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1.
Int J Mol Sci ; 23(23)2022 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-36499300

RESUMO

We previously reported that a novel peptide vaccine platform, based on synthetic melanin nanoaggregates, triggers strong cytotoxic immune responses and significantly suppresses tumor growth in mice. However, the mechanisms underlying such an efficacy remained poorly described. Herein, we investigated the role of dendritic cells (DCs) in presenting the antigen embedded in the vaccine formulation, as well as the potential stimulatory effect of melanin upon these cells, in vitro by coculture experiments and ELISA/flow cytometry analysis. The vaccine efficiency was evaluated in FLT3-L-/- mice constitutively deficient in DC1, DC2, and pDCs, in Zbtb46DTR chimera mice deficient in DC1 and DC2, and in LangerinDTR mice deficient in dermal DC1 and Langerhans cells. We concluded that DCs, and especially migratory conventional type 1 dendritic cells, seem crucial for mounting the immune response after melanin-based vaccination. We also assessed the protective effect of L-DOPA melanin on peptides from enzymatic digestion, as well as the biodistribution of melanin-peptide nanoaggregates, after subcutaneous injection using [18F]MEL050 PET imaging in mice. L-DOPA melanin proved to act as an efficient carrier for peptides by fully protecting them from enzymatic degradation. L-DOPA melanin did not display any direct stimulatory effects on dendritic cells in vitro. Using PET imaging, we detected melanin-peptide nanoaggregates up to three weeks after subcutaneous injections within the secondary lymphoid tissues, which could explain the sustained immune response observed (up to 4 months) with this vaccine technology.


Assuntos
Vacinas Anticâncer , Neoplasias , Camundongos , Animais , Melaninas/metabolismo , Distribuição Tecidual , Células Dendríticas , Peptídeos/farmacologia , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo
2.
Cancer Immunol Immunother ; 69(12): 2501-2512, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32561966

RESUMO

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas Anticâncer/imunologia , Melaninas/imunologia , Neoplasias/terapia , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Adjuvante de Freund/administração & dosagem , Adjuvante de Freund/imunologia , Humanos , Imunogenicidade da Vacina , Lipídeos/administração & dosagem , Lipídeos/imunologia , Melaninas/administração & dosagem , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Receptor Toll-Like 9/agonistas , Receptor Toll-Like 9/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia
3.
Radiol Oncol ; 52(4): 392-398, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30511933

RESUMO

Background Malignant gliomas are rapidly growing tumours that extensively invade the brain and have bad prognosis. Our study was performed to assess the metabolic effects of bevacizumab on the glioma cells carrying the IDH1 mutation, a mutation, associated with better prognosis and treatment outcome. Bevacizumab is known to inhibit tumour growth by neutralizing the biological activity of vascular endothelial growth factor (VEGF). However, the direct effects of bevacizumab on tumour cells metabolism remain poorly known. Materials and methods The immunoassay and MTT assay were used to assess the concentration of secreted VEGF and cell viability after bevacizumab exposure. Metabolomic studies on cells were performed using high resolution magic angle spinning spectroscopy (HRMAS). Results mIDH1-U87 cells secreted VEGF (13 ng/mL). Regardless, bevacizumab had no cytotoxic effect, even after a 72h exposure and with doses as high as 1 mg/mL. Yet, HRMAS analysis showed a significant effect of bevacizumab (0.1 mg/mL) on the metabolic phenotype of mIDH1-U87 cells with elevation of 2-hydroxyglutarate and changes in glutamine group metabolites (alanine, glutamate, glycine) and lipids (polyunsaturated fatty acids [PUFA], glycerophosphocholine, and phosphocholine). Conclusions In mIDH1-U87 cells, changes in glutamine group metabolites and lipids were identified as metabolic markers of bevacizumab treatment. These data support the possibility of a functional tricarboxylic acid cycle that runs in reductive manner, as a probable mechanism of action of bevacizumab in IDH1 mutated gliomas and propose a new target pathway for effective treatment of malignant gliomas.


Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Bevacizumab/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Glioma/tratamento farmacológico , Glioma/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Fenótipo , Análise de Componente Principal
4.
PLoS One ; 12(7): e0181403, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28715455

RESUMO

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8-35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.


Assuntos
Adjuvantes Imunológicos , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Melaninas/imunologia , Neoplasias/terapia , Animais , Antígenos de Neoplasias/química , Vacinas Anticâncer/química , Epitopos de Linfócito T/química , Feminino , Imunização , Memória Imunológica , Linfonodos/imunologia , Linfonodos/patologia , Melaninas/síntese química , Melaninas/química , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias/imunologia , Neoplasias/patologia , Oxirredução , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/imunologia
5.
Cancer Sci ; 106(9): 1212-8, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26094710

RESUMO

TLR-9 agonists are immunostimulating agents that have antitumor effects in animal models. A phase I trial was conducted to define the safety profile of subcutaneous injections, combined with intrathecally administration of CpG-28, a TRL 9 agonist, in patients with neoplastic meningitis (NM). Cohorts of 3-6 patients with NM were treated for 5 weeks with escalating doses of CpG-28. The primary endpoint was tolerance. Secondary endpoints were progression free survival (PFS) and overall survival (OS). Twenty-nine patients were treated with CpG-28. The primary cancers were malignant glioma, lung carcinoma, breast cancer, melanoma or melanocytoma, ependymoma, and colorectal cancer. The median age was 56 years and median Karnovsky Performance status (KPS) was 70%. The treatment was well tolerated. Adverse effects that were possibly or probably related to the studied drug were grade 2 lymphopenia, anemia and neutropenia, local erythema at injection sites, fever and seizure. There were five serious adverse events: two confusions, two infections of ventricular devices and one grade 4 thrombopenia and neutropenia. The median PFS was 7 weeks and median OS was 15 weeks. Interestingly, the median survival was slightly (but not significantly) higher in the eight patients who were concomitantly treated with bevacizumab (19 weeks vs 15 weeks; P = 0.11). CpG-28 was well tolerated at doses up to 0.3 mg/kg subcutaneously and 18 mg intrathecally. Additional trials are warranted.


Assuntos
Antineoplásicos/uso terapêutico , Meningite/terapia , Neoplasias/terapia , Oligodesoxirribonucleotídeos/uso terapêutico , Adulto , Idoso , Antineoplásicos/efeitos adversos , Intervalo Livre de Doença , Feminino , Humanos , Imunoterapia/métodos , Masculino , Meningite/metabolismo , Pessoa de Meia-Idade , Oligodesoxirribonucleotídeos/efeitos adversos , Receptor Toll-Like 9/agonistas , Adulto Jovem
6.
Nanomaterials (Basel) ; 5(4): 1588-1609, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-28347083

RESUMO

The superparamagnetic iron oxide nanoparticles (SPIONs) have great potential in therapeutic and diagnostic applications. Due to their superparamagnetic behavior, they are used clinically as a Magnetic Resonance Imaging (MRI) contrast agent. Iron oxide nanoparticles are also recognized todays as smart drug-delivery systems. However, to increase their specificity, it is essential to functionalize them with a molecule that effectively targets a specific area of the body. Among the molecules that can fulfill this role, peptides are excellent candidates. Oligonucleotides are recognized as potential drugs for various diseases but suffer from poor uptake and intracellular degradation. In this work, we explore four different strategies, based on the electrostatic interactions between the different partners, to functionalize the surface of SPIONs with a phosphorothioate oligonucleotide (ODN) and a cationic peptide labeled with a fluorophore. The internalization of the nanoparticles has been evaluated in vitro on RAW 264.7 cells. Among these strategies, the "«one-step assembly¼", i.e., the direct complexation of oligonucleotides and peptides on iron oxide nanoparticles, provides the best way of coating for the internalization of the nanocomplexes.

7.
PLoS One ; 9(6): e99198, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24922514

RESUMO

BACKGROUND: Glioma cells not only secrete high levels of vascular endothelial growth factor (VEGF) but also express VEGF receptors (VEGFR), supporting the existence of an autocrine loop. The direct impact on glioma cells metabolism of drugs targeting the VEGF pathway, such as Bevacizumab (Bev) or VEGFR Tyrosine Kinase Inhibitor (TKI), is poorly known. MATERIAL AND METHODS: U87 cells were treated with Bev or SU1498, a selective VEGFR2 TKI. VEGFR expression was checked with FACS flow cytometry and Quantitative Real-Time PCR. VEGF secretion into the medium was assessed with an ELISA kit. Metabolomic studies on cells were performed using High Resolution Magic Angle Spinning Spectroscopy (HR-MAS). RESULTS: U87 cells secreted VEGF and expressed low level of VEGFR2, but no detectable VEGFR1. Exposure to SU1498, but not Bev, significantly impacted cell proliferation and apoptosis. Metabolomic studies with HR MAS showed that Bev had no significant effect on cell metabolism, while SU1498 induced a marked increase in lipids and a decrease in glycerophosphocholine. Accordingly, accumulation of lipid droplets was seen in the cytoplasm of SU1498-treated U87 cells. CONCLUSION: Although both drugs target the VEGF pathway, only SU1498 showed a clear impact on cell proliferation, cell morphology and metabolism. Bevacizumab is thus less likely to modify glioma cells phenotype due to a direct therapeutic pressure on the VEGF autocrine loop. In patients treated with VEGFR TKI, monitoring lipids with magnetic resonance spectroscopic (MRS) might be a valuable marker to assess drug cytotoxicity.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Glioma/tratamento farmacológico , Glioma/metabolismo , Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Apoptose/efeitos dos fármacos , Bevacizumab , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Cinamatos/farmacologia , Cinamatos/uso terapêutico , Glioma/patologia , Humanos , Análise dos Mínimos Quadrados , Ácido Linoleico/metabolismo , Lipídeos/análise , Espectroscopia de Ressonância Magnética , Análise de Componente Principal , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
8.
Oncoimmunology ; 1(3): 326-333, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22737608

RESUMO

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) have emerged as a dominant T cell population inhibiting anti-tumor effector T cells. Initial strategies used for Treg-depletion (cyclophosphamide, anti-CD25 mAb…) also targeted activated T cells, as they share many phenotypic markers. Current, ameliorated approaches to inhibit Treg aim to either block their function or their migration to lymph nodes and the tumor microenvironment. Various drugs originally developed for other therapeutic indications (anti-angiogenic molecules, tyrosine kinase inhibitors,etc) have recently been discovered to inhibit Treg. These approaches are expected to be rapidly translated to clinical applications for therapeutic use in combination with immunomodulators.

9.
Nucleic Acid Ther ; 21(4): 231-40, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21787231

RESUMO

Oligonucleotides containing CpG motifs (cytosine-phosphate-guanosine oligodeoxynucleotide [CpG ODN]) display strong immunostimulatory effects, and polycations have been previously reported as cellular delivery system. In the present study, we investigated the adjuvant properties of combinations of a CpG ODN with various polycations (poly-arginine, poly-lysine, poly-histidine, or chitosan) in an ovalbumin vaccination model. We showed that, when combined to CpG ODN, poly-arginine and poly-histidine, but not poly-lysine or chitosan, enhanced efficiently both the IgG antibody production and the number of splenocytes secreting interferon-gamma after stimulation with a CD8+ T cell-restricted peptide. Interestingly, CpG ODN-poly-arginine, which was the most efficient, compared favorably to the complete Freund's adjuvant and aluminium salts and induced no local toxicity, making this combination a very attractive adjuvant for vaccines.


Assuntos
Adjuvantes Imunológicos/farmacologia , Soros Imunes/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Ovalbumina/imunologia , Poliaminas/farmacologia , Vacinação/métodos , Compostos de Alúmen/farmacologia , Animais , Células Cultivadas , Quitosana/farmacologia , Estabilidade de Medicamentos , Ensaio de Desvio de Mobilidade Eletroforética , Adjuvante de Freund/farmacologia , Histidina/farmacologia , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Tamanho da Partícula , Peptídeos/farmacologia , Polieletrólitos , Polilisina/farmacologia
10.
Cancer Immunol Immunother ; 58(10): 1627-34, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19221744

RESUMO

BACKGROUND: CD4+CD25+ regulatory T cells (Treg), which constitute about 2-3% of CD4+ human T cells, are the main contributors to the maintenance of immune tolerance. Cancer patients, including glioblastoma patients, bear increased number of circulating and tumor infiltrating Treg that exert functional inhibition on tumor-specific T cells. Temozolomide (TMZ) is one of the most effective chemotherapeutic agents in glioblastoma (GBM). Lymphopenia is a common side effect of TMZ treatment, but to what extent the Treg compartment is affected by this chemotherapy has been poorly investigated. We therefore studied the impact of various TMZ regimens on Treg cell population in a TMZ-resistant rat model of glioma. METHODS: RG2 glioma cells were implanted s.c. in Fischer rats. Twelve days after tumor implantation, TMZ was administered orally with schedules designed to mimic the TMZ regimens currently used in humans: 30 mg/kg per day for 5 days, or 10 mg/kg per day for 21 days. In addition, two metronomic regimens with low-dose TMZ (2 and 0.5 mg/kg per day for 21 days) were evaluated. Splenocytes and tumor infiltrating lymphocytes were analysed by flow cytometry using CD3, CD4, CD25, and Foxp3 mAbs. Statistical significance was determined by the Mann-Whitney U test, the Student's t test or the ANOVA test. RESULTS: In the spleen of tumor-bearing animals, low-dose TMZ metronomic regimens (0.5 and 2 mg/kg for 21 days) induced a significant decrease of Treg/CD4+ ratios (13 +/- 2; p < 0.01, 14 +/- 3; p < 0.05, respectively, vs. 19 +/- 5 for controls). On the contrary, high-dose TMZ regimen (10 mg/kg per day for 21 days or 30 mg/kg for 5 days) did not significantly modify the percentage of Treg/CD4+. Within tumors, treatment with the 0.5 mg/kg TMZ regimen induced a slight and nearly significant decrease in the percentage of Treg/CD4+ after a 2 to 3-week treatment (24 +/- 9 vs. 35 +/- 11; p = 0.06). Treg depletion induced by the low-dose metronomic TMZ regimen was accompanied by a decreased suppressive function of the remaining Treg cells as assessed by an in vitro functional test. Treatment with 0.5 mg/kg metronomic TMZ reduced tumor progression when compared to untreated animals but the effect did not reach statistical significance, indicating that Treg depletion alone is not sufficient to significantly impact tumor growth in our model of fully established tumor. CONCLUSIONS: A low-dose metronomic TMZ regimen, but not a standard TMZ regimen, reduced the number of circulating Tregs. These results can have clinical applications for immunotherapeutic approaches in GBM.


Assuntos
Antineoplásicos Alquilantes/administração & dosagem , Neoplasias Encefálicas/imunologia , Dacarbazina/análogos & derivados , Glioma/imunologia , Depleção Linfocítica , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Neoplasias Encefálicas/tratamento farmacológico , Dacarbazina/administração & dosagem , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Glioma/tratamento farmacológico , Linfócitos do Interstício Tumoral/imunologia , Ratos , Ratos Endogâmicos F344 , Baço/imunologia , Baço/metabolismo , Baço/patologia , Temozolomida , Células Tumorais Cultivadas
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