gene copy number as a predictive biomarker of response and survival after chemotherapy. PATIENTS AND METHODS: To validate this hypothesis, we investigated MITF gene amplification in tumor tissues obtained from 116 metastatic melanoma patients before an individualized sensitivity-directed chemotherapy using quantitative real-time PCR. MITF amplification rates were correlated with tumor chemosensitivity quantified by an ATP-based luminescence assay and with chemotherapy outcome in terms of response and survival. RESULTS: Of 116 tumor tissues, 104 were evaluable for MITF gene amplification. Strong amplification (> or =4 copies per cell) was detected in 24 of 104 tissues (23%), whereas 62 of 104 tissues (60%) harbored >3 copies per cell. Strong MITF gene amplification was associated with a reduced disease-specific survival (P = 0.031). However, no correlation was found between MITF copy number and in vitro chemosensitivity or in vivo chemotherapy response. CONCLUSION: Our findings suggest that strong amplifications of the melanoma oncogene MITF affects patient survival but does not influence tumor chemosensitivity and chemotherapy response. Thus, the MITF gene copy number seems a useful prognostic marker in metastatic melanoma but could not be confirmed as a predictive marker of chemosensitivity and chemotherapy response.
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Antineoplásicos/farmacología , Amplificación de Genes , Perfilación de la Expresión Génica , Melanoma/patología , Factor de Transcripción Asociado a Microftalmía/biosíntesis , Factor de Transcripción Asociado a Microftalmía/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Melanoma/metabolismo , Persona de Mediana Edad , Pronóstico , Resultado del TratamientoRESUMEN
BACKGROUND: It was conclusively demonstrated that the cell surface glycoprotein CD147 on tumor cells mediates induction of matrix metalloproteinases (MMPs) by stromal cells in humans. However, for murine models such evidence remains elusive. METHODS AND RESULTS: To address the impact of CD147 on MMP expression in the murine B16 melanoma model, we consequently stably knocked down CD147 expression in two B16 sublines. The CD147 knockdown remained stable under in vivo conditions as confirmed by immunohistochemistry. However, no differences in MMP-2, MMP-9 and MT1-MMP expression by stromal and tumor cells were detectable in CD147+ and CD147- tumors. Since the tumor microenvironment is a complex system, involving several cell types, the extracellular matrix and plethora soluble factors, we subsequently studied the role of murine CD147 in vitro. Coculture of melanoma cells with different fibroblast cell lines demonstrated that neither CD147+ nor CD147- B16 tumor cells altered the expression of MMP-2 or MMP-9 by the fibroblasts, although we could confirm the susceptibility of these fibroblasts for MMP induction. CONCLUSIONS: At least for the murine B16 melanoma model, CD147 expression on tumor cells seems not to be crucial for MMP-2, MMP-9 and MT1-MMP induction on tumor-associated stromal cells.
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Basigina/biosíntesis , Regulación Neoplásica de la Expresión Génica , Metaloproteinasas de la Matriz/biosíntesis , Melanoma Experimental/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 14 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIHRESUMEN
BACKGROUND: Recent reports have demonstrated that the enzyme indoleamine 2,3-dioxygenase (IDO) is upregulated in human dendritic cells (DCs) upon in vitro maturation. IDO is supposed to convey immunosuppressive effects by degrading the essential amino acid tryptophan, thereby downregulating T-cell functions. Hence, we evaluated IDO expression in DC preparations used for therapeutic DC vaccination and its in vivo effects. PATIENTS, METHODS AND RESULTS: IDO expression was detected by real-time-PCR in a series of human clinical grade DCs (n = 28) prior to vaccination of advanced melanoma patients (n = 11). These analyses revealed an intra- and interpersonal variation in IDO mRNA levels. IDO was strongly upregulated in human DCs on RNA and on protein level upon in vitro maturation by Interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), Interleukin-6 (IL-6) and Prostaglandin E2 (PGE2) over a time course of 24 h. The enzymatic activity of induced IDO was demonstrated by measuring tryptophan degradation. Moreover, in biopsies obtained 24 h after application of the DC vaccine a prominent infiltrate of IDO-positive cells was observed by immunohistochemistry. The inflammatory infiltrate of these sites stained positive for the transcription factor Forkhead box P3 (FoxP3), suggesting an IDO-mediated induction of regulatory T-cells. All analysed melanoma patients (n = 11) receiving DC based immunotherapy exhibited rapid disease progression with a short overall survival due to advanced tumour stage. CONCLUSION: The presented observations suggest a potential clinical relevance of IDO expression in DC-based therapeutic vaccines via the attraction or induction of FoxP3(+) T-cells.
Asunto(s)
Vacunas contra el Cáncer/metabolismo , Células Dendríticas/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Melanoma/terapia , Adulto , Animales , Western Blotting , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Inmunohistoquímica , Inmunoterapia , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Melanoma/inmunología , Persona de Mediana Edad , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Triptófano/metabolismoRESUMEN
An effective immunological eradication of tumors by the adaptive immune system depends on T cell priming, expansion of specific T cells and their effector function. It has been shown that either step may be impaired in the tumor-bearing host, and several strategies have been used to improve antitumor immune responses. In this regard, tumor-targeted IL2 therapy leads to the destruction of established melanoma metastases in fully immune competent mice as previously demonstrated. This effect has been attributed, but never directly confirmed, to the boost of antigen-experienced T cells. To this end, we demonstrate the absence of any antitumor effect of targeted IL2 in mice characterized by an impaired priming of T cell responses. Notably, in these animals tumor-targeted IL2 therapy induced tumor regression only after adoptive transfer of tumor-conditioned splenocytes. A detailed analysis revealed that T cells present within the transferred splenocytes were actively participating in the immune response as these were clonally expanded after targeted IL2 therapy. In summary, we demonstrate here that in LTalpha(-/-) mice lacking sufficient numbers of tumor-specific T cells only the passive transfer of such cells prior to therapy restores the efficacy of tumor-targeted IL2 therapy. Thus, the antitumor effect of tumor-targeted IL2 is indeed based on the boost of pre-existing T cell responses.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunoterapia Adoptiva , Interleucina-2/uso terapéutico , Linfotoxina-alfa/deficiencia , Melanoma Experimental/tratamiento farmacológico , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Células Clonales/inmunología , Citotoxicidad Inmunológica , Sistemas de Liberación de Medicamentos , Interleucina-2/administración & dosificación , Interleucina-2/farmacología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Linfotoxina-alfa/genética , Melanoma Experimental/inmunología , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Organismos Libres de Patógenos Específicos , Bazo/citología , Tejido Subcutáneo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/trasplanteRESUMEN
Immunogenic peptide epitopes from tumor-associated antigens serve as targets for cellular immune responses in numerous clinical trials for therapeutic vaccinations. From these it became evident that prevailing questions can only be addressed in animal models. Hence, problems arise from the fact that while for human melanoma many different immunogenic peptide epitopes are known, for mouse melanoma the available selection is very restricted. To overcome this limitation, we applied reverse immunology to identify Kb-restricted epitopes derived of mouse MAGE. Two epitopes which bind strongly to Kb were selected to test for their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature dendritic cells pulsed with these peptides displayed reactivity to the respective epitopes as measured by enzyme-linked immunospot assays and tetramer staining. The processing and presentation of these epitopes was evident by the killing of melanoma cells by the vaccination-induced T cells. Moreover, intravenous challenge with syngeneic melanoma cells demonstrated the protective immunity induced by this vaccination. In summary, we demonstrate the immunogenicity of two Kb-restricted peptide epitopes derived from mouse MAGE proteins which may serve as valuable tool for preclinical evaluation of vaccination strategies.
Asunto(s)
Antígenos de Neoplasias/inmunología , Epítopos de Linfocito T/inmunología , Melanoma Experimental/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Antígenos H-2/inmunología , Inmunización , Técnicas para Inmunoenzimas , Interferón gamma/inmunología , Melanoma Experimental/patología , Antígenos Específicos del Melanoma , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Fragmentos de Péptidos/inmunología , Bazo/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
The human parasite Entamoeba histolytica is an ancient protozoan that expresses only one unconventional myosin, which has homology with myosin IB from other amoebae. Myosin IB is involved in phagocytosis of human cells by E. histolytica. In this work, we developed a microrheological technique, analysing magnetic phagosomes, which allowed us to probe the density of the F-actin network in living cells. Using this technique, we showed that overexpression of myosin IB led to an increase in cytoplasm viscosity, which correlated with a delay in initiating human cell phagocytosis. To investigate which myosin IB domains sustain cell viscosity changes, we overexpressed truncated forms of the protein. Our results demonstrate that both actin-binding sites that are present in the heavy chain but not the SH3 domain are required to modulate the density of the actin network. These data suggested that, as well as the motor activity, myosin IB in E. histolytica plays a structural role on the actin network owing to its ability to cross-link filaments. The gelation state of cell cytoplasm and the dynamics of cortical F-actin during phagocytosis seem to be modulated by the myosin IB structuring cytoskeleton activity.
Asunto(s)
Citoplasma/metabolismo , Entamoeba histolytica/metabolismo , Miosina Tipo I/biosíntesis , Fagocitosis , Actinas/metabolismo , Animales , Sitios de Unión , Citoesqueleto/metabolismo , Hemoglobinas/química , Humanos , Magnetismo , Miosina Tipo I/química , Fagosomas/metabolismo , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Factores de Tiempo , Viscosidad , Dominios Homologos srcRESUMEN
The murine model of infection with Leishmania major has allowed the demonstration in vivo of the importance CD4+ T cell subsets, distinguishable by the pattern of cytokines they produce, on the outcome of infectious diseases. Genetically determined resistance and susceptibility to infection with this parasite are the result of the development of Th1 and Th2 response, respectively. In this short paper, we present some results obtained in our group pertaining to the analysis of the mechanisms, operational during the early phase of this infection, responsible for the maturation of these functionally distinct CD4+ responses.