RESUMEN
Immunotherapy is a promising approach for specific targeting of cancer cells. Leukemia inhibitory factor (LIF) regulates several features of cancers and cancer stem cells (CSCs) through binding to LIF receptor (LIFR). In this study, we investigated the consensus of LIF and LIFR immunization on the growth of mouse mammary tumors. For this purpose, mouse LIF and LIFR were designed as truncated proteins, expressed in E. coli and then injected to mice as individual and mixed antigens. The results showed the production of neutralizing antibodies and secretion of interferon-γ and interleukin-2 in response to immunization. In continue, the immunized mice were subjected for tumor formation challenge by inoculation of the breast CSCs derived from MC4-L2 cells. Development of the breast tumors was observed in all the control mice, while the tumors appeared in 75% of animals in the LIF group. LIFR injection, individually or in combination with LIF, strongly inhibited the tumor growth to only 25% of the mice. Moreover, a delay in tumor appearance was observed in the immunized mice compared to the controls. Immunostaining of the tumor sections confirmed the expression of LIF and LIFR. In conclusion, LIF and LIFR might be effective targets for immunotherapy of the tumors that express these proteins.
Asunto(s)
Neoplasias de la Mama/genética , Factor Inhibidor de Leucemia/genética , Células Madre Neoplásicas/inmunología , Receptores OSM-LIF/genética , Animales , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Femenino , Inhibidores de Crecimiento/inmunología , Humanos , Inmunización , Interleucina-6/genética , Factor Inhibidor de Leucemia/inmunología , Ratones , Unión Proteica/genética , Receptores OSM-LIF/inmunologíaRESUMEN
Recently cancer/testis antigens (CTA) have gained lots of attention as targets of immune therapy. However, the therapeutic efficacy of the CTAs single-antigen vaccines is not satisfying due to tumor heterogenicity. Therefore, many studies have focused on the enhancement of their efficacy by utilizing rich sources of tumor-associated antigens for anti-cancer vaccination. In the present study, the testicular germ cells and sperm cells as well-known sources of cancer/testis antigens were investigated for anti-4T1 breast cancer vaccination in BALB/c mice. The testicular germ cells (TGCs) and sperm cells were isolated from male BALB/c mice. The definite number of cells were homogenized and mixed with Bacillus Calmette-Guerin (BCG) for vaccination of female BALB/c mice. The treatment groups underwent 3 times of immunizations with one-week intervals and one week after the last injection, all groups were injected with 4T1 cancer cells. The TGCs + BCG (259.7⯱â¯39â¯mm3) and Sperm + BCG (426⯱â¯52â¯mm3) groups exhibited a significant decrease in the tumors' volume in comparison with BCG (641.3⯱â¯102â¯mm3) and no-treatment (788.1⯱â¯117â¯mm3) groups. Therefore, the TGCs + BCG immunized mice had the smallest tumors in comparison with all groups (Pâ¯<â¯0.05). Also, the vital organs of TGCs + BCG (lungs: 6.8⯱â¯2, liver: 10.1⯱â¯2) immunized mice exhibited lowest metastatic burden in comparison with the Sperm + BCG (lungs: 13.5⯱â¯3, liver: 21.1⯱â¯4), BCG (lungs: 24.3⯱â¯4, liver: 33⯱â¯4), and no-treatment (lungs: 26.5⯱â¯6, liver: 37.3⯱â¯3) groups. These observations were inconsistent with the tumor-bearing mice survival evaluations as the TGCs + BCG group had longer mean survival time (79.6⯱â¯12â¯days) in comparison with other groups (no-treatment: 49.8⯱â¯8, BCG: 50.5⯱â¯10, BCGâ¯+â¯Sperm: 64.6⯱â¯7â¯days). Therefore, TGCs can be a potential source of antigens for the anti-breast cancer immunization and more investigations are necessary.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/terapia , Vacunas contra el Cáncer/inmunología , Genitales Masculinos/patología , Células Germinativas/inmunología , Inhibidores de Crecimiento/inmunología , Neoplasias Testiculares/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunización , Masculino , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Neoplasias Testiculares/inmunología , Carga Tumoral , VacunaciónRESUMEN
One of the prime objectives of cancer immunology and immunotherapy is to study the issues related to rescue and/or maintenance of the optimum effector CD8(+) T cell functions by minimizing tumor-induced negative factors. In this regard the influence of host intrinsic CD4(+) helper T cells towards generation and maintenance of CD8(+) effector T cells appears controversial in different experimental settings. Therefore, the present study was aimed to re-analyze the influence of CD4(+) helper T cells towards effector T cells during neem leaf glycoprotein (NLGP)-vaccine-mediated tumor growth restriction. CD4 depletion (mAb; Clone GK1.5) surprisingly resulted in significant increase in CD8(+) T cells in different immune organs from NLGP-treated sarcoma-bearing mice. However, such CD8 surge could not restrict the sarcoma growth in NLGP-treated CD4-depleted mice. Furthermore, CD4 depletion in early phase hinders CD8(+) T cell activation and terminal differentiation by targeting crucial transcription factor Runx3. CD4 depletion decreases accumulation of CD8α(+) dendritic cells within tumor draining lymph node, hampers antigen cross priming and CD86-CD28 interactions for optimum CD8(+) T cell functions. In order to search the mechanism of CD4(+) T cell help on NLGP-mediated CD8 effector functions, the role of CD4(+) helper T cell-derived IL-2 on optimization of CD8 functions was found using STAT5 signaling, but complete response requires physical contact of CD4(+) helper T cells with its CD8 counterpart. In conclusion, it was found that CD4(+) T cell help is not required to generate CD8(+) T cells but was found to be an integral phenomenon in maintenance of its anti-tumor functions even in NLGP-vaccine-mediated sarcoma growth restriction.
Asunto(s)
Antineoplásicos/inmunología , Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Inhibidores de Crecimiento/inmunología , Sarcoma/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Azadirachta/inmunología , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Procesos de Crecimiento Celular , Línea Celular Tumoral , Células Cultivadas , Citotoxicidad Inmunológica , Femenino , Interleucina-2/metabolismo , Depleción Linfocítica , Ratones , Neoplasias ExperimentalesRESUMEN
Carcinoembryonic antigen (CEA) is one of the promising tumor antigens mainly associated with carcinoma of the colon, lung, breast, etc. and received wide attention for cancer immunotherapy. Neem leaf glycoprotein (NLGP), an effective immunomodulator, is able to generate humoral and cellular immune responses in murine tumor models. We have generated a monoclonal antibody (mAb) against NLGP by fusing NLGP-immunized mice splenocytes with nonsecretory myeloma cells. A highly anti-NLGP mAb secreting clone (1C8; IgG2a in nature) has been identified and propagated in culture. 1C8 recognizes human CEA as good as NLGP by enzyme linked immunosorbent assay, Western blotting, and immunoprecipitation. 1C8 detects CEA on colon cancer tissues by immunochistochemistry. By flow cytometry, 1C8 specifically reacts with CEA(+) human (Colo-205, HCT-116, and HT-29) and mouse (CT-26) colon cancer cells, but it showed minimum reactivity with CEA(-) human (MCF7, SiHa, and SCC084) and mouse (B16MelF10) cancer cells. This anti-NLGP 1C8 mAb revealed significant antitumor activity and better survivability in vivo in animals bearing mouse (CT-26 in BALB/c) and human (Colo-205 in athymic nude) CEA(+) cancer cells. 1C8 has no direct influence on proliferation and migration of CEA(+) cells, however, NK cell-dependent strong antibody-dependent cellular cytotoxicity reaction toward CEA(+) cells and normalization of angiogenesis are chiefly associated with tumor growth restriction. Obtained results provided a new immunotherapeutic approach for the effective management of CEA(+) tumors.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Azadirachta/inmunología , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/terapia , Glicoproteínas/inmunología , Inmunomodulación , Proteínas de Plantas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Plantas/inmunología , Fusión Celular , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/inmunología , Inhibidores de Crecimiento/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Neovascularización Patológica/terapia , Hojas de la Planta/inmunologíaRESUMEN
Patients with the monogenic disease autoimmune polyendocrine syndrome type I (APSI) develop autoimmunity against multiple endocrine organs and suffer from chronic mucocutaneous candidiasis (CMC), a paradoxical complication with an unknown mechanism. We report here that saliva from APSI patients with CMC is defective in inhibiting growth of Candida albicans in vitro and show reduced levels of a salivary protein identified as cystatin SA1. In contrast, APSI patients without CMC express salivary cystatin SA1 and can inhibit C. albicans to the same extent as healthy controls. We evaluated the anti-fungal activity of cystatin SA1 and found that synthesized full length cystatin SA1 efficiently inhibits growth of C. albicans in vitro. Moreover, APSI patients exhibit salivary IgA autoantibodies recognizing myosin-9, a protein expressed in the salivary glands, thus linking autoimmunity to cystatin SA1 deficiency and CMC. This data suggests an autoimmune mechanism behind CMC in APSI and provides rationale for evaluating cystatin SA1 in antifungal therapy.
Asunto(s)
Candidiasis Mucocutánea Crónica/inmunología , Inhibidores de Crecimiento/metabolismo , Poliendocrinopatías Autoinmunes/inmunología , Cistatinas Salivales/metabolismo , Adulto , Autoanticuerpos/metabolismo , Autoinmunidad , Candidiasis Mucocutánea Crónica/etiología , Candidiasis Mucocutánea Crónica/genética , Femenino , Predisposición Genética a la Enfermedad , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/inmunología , Humanos , Inmunoglobulina A/metabolismo , Masculino , Proteínas Motoras Moleculares/inmunología , Cadenas Pesadas de Miosina/inmunología , Poliendocrinopatías Autoinmunes/complicaciones , Poliendocrinopatías Autoinmunes/genética , Saliva/metabolismo , Cistatinas Salivales/genética , Cistatinas Salivales/inmunología , Adulto JovenRESUMEN
αß and γδ lineage T cells are thought to arise from a common CD4(-)CD8(-) progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We demonstrated recently that a ubiquitously expressed ribosomal protein, Rpl22, is selectively required for the development of αß lineage T cells. Germline ablation of Rpl22 impairs development of αß lineage, but not γδ lineage, T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors used by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22(-/-) thymocytes, including miR-34a, PUMA, p21(waf), Bax, and Noxa. Notably, the proapoptotic factor Bim, while not a direct p53 target, was also strongly induced in Rpl22(-/-) T cells. Gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in Rpl22-deficient T cells; however, only a few p53 targets alleviated developmental arrest when individually ablated by gene targeting or knockdown. Co-elimination of PUMA and Bim resulted in a nearly complete restoration of development of Rpl22(-/-) thymocytes, indicating that p53-mediated arrest is enforced principally through effects on cell survival. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21(waf)) and apoptosis (PUMA) actually abrogated the partial rescue caused by loss of PUMA alone, suggesting that the G1 checkpoint protein p21(waf) facilitates thymocyte development in some contexts.
Asunto(s)
Diferenciación Celular/inmunología , Marcación de Gen , Inhibidores de Crecimiento/inmunología , Proteínas Ribosómicas/deficiencia , Subgrupos de Linfocitos T/inmunología , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/genética , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteína 11 Similar a Bcl2 , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Marcación de Gen/métodos , Inhibidores de Crecimiento/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Timo/inmunología , Timo/metabolismo , Timo/patología , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/deficiencia , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/deficiencia , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
A fundamental problem in immunoregulation is how CD4(+) T cells react to immunogenic peptides derived from the V region of the BCR that are created by somatic mechanisms, presented in MHC II, and amplified to abundance by B cell clonal expansion during immunity. BCR neo Ags open a potentially dangerous avenue of T cell help in violation of the principle of linked Ag recognition. To analyze this issue, we developed a murine adoptive transfer model using paired donor B cells and CD4 T cells specific for a BCR-derived peptide. BCR peptide-specific T cells aborted ongoing germinal center reactions and impeded the secondary immune response. Instead, they induced the B cells to differentiate into short-lived extrafollicular plasmablasts that secreted modest quantities of Ig. These results uncover an immunoregulatory process that restricts the memory pathway to B cells that communicate with CD4 T cells via exogenous foreign Ag.
Asunto(s)
Subgrupos de Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito B/inmunología , Centro Germinal/inmunología , Región Variable de Inmunoglobulina/inmunología , Memoria Inmunológica , Péptidos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Subgrupos de Linfocitos B/patología , Subgrupos de Linfocitos B/trasplante , Linfocitos T CD4-Positivos/clasificación , Linfocitos T CD4-Positivos/patología , Comunicación Celular/genética , Comunicación Celular/inmunología , Epítopos de Linfocito B/administración & dosificación , Femenino , Centro Germinal/patología , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/inmunología , Región Variable de Inmunoglobulina/administración & dosificación , Cadenas kappa de Inmunoglobulina/genética , Memoria Inmunológica/genética , Ratones , Ratones Endogámicos A , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Péptidos/administración & dosificación , Péptidos/genética , Células Plasmáticas/inmunología , Células Plasmáticas/patología , Receptores de Antígenos de Linfocitos B/administración & dosificaciónRESUMEN
Inflammatory mediators have the potential to impact a surprising range of diseases, including obesity and its associated metabolic syndrome. In this paper, we show that the proinflammatory cytokine IL-17 inhibits adipogenesis, moderates adipose tissue (AT) accumulation, and regulates glucose metabolism in mice. IL-17 deficiency enhances diet-induced obesity in mice and accelerates AT accumulation even in mice fed a low-fat diet. In addition to potential systemic effects, IL-17 is expressed locally in AT by leukocytes, predominantly by γδ T cells. IL-17 suppresses adipocyte differentiation from mouse-derived 3T3-L1 preadipocytes in vitro, and inhibits expression of genes encoding proadipogenic transcription factors, adipokines, and molecules involved in lipid and glucose metabolism. IL-17 also acts on differentiated adipocytes, impairing glucose uptake, and young IL-17-deficient mice show enhanced glucose tolerance and insulin sensitivity. Our findings implicate IL-17 as a negative regulator of adipogenesis and glucose metabolism in mice, and show that it delays the development of obesity.
Asunto(s)
Adipogénesis/inmunología , Tejido Adiposo/citología , Tejido Adiposo/inmunología , Glucosa/metabolismo , Homeostasis/inmunología , Interleucina-17/fisiología , Obesidad/inmunología , Células 3T3-L1 , Adipogénesis/genética , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Glucosa/antagonistas & inhibidores , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/inmunología , Resistencia a la Insulina/inmunología , Interleucina-17/biosíntesis , Interleucina-17/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , Obesidad/patología , Receptores de Antígenos de Linfocitos T gamma-delta/biosíntesis , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patologíaRESUMEN
As immune effector molecules, antimicrobial peptides (AMPs) play an important role in the invertebrate immune system. Here, we present two novel AMPs, named centrocins 1 (4.5kDa) and 2 (4.4kDa), purified from coelomocyte extracts of the green sea urchin, Strongylocentrotus droebachiensis. The native peptides are cationic and show potent activities against Gram-positive and Gram-negative bacteria. The centrocins have an intramolecular heterodimeric structure, containing a heavy chain (30 amino acids) and a light chain (12 amino acids). The cDNA encoding the peptides and genomic sequences were cloned and sequenced. One putative isoform (centrocin 1b) was identified and one intron was found in the genes coding for the centrocins. The full length protein sequence of centrocin 1 consists of 119 amino acids, whereas centrocin 2 consists of 118 amino acids which both include a preprosequence of 51 or 50 amino acids for centrocins 1 and 2, respectively, and an interchain of 24 amino acids between the heavy and light chain. The difference of molecular mass between the native centrocins and the deduced sequences from cDNA indicates that the native centrocins contain a post-translational brominated tryptophan. In addition, two amino acids at the C-terminal, Gly-Arg, were removed from the light chains during the post-translational processing. The separate peptide chains of centrocin 1 were synthesized and the heavy chain alone was shown to be sufficient for antimicrobial activity. The genome of the closely related species, the purple sea urchin (S. purpuratus), was shown to contain two putative proteins with high similarity to the centrocins.
Asunto(s)
Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Bacterias/inmunología , Infecciones Bacterianas/inmunología , Hongos/inmunología , Inhibidores de Crecimiento/metabolismo , Micosis/inmunología , Secuencia de Aminoácidos , Animales , Antiinfecciosos/inmunología , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Bacterias/crecimiento & desarrollo , Bacterias/patogenicidad , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Clonación Molecular , Hongos/crecimiento & desarrollo , Hongos/patogenicidad , Inhibidores de Crecimiento/inmunología , Inmunidad Innata/genética , Datos de Secuencia Molecular , Micosis/genética , Micosis/metabolismo , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Erizos de Mar , Homología de Secuencia , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The functional importance of positively charged residues in antibacterial peptides (ABPs) has been well documented. However, their role in antifungal peptides (AFPs) has not been extensively evaluated. To address this question, we investigated the functional role of charged residues in drosomycin, a specific Drosophila antifungal peptide, using site-directed mutagenesis in combination with circular dichroism (CD) analysis and antifungal assays. As a result, we firstly identified five cationic residues (R6, K8, R20, R21 and K38) whose alterations significantly affected the antifungal activity. Intriguingly, two negatively charged residues (D1 and E25) are also recognized as functional determinants of drosomycin. This indicates that it is the location of these charged residues rather than net charges that is crucial for activity. These functional sites are located in different secondary structure elements, including the N-loop, alpha-helix and gamma-core regions, all highly exposed on the molecular surface, suggesting that drosomycin may bind to fungal targets through electrostatic interactions. Our work has implications for further modification of drosomycin to obtain new antifungal peptides with enhanced activity.
Asunto(s)
Aminoácidos Básicos/química , Antifúngicos/metabolismo , Proteínas de Drosophila/metabolismo , Hongos/inmunología , Micosis/inmunología , Fragmentos de Péptidos/metabolismo , Aminoácidos Básicos/genética , Aminoácidos Básicos/metabolismo , Animales , Antifúngicos/química , Antifúngicos/inmunología , Dicroismo Circular , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/inmunología , Hongos/crecimiento & desarrollo , Hongos/patogenicidad , Inhibidores de Crecimiento/química , Inhibidores de Crecimiento/inmunología , Inhibidores de Crecimiento/metabolismo , Inmunidad/genética , Mutagénesis Sitio-Dirigida , Mutación/genética , Micosis/genética , Micosis/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Unión Proteica/genética , Unión Proteica/inmunología , Conformación ProteicaRESUMEN
BACKGROUND AIMS: Because of the inflammatory nature and extensive stromal compartment in pancreatic tumors, we investigated the role of mesenchymal stromal cells (MSC) to engraft selectively in pancreatic carcinomas and serve as anti-tumor drug delivery vehicles to control pancreatic cancer progression. METHODS: Human pancreatic carcinoma cells, PANC-1, expressing renilla luciferase were orthotopically implanted into SCID mice and allowed to develop for 10 days. Firefly luciferase-transduced MSC or MSC expressing interferon (IFN)-beta were then injected intraperitoneally weekly for 3 weeks. Mice were monitored by bioluminescent imaging for expression of renilla (PANC-1) and firefly (MSC) luciferase. RESULTS: MSC selectively homed to sites of primary and metastatic pancreatic tumors and inhibited tumor growth (P=0.032). The production of IFN-beta within the tumor site by MSC-IFN-beta further suppressed tumor growth (P=0.0000083). Prior studies indicated that MSC home to sites of inflammation; therefore, we sought to alter the tumor microenvironment through treatment with a potent anti-inflammatory agent. After treatment, inflammation-associated mediators were effectively down-regulated, including NFkappaB, vascular endothelial growth factor (VEGF) and interleukin (IL)-6 as well as chemokines involved in MSC migration (CCL3 and CCL25). Treatment with the anti-inflammatory agent CDDO-Me before and after MSC-IFN-beta injections resulted in reduction of MSC in the tumors and reversed the positive effect of tumor inhibition by MSC-IFN-beta alone (P=0.041). CONCLUSIONS: These results suggest that MSC exhibit innate anti-tumor effects against PANC-1 cells and can serve as delivery vehicles for IFN-beta for the treatment of pancreatic cancer. However, these beneficial effects may be lost in therapies combining MSC with anti-inflammatory agents.
Asunto(s)
Interferón beta/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Neoplasias Pancreáticas/terapia , Células del Estroma/metabolismo , Animales , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Procesos de Crecimiento Celular/inmunología , Línea Celular Tumoral , Terapia Genética , Inhibidores de Crecimiento/inmunología , Inhibidores de Crecimiento/uso terapéutico , Humanos , Terapia de Inmunosupresión , Inflamación , Interferón beta/genética , Interferón beta/inmunología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/patología , Ratones , Ratones SCID , Trasplante de Neoplasias , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Células del Estroma/patología , Células del Estroma/trasplante , Transgenes/genéticaRESUMEN
Targeted therapy to the epidermal growth factor receptor (EGFR) seems to be related to its expression level on tumor cells. Interferon-alpha (IFN-alpha) induces growth inhibition but also may up-regulate the EGFR expression in some cancer cell lines. We aimed to determine whether the IFN-alpha combined with an EGFR-specific monoclonal antibody (nimotuzumab) may affect the growth of human tumor epithelial cell lines with different EGFR expression levels. H125, a lung adenosquamous carcinoma, and A431, a vulvar epidermoid carcinoma, cell lines express intermediate and high levels of EGFR, respectively, whereas MDA MB231, a breast adenocarcinoma cell line expresses undetectable levels of EGFR measured by flow cytometry/FACS. We found that IFN-alpha alone inhibited in a dose-dependent fashion the growth of all cell lines, but only up-regulated the EGFR expression in the lung carcinoma-derived cell line. Noteworthy, the combined treatment did not modify the complement-mediated cytotoxicity of the antibody although the antiproliferative activity of nimotuzumab in H125 cells in vitro increased when an IFN-alpha-conditioning treatment was used. In conclusion, this study may provide insights about the rational use of EGFR inhibitors into the immunopharmacological management of targeted therapies including the IFN-alpha for lung cancer.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Células Epiteliales/inmunología , Receptores ErbB/inmunología , Inhibidores de Crecimiento/metabolismo , Factores Inmunológicos/farmacología , Inmunoterapia , Interferón-alfa/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/terapia , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Separación Celular , Proteínas del Sistema Complemento/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Relación Dosis-Respuesta Inmunológica , Sinergismo Farmacológico , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Inhibidores de Crecimiento/inmunología , Humanos , Factores Inmunológicos/uso terapéutico , Interferón-alfa/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias de la Vulva/inmunología , Neoplasias de la Vulva/metabolismo , Neoplasias de la Vulva/patología , Neoplasias de la Vulva/terapiaRESUMEN
Thirty years after angiogenesis was shown to play an enabling role in cancer, modern medicine is still trying to develop novel compounds and therapeutics to target the tumor vasculature. However, most therapeutics require multiple rounds of administration and can have toxic side effects. In this study, we use anti-angiogenesis immunotherapy to target cells actively involved in forming new blood vessels that support the growth and spread of breast cancer. Targeting a central cell type involved in angiogenesis, endothelial cells, we immunized against host vascular endothelial growth factor receptor 2 to fight the growth of Her-2/neu(+) breast tumors. Using the bacterial vector, Listeria monocytogenes (Lm), we fused polypeptides from the mouse vascular endothelial growth factor receptor 2 molecule (fetal liver kinase-1) to the microbial adjuvant, listeriolysin-O, and used Lm to deliver the Ags and elicit potent antitumor CTL responses. Lm-listeriolysin-O-fetal liver kinase-1 was able to eradicate some established breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumor-associated Ag Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to HER-2/neu and was not solely due to the reduction of tumor vasculature. However, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy. We show that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.
Asunto(s)
Inhibidores de la Angiogénesis/inmunología , Vacunas contra el Cáncer/inmunología , Listeria monocytogenes/inmunología , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/prevención & control , Neovascularización Patológica/prevención & control , Receptor ErbB-2/biosíntesis , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/genética , Animales , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/genética , Toxinas Bacterianas/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/genética , Línea Celular Tumoral , Femenino , Inhibidores de Crecimiento/administración & dosificación , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/inmunología , Proteínas de Choque Térmico/administración & dosificación , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/inmunología , Proteínas Hemolisinas/administración & dosificación , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Listeria monocytogenes/genética , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/prevención & control , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Datos de Secuencia Molecular , Neovascularización Patológica/inmunología , Neovascularización Patológica/fisiopatología , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/administración & dosificación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
CD1d is expressed on APCs and presents glycolipids to CD1d-restricted NKT cells. For the first time, we demonstrate the ability of anti-CD1d mAbs to inhibit the growth of different CD1d-negative experimental carcinomas in mice. Anti-CD1d mAbs systemically activated CD1d(+) APC, as measured by production of IFN-gamma and IL-12. Tumor growth inhibition was found to be completely dependent on IFN-gamma and IL-12 and variably dependent on CD8(+) T cells and NK cells, depending upon the tumor model examined. Anti-CD1d mAb induced greater CD8(+) T cell-dependent tumor suppression where regulatory CD1d-restricted type II NKT cells have been implicated, and were less effective in a NK cell-dependent manner against tumors where T regulatory cells were immunosuppressive. The ability of anti-CD1d mAbs to coincidently activate CD1d(+) APCs to release IL-12 and inhibit CD1d-restricted type II NKT cells makes CD1d an exciting new target for immunotherapy of cancer based on tumor immunoregulation.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Antígenos CD1d/inmunología , Antígenos CD1d/metabolismo , Antineoplásicos/uso terapéutico , Inhibidores de Crecimiento/uso terapéutico , Animales , Anticuerpos Bloqueadores/fisiología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Antígenos CD1d/biosíntesis , Antineoplásicos/inmunología , Antineoplásicos/metabolismo , Línea Celular Tumoral , Células Cultivadas , Femenino , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/inmunología , Interferón gamma/biosíntesis , Interferón gamma/fisiología , Interleucina-12/biosíntesis , Interleucina-12/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones SCID , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/patologíaRESUMEN
During viral infection, dendritic cells (DCs) capture infected cells and present viral Ags to CD8(+) T cells. However, activated DCs might potentially present cell-associated Ags derived from captured dead cells. In this study, we find that human DCs that captured dead cells containing the TLR3 agonist poly(I:C) produced cytokines and underwent maturation, but failed to elicit autologous CD8(+) T cell responses against Ags of dead cells. Accordingly, DCs that captured dead cells containing poly(I:C), or influenza virus, are unable to activate CD8(+) T cell clones specific to cell-associated Ags of captured dead cells. CD4(+) T cells are expanded with DCs that have captured poly(I:C)-containing dead cells, indicating the inhibition is specific for MHC class I-restricted cross-presentation. Furthermore, these DCs can expand naive allogeneic CD8(+) T cells. Finally, soluble or targeted Ag is presented when coloaded onto DCs that have captured poly(I:C)-containing dead cells, indicating the inhibition is specific for dead cell cargo that is accompanied by viral or poly(I:C) stimulus. Thus, DCs have a mechanism that prevents MHC class I-restricted cross-presentation of cell-associated Ag when they have captured dead infected cells.
Asunto(s)
Reactividad Cruzada/inmunología , Células Dendríticas/virología , Inhibidores de Crecimiento/inmunología , Antígeno HLA-A2/inmunología , Terapia de Inmunosupresión , Virus de la Influenza A/inmunología , Melanoma/virología , Poli I-C/inmunología , Antígenos de Neoplasias/inmunología , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/inmunología , Células Dendríticas/patología , Antígeno HLA-A2/metabolismo , Humanos , Terapia de Inmunosupresión/métodos , Activación de Linfocitos/inmunología , Melanoma/inmunología , Melanoma/patología , Necrosis , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/virologíaRESUMEN
The reduced ability of central axons to regenerate after injury is significantly influenced by the presence of several molecules that inhibit axonal growth. Nogo-A is one of the most studied and most potent of the myelin-associated growth inhibitory molecules. Its neutralization, as well as interference with its signalling, allows for enhanced axonal sprouting and growth following injury. Using differentiated rat organotypic hippocampal slice cultures treated for 5 days with either of two different function-blocking anti-Nogo-A antibodies, we show an increase in CA3 fibre regeneration after lesion. In intact slices, 5 days of anti-Nogo-A antibody treatment led to increased sprouting of intact CA3 fibres that are positive for neurofilament 68. A transcriptomic approach confirmed the occurrence of a growth response on the molecular level upon Nogo-A neutralization in intact cultures. Our results demonstrate that Nogo-A neutralization for 5 days is sufficient for the induction of growth in mature CNS tissue without the prerequisite of an injury. Nogo-A may therefore act as a tonic growth suppressor/stabilizer in the adult intact hippocampus.
Asunto(s)
Anticuerpos/farmacología , Conos de Crecimiento/metabolismo , Inhibidores de Crecimiento/metabolismo , Hipocampo/metabolismo , Proteínas de la Mielina/metabolismo , Regeneración Nerviosa/fisiología , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Biomarcadores/análisis , Biomarcadores/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/ultraestructura , Inhibidores de Crecimiento/antagonistas & inhibidores , Inhibidores de Crecimiento/inmunología , Hipocampo/citología , Hipocampo/efectos de los fármacos , Proteínas de la Mielina/antagonistas & inhibidores , Proteínas de la Mielina/inmunología , Regeneración Nerviosa/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas de Neurofilamentos/metabolismo , Neurogénesis/efectos de los fármacos , Neurogénesis/inmunología , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/inmunología , Proteínas Nogo , Análisis de Secuencia por Matrices de Oligonucleótidos , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunologíaRESUMEN
Oncostatin M (OSM), a member of the interleukin-6 family, is produced by monocytes and macrophages in the peripheral blood and microglia in the brain. The present study aimed to elucidate a regulatory role of OSM in the functions of blood-brain barrier (BBB) comprised of rat brain capillary endothelial cells (RBECs). OSM decreased the transendothelial electrical resistance of RBEC monolayers in a concentration- and time-dependent manner. Immunocytochemical observations of ZO-1 and claudin-5 in OSM-treated RBECs showed a zipper-like and/or zigzag shape along the junctions between cells, in contrast with the smooth and linear shape in vehicle-treated cultures. When RBECs were pre-treated with anti-OSM antibody, OSM failed to evoke these changes. The cellular constituents producing OSM in the brain and peripheral blood could be implicated in the functional and structural impairment of the BBB.
Asunto(s)
Barrera Hematoencefálica/citología , Células Endoteliales/efectos de los fármacos , Inhibidores de Crecimiento/toxicidad , Oncostatina M/toxicidad , Animales , Anticuerpos/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Impedancia Eléctrica , Células Endoteliales/patología , Inhibidores de Crecimiento/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Oncostatina M/inmunología , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Previous studies have shown that the Notch1 and TGF-beta signaling pathways are mutually re-enforcing. Given recent evidence that regulatory T cell (Treg) effector function is mediated by TGF-beta signaling, we investigated whether Notch1 signaling also participated in Treg effector function. Initial studies showed that Notch1 ligands, particularly Jagged1, are present on Tregs and that, indeed, blockade of Notch1 signaling with an anti-Jagged1 or a blocking anti-Notch1 Ab inhibits Treg suppressor function in vitro. We then showed that a signaling component generated by Notch1 activation (Notch1 intracellular domain) of dendritic cells physically interacts with a signaling component generated by TGF-beta signaling (pSmad3). Furthermore, this interaction has functional downstream effects because over-expression of Notch1 intracellular domain facilitates pSmad3 translocation to the nucleus and enhances pSmad3 transcriptional activity of a Smad-sensitive promoter linked to a luciferase reporter. Finally, we showed that blockade of TGF-beta signaling and Notch signaling did not have additive inhibitory effects on Treg suppressor function. These results are consistent with the conclusion that Notch1 signaling facilitates TGF-beta-mediated effector function of Tregs.
Asunto(s)
Receptor Notch1/fisiología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Femenino , Inhibidores de Crecimiento/antagonistas & inhibidores , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/inmunología , Inhibidores de Crecimiento/fisiología , Células HT29 , Humanos , Sueros Inmunes/farmacología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Subunidad alfa del Receptor de Interleucina-2/biosíntesis , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Proteína Jagged-1 , Células Jurkat , Ligandos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Visón , Receptor Notch1/antagonistas & inhibidores , Receptor Notch1/biosíntesis , Receptor Notch1/inmunología , Proteínas Serrate-Jagged , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
BACKGROUND: Myelin-associated proteins contribute to failure of axon regeneration in the injured central nervous system of the adult. METHODS: In this study, we employed a recombinant DNA vaccine encoding the myelin-derived inhibitors NogoA, myelin-associated glycoprotein (MAG) and tenascin-R (TnR), so as to effect the production of antibodies against these myelin-related antigens, in a rodent head injury model and ascertained its potential for promoting axonal plasticity and functional recovery. Adult rats underwent lateral fluid percussion at the left sensorimotor cortex (SMC) and treatment with the DNA vaccine before or after injury. Behavioral tests and neuroanatomical tract tracing was carried out. FINDINGS: The vaccinated rats showed improved corticorubral plasticity and functional recovery compared to control groups. CONCLUSIONS: This suggests that a DNA vaccination approach may provide a promising strategy for promoting repair after traumatic brain injury.
Asunto(s)
Lesiones Encefálicas/inmunología , Lesiones Encefálicas/prevención & control , Inhibidores de Crecimiento/inmunología , Recuperación de la Función/fisiología , Vacunas de ADN/uso terapéutico , Análisis de Varianza , Animales , Axones/inmunología , Axones/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/inmunología , Corteza Cerebral/metabolismo , Modelos Animales de Enfermedad , Femenino , Inhibidores de Crecimiento/genética , Proteínas de la Mielina/genética , Proteínas de la Mielina/inmunología , Glicoproteína Asociada a Mielina/genética , Glicoproteína Asociada a Mielina/inmunología , Regeneración Nerviosa/efectos de los fármacos , Regeneración Nerviosa/inmunología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/inmunología , Vías Nerviosas/metabolismo , Examen Neurológico/métodos , Proteínas Nogo , Desempeño Psicomotor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Tenascina/genética , Tenascina/inmunología , Factores de Tiempo , Vacunación/métodos , Vacunas de ADN/inmunologíaRESUMEN
Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well-established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.