Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 305
Filtrar
1.
Nat Commun ; 12(1): 440, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33469052

RESUMO

The main challenges for programmed cell death 1(PD-1)/PD-1 ligand (PD-L1) checkpoint blockade lie in a lack of sufficient T cell infiltration, tumor immunosuppressive microenvironment, and the inadequate tumor accumulation and penetration of anti-PD-1/PD-L1 antibody. Resetting tumor-associated macrophages (TAMs) is a promising strategy to enhance T-cell antitumor immunity and ameliorate tumor immunosuppression. Here, mannose-modified macrophage-derived microparticles (Man-MPs) loading metformin (Met@Man-MPs) are developed to efficiently target to M2-like TAMs to repolarize into M1-like phenotype. Met@Man-MPs-reset TAMs remodel the tumor immune microenvironment by increasing the recruitment of CD8+ T cells into tumor tissues and decreasing immunosuppressive infiltration of myeloid-derived suppressor cells and regulatory T cells. More importantly, the collagen-degrading capacity of Man-MPs contributes to the infiltration of CD8+ T cells into tumor interiors and enhances tumor accumulation and penetration of anti-PD-1 antibody. These unique features of Met@Man-MPs contribute to boost anti-PD-1 antibody therapy, improving anticancer efficacy and long-term memory immunity after combination treatment. Our results support Met@Man-MPs as a potential drug to improve tumor resistance to anti-PD-1 therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Micropartículas Derivadas de Células/imunologia , Portadores de Fármacos/farmacologia , Neoplasias/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , /uso terapêutico , Memória Imunológica , Masculino , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Células RAW 264.7 , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , /imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Nat Commun ; 11(1): 4909, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32999291

RESUMO

Effectively activating macrophages against cancer is promising but challenging. In particular, cancer cells express CD47, a 'don't eat me' signal that interacts with signal regulatory protein alpha (SIRPα) on macrophages to prevent phagocytosis. Also, cancer cells secrete stimulating factors, which polarize tumor-associated macrophages from an antitumor M1 phenotype to a tumorigenic M2 phenotype. Here, we report that hybrid cell membrane nanovesicles (known as hNVs) displaying SIRPα variants with significantly increased affinity to CD47 and containing M2-to-M1 repolarization signals can disable both mechanisms. The hNVs block CD47-SIRPα signaling axis while promoting M2-to-M1 repolarization within tumor microenvironment, significantly preventing both local recurrence and distant metastasis in malignant melanoma models. Furthermore, by loading a stimulator of interferon genes (STING) agonist, hNVs lead to potent tumor inhibition in a poorly immunogenic triple negative breast cancer model. hNVs are safe, stable, drug loadable, and suitable for genetic editing. These properties, combined with the capabilities inherited from source cells, make hNVs an attractive immunotherapy.


Assuntos
Micropartículas Derivadas de Células/imunologia , Imunoterapia/métodos , Macrófagos/imunologia , Melanoma/terapia , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias de Mama Triplo Negativas/terapia , Animais , Antígeno CD47/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Melanoma/imunologia , Melanoma/secundário , Proteínas de Membrana/agonistas , Proteínas de Membrana/imunologia , Camundongos , Nanopartículas/administração & dosagem , Recidiva Local de Neoplasia/imunologia , Nucleotídeos Cíclicos/administração & dosagem , Receptores Imunológicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Evasão Tumoral/efeitos dos fármacos , Evasão Tumoral/imunologia , Microambiente Tumoral/imunologia
3.
Life Sci ; 261: 118357, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32861794

RESUMO

In our previous study, T lymphocyte microparticles (TLMPs) originated from CEM T lymphoblast-like cell line induced enhanced production of inflammation-associated cytokines and apoptosis in human bronchial epithelial cells (HBEs). To measure TLMP subpopulations in bronchoalveolar lavage fluids (BALF) from patients with chronic obstructive pulmonary disease (COPD), and to explore the effects of MPs derived from different T cell subpopulations on airway epithelium, this study was conducted. A hospital-based case-control study including 47 COPD patients and 28 healthy volunteers was performed. The cellular origins of MPs from airway in COPD and controls were evaluated using flow cytometry. CD4+ or CD8+ TLMPs were isolated by MACS to investigate their effects on HBEs in vitro. The numbers of MPs derived from T lymphocytes in BALF as well as these subpopulations (CD4+ and CD8+ T lymphocytes) were significantly upregulated in COPD patients compared with healthy volunteers. However, there was no significant difference between stable COPD and patients with acute exacerbation. Additionally, significant correlation between CD4+ and CD8+ TLMPs was observed, however neither type nor total level of TLMPs was correlated with any base parameter. Furthermore, isolated CD4+ and CD8+ TLMPs reduced cell viability and induced significant production of inflammatory cytokines including interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, MCP-2, matrix metallopeptidase (MMP)-9 and tumor necrosis factor-alpha (TNF-α) in HBEs, while the levels of anti-inflammatory cytokine IL-10 were decreased. TLMPs in the airways, as putative biomarkers, may lead to airway epithelial injury and inflammation and serve essential roles in the pathophysiology of COPD.


Assuntos
Micropartículas Derivadas de Células/imunologia , Epitélio/patologia , Pulmão/patologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Linfócitos T/imunologia , Idoso , Brônquios/patologia , Líquido da Lavagem Broncoalveolar , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Estudos de Casos e Controles , Comunicação Celular , Citocinas/biossíntese , Progressão da Doença , Células Epiteliais/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
4.
Sci Rep ; 10(1): 7396, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32355188

RESUMO

A vaccine based on outer membrane vesicles of pertussis (omvPV) is protective in a mouse-challenge model and induces a broad antibody and mixed Th1/Th2/Th17 response against multiple antigens following subcutaneous immunization. However, this route did not result in mucosal immunity and did not prevent nasopharyngeal colonization. In this study, we explored the potential of intranasal immunization with omvPV. Only intranasal immunization induced strong mucosal immune responses that encompasses enhanced pulmonary and nasal IgA antibody levels, mainly directed against Vag8 and LPS. Furthermore, high numbers of IgA- and IgG-producing plasma cells were detected as well as lung-resident IgA memory B-cells. Finally, only intranasal immunization induced pulmonary Th1/Th17-related cytokine responses. The magnitude and type of systemic immunity was comparable between both routes and included high systemic IgG antibody levels, strong IgG-producing plasma cell responses, memory B-cells residing in the spleen and systemic Th1/Th2/Th17-related cytokine responses. Importantly, only intranasal immunization prevented colonization in both the lungs and the nasal cavity. In conclusion, intranasal omvPV immunization induces mucosal IgA and Th17-mediated responses without influencing the systemic immunity profile. These responses resulted in prevention of Bordetella pertussis colonization in the respiratory tract, including the nasal cavity, thereby potentially preventing transmission.


Assuntos
Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Micropartículas Derivadas de Células/imunologia , Imunidade nas Mucosas , Imunoglobulina A/imunologia , Vacina contra Coqueluche/imunologia , Células Th17/imunologia , Coqueluche/prevenção & controle , Administração Intranasal , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Feminino , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologia , Células Th1/patologia , Células Th17/patologia , Coqueluche/imunologia , Coqueluche/patologia
5.
Sci Rep ; 10(1): 92, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919420

RESUMO

Melanoma patients' plasma contains exosomes produced by malignant and normal cells. Plasma exosomes were isolated and separated by immunocapture into two fractions: melanoma cell-derived exosomes (MTEX) and normal cell-derived exosomes (non-MTEX). Immunosuppressive effects of MTEX on primary human immune cells were evaluated. Exosomes were isolated from plasma of 12 melanoma patients and six healthy donors (HDs). Expression levels of 19 immunoregulatory proteins in MTEX, non-MTEX and HDs exosomes were evaluated by on-bead flow cytometry. Functional/phenotypic changes induced in CD8+ T or natural killer (NK) cells by MTEX or non-MTEX were compared. Plasma protein levels were higher in patients than HDs (P < 0.0009). In patients, MTEX accounted for 23-66% of total exosomes. MTEX were enriched in immunosuppressive proteins (P = 0.03). MTEX, but not HDs exosomes, inhibited CD69 expression (P ≤ 0.0008), induced apoptosis (P ≤ 0.0009) and suppressed proliferation (P ≤ 0.002) in CD8+ T cells and downregulated NKG2D expression in NK cells (P = 0.001). Non-MTEX were enriched in immunostimulatory proteins (P = 0.002) and were only weakly immunosuppressive. Elevated MTEX/total exosome ratios and, surprisingly, non-MTEX ability to induce apoptosis of CD8+ T cells emerged as positive correlates of disease stage. MTEX emerge as the major mechanism of tumor-induced immune suppression and as an underestimated barrier to successful melanoma immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Micropartículas Derivadas de Células/imunologia , Exossomos/patologia , Tolerância Imunológica/imunologia , Melanoma/imunologia , Estudos de Casos e Controles , Exossomos/genética , Exossomos/imunologia , Humanos , Células Matadoras Naturais/imunologia , Melanoma/genética , Melanoma/patologia
6.
Exp Cell Res ; 386(2): 111737, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31759058

RESUMO

The presence of elevated T lymphocytic microparticles (TLMPs) during respiratory illness is associated with airway and lung inflammation and epithelial injuries. Although inflammasome and IL-1ß signaling are crucial in airway inflammation, little was known about their regulatory mechanism. We hypothesized that TLMPs trigger inflammasome activation and IL-1ß production in bronchial and alveolar epithelial cells to induce airway and lung inflammation. In this study, TLMPs induced IL-1ß and IL-18 secretion through NLRP3 inflammasome activation and upregulated TLR4 mRNA and protein expression in alveolar (A549) and human airway epithelial (16HBE) cells. Pretreatment with CLI-095, a specific inhibitor of TLR4 signaling, dramatically diminished the TLMP-induced release of IL-1ß and IL-18 by inhibiting the formation of NLRP3/ASC/pro-caspase-1 inflammasome in a dose-dependent manner. The TLMP-induced autophagy inhibition in epithelial cells was dependent on the PI3K/Akt signaling pathway, which significantly increased NLRP3 expression and enhanced TLMP-induced inflammation. TLR4, IL-1ß, and IL-18 proteins harbored in TLMPs were nonessential for the pro-inflammatory effect. In conclusion, TLMPs induce bronchial and alveolar epithelial cell secretion of IL-1ß and IL-18 cytokines by activating the TLR4 and PI3K/Akt signaling pathways and inhibiting autophagy. These effects lead to NLRP3 inflammasome formation and accumulation. TLMPs may be regarded as deleterious markers of airway and lung damage in respiratory diseases.


Assuntos
Micropartículas Derivadas de Células/imunologia , Meios de Cultivo Condicionados/farmacologia , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Células A549 , Anti-Inflamatórios/farmacologia , Brônquios/citologia , Brônquios/imunologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Caspase 1/genética , Caspase 1/imunologia , Linhagem Celular , Micropartículas Derivadas de Células/química , Meios de Cultivo Condicionados/química , Dactinomicina/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Regulação da Expressão Gênica , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Inflamação , Interleucina-18/genética , Interleucina-18/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Sulfonamidas/farmacologia , Receptor 4 Toll-Like/imunologia
7.
Platelets ; 31(3): 322-328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31280643

RESUMO

Immune thrombocytopenia (ITP) patients have thrombocytopenia and increased bleeding risk, but, conversely, they also have increased thrombotic risk which appears to be exacerbated by thrombopoietin-receptor agonist (TPO-RA)-treatment. Microvesicles (MVs) released from activated/apoptotic cells are prothrombotic due to exposure of phosphatidylserine (PS) and tissue factor (TF). MVs are increased in ITP patients, but their prothrombotic effect, before and during treatment with TPO-RAs, is unclear.We studied the effect of TPO-RAs on the procoagulant activity of MVs in 11 ITP patients, before, and two and six weeks after initiation of treatment, and in 15 healthy controls. MV-associated PS-activity, TF-activity and the capacity of isolated MVs and plasma to generate thrombin in a phospholipid-dependent manner were measured.Before treatment with TPO-RAs, prothrombotic markers in ITP patients were comparable to levels found in healthy controls. After both two and six weeks of TPO-RA-treatment, ITP patients had higher MV-associated PS-activity and phospholipid-dependent thrombin generation in plasma than controls. In addition, ITP patients had increased phospholipid-dependent MV-associated thrombin generation two weeks after initiation of TPO-RA-treatment compared with controls and pre-treatment levels. MV-associated TF-activity was low in controls and in ITP patients before and after initiation of TPO-RA-treatment.In conclusion, TPO-RAs increase phospholipid-dependent MV-associated thrombin generation in ITP patients. This could contribute to or exacerbate a pre-existing hypercoagulable state. Phospholipid-dependent thrombin generation generated by isolated MVs, or measured directly in plasma, may be potential tools that could help in the risk-assessment of future thromboembolic events in ITP patients, both before and after initiation of TPO-RA-treatment.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Púrpura Trombocitopênica Idiopática/etiologia , Púrpura Trombocitopênica Idiopática/metabolismo , Trombina/biossíntese , Biomarcadores , Estudos de Casos e Controles , Micropartículas Derivadas de Células/imunologia , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , Humanos , Imunoglobulinas Intravenosas , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/diagnóstico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Receptores de Trombopoetina/agonistas , Trombopoetina/farmacologia , Trombopoetina/uso terapêutico
8.
Oncogene ; 39(1): 187-203, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31467431

RESUMO

Radiotherapy induces immune-related responses in cancer patients by various mechanisms. Here, we investigate the immunomodulatory role of tumor-derived microparticles (TMPs)-extracellular vesicles shed from tumor cells-following radiotherapy. We demonstrate that breast carcinoma cells exposed to radiation shed TMPs containing elevated levels of immune-modulating proteins, one of which is programmed death-ligand 1 (PD-L1). These TMPs inhibit cytotoxic T lymphocyte (CTL) activity both in vitro and in vivo, and thus promote tumor growth. Evidently, adoptive transfer of CTLs pre-cultured with TMPs from irradiated breast carcinoma cells increases tumor growth rates in mice recipients in comparison with control mice receiving CTLs pre-cultured with TMPs from untreated tumor cells. In addition, blocking the PD-1-PD-L1 axis, either genetically or pharmacologically, partially alleviates TMP-mediated inhibition of CTL activity, suggesting that the immunomodulatory effects of TMPs in response to radiotherapy is mediated, in part, by PD-L1. Overall, our findings provide mechanistic insights into the tumor immune surveillance state in response to radiotherapy and suggest a therapeutic synergy between radiotherapy and immune checkpoint inhibitors.


Assuntos
Antígeno B7-H1/genética , Neoplasias da Mama/radioterapia , Micropartículas Derivadas de Células/imunologia , Imunomodulação/imunologia , Animais , Antígeno B7-H1/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/genética , Micropartículas Derivadas de Células/efeitos da radiação , Feminino , Xenoenxertos , Humanos , Evasão da Resposta Imune/imunologia , Evasão da Resposta Imune/efeitos da radiação , Imunomodulação/efeitos da radiação , Camundongos , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais/efeitos da radiação , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos da radiação
9.
Int J Mol Sci ; 20(21)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31683793

RESUMO

Rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), and psoriatic arthritis (PsA) make up a group of chronic immune-mediated inflammatory diseases (IMIDs). The course of these diseases involves chronic inflammation of joints and enthesopathies, which can result in joint damage and disability. Microparticles (MPs) are a group of small spherical membranous vesicles. The structure and cellular origin of MPs, mechanisms that stimulate their secretion and the place of their production, determine their biological properties, which could become manifest in the pathogenesis of immune-mediated inflammatory diseases. Microparticles can stimulate synovitis with proinflammatory cytokines and chemokines. MPs may also contribute to the pathogenesis of rheumatic diseases by the formation of immune complexes and complement activation, pro-coagulation activity, activation of vascular endothelium cells, and stimulation of metalloproteinase production. It seems that in the future, microparticles can become a modern marker of disease activity, a response to treatment, and, possibly, they can be used in the prognosis of the course of arthritis. The knowledge of the complexity of MPs biology remains incomplete and it requires further comprehensive studies to explain how they affect the development of rheumatic diseases. This review focuses on the immunopathogenic and therapeutic role of MPs in chronic immune-mediated inflammatory joint diseases.


Assuntos
Micropartículas Derivadas de Células/imunologia , Entesopatia/imunologia , Inflamação/imunologia , Artropatias/imunologia , Artrite Juvenil/imunologia , Artrite Juvenil/metabolismo , Artrite Psoriásica/imunologia , Artrite Psoriásica/metabolismo , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Micropartículas Derivadas de Células/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Entesopatia/metabolismo , Humanos , Inflamação/metabolismo , Artropatias/metabolismo , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/metabolismo
10.
Front Immunol ; 10: 2391, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31681284

RESUMO

Background: Microvesicles (MVs) expressing the type 1 interferon (IFN)-inducible protein galectin-3 binding protein (G3BP) may play a pathogenic role in systemic lupus erythematosus (SLE). Co-expression of double-stranded DNA (dsDNA) on such MVs may render them immunogenic and targets for anti-dsDNA antibodies. Little is known about the mechanisms underlying generation of this MV population. In this study, we investigated how Toll-like receptors (TLRs), IFN-α, and T cells are involved in this process in healthy subjects. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from 12 healthy donors were stimulated in-vitro for 24 h with a series of TLR-agonists or the T cell activating antibody OKT3 or were subjected to apoptosis by incubation with staurosporine. MVs in the supernatants were subsequently isolated by differential centrifugation and were quantified and characterized with respect to expression of G3BP and dsDNA by flow cytometry. Results: Stimulation of PBMCs with the TLR9-agonist and strong IFN-α inducer ODN2395 significantly increased the release of MVs expressing G3BP. The production of MVs with this phenotype was markedly enhanced by co-stimulation of T cells. Furthermore, dependency on IFN-α in the generation of G3BP-expressing MVs was indicated by a marked reduction following addition of the IFN-α inhibitor IFN alpha-IFNAR-IN-1 hydrochloride. Conclusion: Release of G3BP-expressing MVs from healthy donor PBMCs is induced by stimulation of TLR9 in an IFN-α-dependent manner and is enhanced by co-stimulation of T cells.


Assuntos
Micropartículas Derivadas de Células/imunologia , DNA/imunologia , Interferon-alfa/imunologia , Leucócitos Mononucleares/imunologia , Receptor Toll-Like 9/imunologia , Adulto , Antígenos de Neoplasias , Biomarcadores Tumorais , Feminino , Humanos , Masculino , Muromonab-CD3/farmacologia , Receptor de Interferon alfa e beta/imunologia
11.
J Cell Sci ; 132(20)2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615844

RESUMO

Extracellular vesicles (EVs) are a heterogeneous collection of membrane-bound vesicles released by cells that contain bioactive cargoes including proteins, lipids and nucleic acids. Multiple subpopulations of EVs have now been recognized and these include exosomes and microvesicles. EVs have been thought to facilitate intercellular and distal communication to bring about various processes that enable tumor progression and metastases. Here, we describe the current knowledge of the functional cargo contained within EVs, with a focus on tumor microvesicles, and review the emerging theory of how EVs support immune suppression in cancer.


Assuntos
Comunicação Celular/imunologia , Micropartículas Derivadas de Células/imunologia , Vesículas Extracelulares/imunologia , Neoplasias/imunologia , Animais , Micropartículas Derivadas de Células/patologia , Vesículas Extracelulares/patologia , Humanos , Metástase Neoplásica , Neoplasias/patologia
12.
Vaccine ; 37(41): 6085-6092, 2019 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-31477437

RESUMO

Prostate Cancer specific immunotherapy in combination with immune stimulating adjuvants may serve as a viable strategy for facilitating tumor regression and preventing recurrence. In this study, an oral microparticulate vaccine encapsulating tumor associated antigens (TAA) extracted from a murine prostate cancer cell line, TRAMP-C2, was formulated with the help of a spray dryer. Microparticles were characterized in vitro to determine their physicochemical properties and antigenicity. Formulated microparticles had an average size of 4.92 ±â€¯0.5 µm with a zeta potential of 7.92 ±â€¯1.2 mV. In order to test our formulation for its ability to demonstrate adequate antigen presentation and co-stimulation, microparticles were tested in vitro on murine dendritic cells. In vitro biological characterization demonstrated the activation of specific immune system markers such as CD80/86, CD40, MHC-I and MHC-II. Following in vitro characterization, in vivo anti-tumor efficacy of the oral microparticulate vaccine was evaluated in C57BL/6 male mice. Combination therapy of vaccine microparticles with cyclophosphamide and granulocyte macrophage-colony stimulating factor (GM-CSF) demonstrated a five-fold reduction in tumor volume as compared to non-vaccinated mice. At the cellular level, cyclophosphamide and GM-CSF augmented the vaccine response as indicated by the reduced tumor volume and significant elevation of cytotoxic T-cell (CTL) CD8+ and (T-helper) CD4+ T-cells compared to mice receiving vaccine microparticles alone. Furthermore, our studies indicate a significant reduction in T-regulatory cells (T-regs) in mice receiving vaccine along with GM-CSF and cyclophosphamide, one of the immune escape mechanisms linked to tumor growth and progression. Thus, oral microparticulate vaccines have the potential to trigger a robust anti-tumor cellular response, and in combination with clinically relevant agents, significantly resist tumor growth and progression.


Assuntos
Vacinas Anticâncer/imunologia , Micropartículas Derivadas de Células/imunologia , Imunoterapia/métodos , Neoplasias da Próstata/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Apresentação do Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Contagem de Linfócito CD4 , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Ciclofosfamida/uso terapêutico , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia
13.
Methods Mol Biol ; 2032: 203-211, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31522421

RESUMO

Flow-cytometric detection of circulating endothelial cells and endothelial microparticles is an essential tool in studies of vascular diseases. Here we describe the principles and detailed methods for human blood sample processing, storage, labeling, and gating of circulating endothelial elements.


Assuntos
Células Endoteliais/imunologia , Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Doenças Vasculares/diagnóstico , Plaquetas/imunologia , Contagem de Células , Micropartículas Derivadas de Células/imunologia , Humanos , Doenças Vasculares/imunologia
14.
Pathog Dis ; 77(5)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31504509

RESUMO

As the trend of antibiotic resistance has increased, prevention and treatment of Helicobacter pylori infection have been challenged by the fact that no vaccines preventing H. pylori infection are available. Scientists continue to make sustained efforts to find better vaccine formulations and adjuvants to eradicate this chronic infection. In this study, we systemically analyzed the protein composition and potential vaccine function of outer-membrane vesicles (OMVs) derived from gerbil-adapted H. pylori strain 7.13. In total, we identified 169 proteins in H. pylori OMVs and found that outer-membrane, periplasmic and extracellular proteins (48.9% of the total proteins) were enriched. Furthermore, we evaluated the immune protective response of H. pylori OMVs in a C57BL/6 mouse model, and mice were orally immunized with OMVs or the H. pylori whole cell vaccine (WCV) alone, with or without cholera toxin (CT) as an adjuvant. The data demonstrated that oral immunization with OMVs can elicit a strong humoral and significantly higher mucosal immune response than the group immunized with the WCV plus the CT adjuvant. Moreover, our results also confirmed that OMVs predominantly induced T helper 2 (Th2)-biased immune responses that can significantly reduce bacterial loads after challenging with the H. pylori Sydney Strain 1 (SS1). In summary, OMVs as new antigen candidates in vaccine design would be of great value in controlling H. pylori infection.


Assuntos
Antígenos de Bactérias/administração & dosagem , Micropartículas Derivadas de Células/imunologia , Infecções por Helicobacter/terapia , Helicobacter pylori/imunologia , Imunoterapia/métodos , Células Th2/imunologia , Administração Oral , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Modelos Animais de Doenças , Imunidade Humoral , Imunidade nas Mucosas , Camundongos , Camundongos Endogâmicos C57BL , Resultado do Tratamento
15.
Sci Rep ; 9(1): 11631, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31406171

RESUMO

Tiotropium is a muscarinic antagonist that reduces the risk of acute exacerbations of chronic obstructive pulmonary disease, possibly through an as yet incompletely characterized anti-inflammatory activity. We hypothesized that muscarinic activation of bronchial epithelial cells and endothelial cells causes the release of proinflammatory microparticles and that tiotropium inhibits the phenomenon. Microparticle generation was assessed by a functional assay, by flow cytometry and by NanoSight technology. Immortalized bronchial epithelial cells (16HBE) and umbilical vein endothelial cells were treated with acetylcholine in the presence of varying concentrations of tiotropium. Intracellular calcium concentration, extracellular regulated kinase phosphorylation and chemokine content in the conditioned media were assessed by commercial kits. Acetylcholine causes microparticle generation that is completely inhibited by tiotropium (50 pM). Microparticles generated by acetylcholine-stimulated cells increase the synthesis of proinflammatory mediators in an autocrine fashion. Acetylcholine-induced upregulation of microparticle generation is inhibited by an inhibitor of extracellular regulated kinase phosphorylation and by a phospholipase C inhibitor. Tiotropium blocks both extracellular regulated kinase phosphorylation and calcium mobilization, consistent with the hypothesis that the drug prevents microparticle generation through inhibition of these critical pathways. These results might contribute to explain the effect of tiotropium in reducing acute exacerbations of chronic obstructive pulmonary disease.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Antagonistas Muscarínicos/farmacologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Brometo de Tiotrópio/farmacologia , Brônquios/citologia , Brônquios/imunologia , Cálcio/metabolismo , Linhagem Celular , Micropartículas Derivadas de Células/imunologia , Quimiocinas/imunologia , Quimiocinas/metabolismo , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Antagonistas Muscarínicos/uso terapêutico , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Brometo de Tiotrópio/uso terapêutico
16.
Front Immunol ; 10: 1753, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31417549

RESUMO

Communication between cells is essential for multicellular life. During cognate immune interactions, T cells communicate with antigen-presenting cells (APC) via direct cell-cell contact or the release of molecules and vesicles containing T cell messages. A wide variety of mechanisms have been reported and among them a process called "trogocytosis" has traditionally been thought to be the fastest way to directly transfer membrane portions containing intact proteins from one cell to another; however, the mechanism is unverified. Trogocytosis has been distinguished from the generation of extracellular vesicles (EVs), a term that encompasses exosomes and microvesicles, as EVs are released via a contact-independent manner and are suggested to potentially send molecular messages over a distance. However, some previous reports regarding EVs in T cells may be misleading in terms of explaining their cellular origins. In addition, there is little evidence on how EVs are generated from T cells in vivo and function to regulate complex immune responses. A recent work demonstrated that T cell microvilli-thin and finger-like membrane protrusions-are highly fragile and easily separated as membrane particles by trogocytosis, forming a new class of EVs. Surprisingly, released T cell microvilli-derived particles act as vectors, transmitting T cell messages to cognate APCs. This review focuses on how T cell microvilli vesicles are connected with immune regulation mechanisms discovered previously.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Comunicação Celular/imunologia , Micropartículas Derivadas de Células/imunologia , Exossomos/imunologia , Microvilosidades/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/citologia , Humanos , Linfócitos T/citologia
17.
Thorax ; 74(9): 865-874, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31331947

RESUMO

BACKGROUND: Uncontrolled lung inflammation is one of the prominent features in the pathogenesis of lung infection- associated acute lung injury (ALI). Microvesicles (MVs) are extracellular nanovesicles that are generated via direct membrane budding. METHODS: Bronchoalveolar lavage fluid (BALF) samples were collected from mice with or without intratracheal lipopolysaccharide (LPS) instillation. BALF MVs were characterised and MV-containing microRNA (miRNA) profiles were assessed and confirmed. Secretion and function of MV-containing miR-223/142 (MV-miR-223/142) were analysed in vivo. RESULTS: In BALF, MVs are mainly derived from macrophages in response to LPS. After intratracheal instillation (i.t.) of LPS or Klebsiella pneumoniae, MV-containing miR-223/142 are dramatically induced in both BALF and serum. Mechanistically, miRNA 3' end uridylation mediates the packing of miR-223/142 into MVs. To investigate the functional role of MV-miR-223/142, we loaded miR-223/142 mimics into unstimulated MVs and delivered them into the murine lungs via i.t. The miR-223/142 mimics-enriched MVs selectively targeted lung macrophages and suppressed the inflammatory lung responses that were triggered by LPS or K. pneumoniae. Mechanistically, miR-223 and miR-142 synergistically suppress Nlrp3 inflammasome activation in macrophages via inhibition of Nlrp3 and Asc, respectively. CONCLUSIONS: In the pathogenesis of lung macrophage-mediated inflammatory responses, MV-miR-223/142 secretion is robustly enhanced and detectable in BALF and serum. Furthermore, restoration of intracellular miR-223/142 via vesicle-mediated delivery suppresses macrophage activation and lung inflammation via inhibition of Nlrp3 inflammasome activation.


Assuntos
Micropartículas Derivadas de Células/metabolismo , MicroRNAs/metabolismo , Pneumonia/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Caspase 1/imunologia , Micropartículas Derivadas de Células/imunologia , Modelos Animais de Doenças , Inflamassomos/imunologia , Klebsiella pneumoniae , Lipopolissacarídeos , Ativação de Macrófagos , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/imunologia , Pneumonia/imunologia
18.
Proc Natl Acad Sci U S A ; 116(29): 14714-14723, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31262819

RESUMO

Conventional dendritic cells (cDCs) derive from bone marrow (BM) precursors that undergo cascades of developmental programs to terminally differentiate in peripheral tissues. Pre-cDC1s and pre-cDC2s commit in the BM to each differentiate into CD8α+/CD103+ cDC1s and CD11b+ cDC2s, respectively. Although both cDCs rely on the cytokine FLT3L during development, mechanisms that ensure cDC accessibility to FLT3L have yet to be elucidated. Here, we generated mice that lacked a disintegrin and metalloproteinase (ADAM) 10 in DCs (Itgax-cre × Adam10-fl/fl; ADAM10∆DC) and found that ADAM10 deletion markedly impacted splenic cDC2 development. Pre-cDC2s accumulated in the spleen with transcriptomic alterations that reflected their inability to differentiate and exhibited abrupt failure to survive as terminally differentiated cDC2s. Induced ADAM10 ablation also led to the reduction of terminally differentiated cDC2s, and restoration of Notch signaling, a major pathway downstream of ADAM10, only modestly rescued them. ADAM10∆DC BM failed to generate cDC2s in BM chimeric mice with or without cotransferred ADAM10-sufficient BM, indicating that cDC2 development required cell-autonomous ADAM10. We determined cDC2s to be sources of soluble FLT3L, as supported by decreased serum FLT3L concentration and the retention of membrane-bound FLT3L on cDC2 surfaces in ADAM10∆DC mice, and by demonstrating the release of soluble FLT3L by cDC2 in ex vivo culture supernatants. Through in vitro studies utilizing murine embryonic fibroblasts, we determined FLT3L to be a substrate for ADAM10. These data collectively reveal cDC2s as FLT3L sources and highlight a cell-autonomous mechanism that may enhance FLT3L accessibility for cDC2 development and survival.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Diferenciação Celular/imunologia , Células Dendríticas/fisiologia , Proteínas de Membrana/metabolismo , Baço/citologia , Proteína ADAM10/genética , Proteína ADAM10/imunologia , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/imunologia , Animais , Transplante de Medula Óssea , Membrana Celular/imunologia , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Fibroblastos , Células-Tronco Hematopoéticas/fisiologia , Imunidade Celular , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Baço/imunologia , Quimeras de Transplante
19.
Nat Commun ; 10(1): 2402, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160588

RESUMO

Platelet-leukocyte interactions amplify inflammatory reactions, but the underlying mechanism is still unclear. CLEC5A and CLEC2 are spleen tyrosine kinase (Syk)-coupled C-type lectin receptors, abundantly expressed by leukocytes and platelets, respectively. Whereas CLEC5A is a pattern recognition receptor (PRR) to flaviviruses and bacteria, CLEC2 is the receptor for platelet-activating snake venom aggretin. Here we show that dengue virus (DV) activates platelets via CLEC2 to release extracellular vesicles (EVs), including exosomes (EXOs) and microvesicles (MVs). DV-induced EXOs (DV-EXOs) and MVs (DV-MVs) further activate CLEC5A and TLR2 on neutrophils and macrophages, thereby induce neutrophil extracellular trap (NET) formation and proinflammatory cytokine release. Compared to  stat1-/- mice, simultaneous blockade of CLEC5A and TLR2 effectively attenuates DV-induced inflammatory response and increases survival rate from 30 to 90%. The identification of critical roles of CLEC2 and CLEC5A/TLR2 in platelet-leukocyte interactions will support the development of novel strategies to treat acute viral infection in the future.


Assuntos
Plaquetas/metabolismo , Vírus da Dengue/imunologia , Dengue/imunologia , Vesículas Extracelulares/imunologia , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Receptores de Superfície Celular/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Citocinas/imunologia , Dengue/virologia , Exossomos/imunologia , Exossomos/metabolismo , Armadilhas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Humanos , Inflamação , Lectinas Tipo C/genética , Camundongos , Camundongos Knockout , Ativação Plaquetária , Receptores de Superfície Celular/genética , Fator de Transcrição STAT1/genética , Taxa de Sobrevida
20.
Mol Ther ; 27(9): 1612-1620, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31204210

RESUMO

Glioblastoma is the most common and malignant tumor of the CNS, with a mean survival of 14 months after diagnosis. Its unfavorable prognosis reveals the need for novel therapies. It is known that radiation can induce a systemic antitumor effect. Tumor cells produce and release microvesicles in response to cell damage such as radiation. Microvesicles contain a plethora of bioactive molecules, including antigens involved in modulation of the immune response. In this study, we characterized and evaluated irradiated C6 cell-derived microvesicles as a therapeutic vaccination in C6 malignant glioma. Cultured C6 glioma cells were irradiated with a single dose of 50 Gy to obtain the microvesicles. Subcutaneous implantation of C6 cells was performed when the tumor reached 2 cm in diameter, and non-irradiated and irradiated C6 cell-derived microvesicles were administered subcutaneously. Tumor growth, apoptosis, and immunophenotypes were determined. Reduction of tumor volume (more than 50%) was observed in the group treated with irradiated C6 cell-derived microvesicles compared with the control (p = 0.03). The percentages of infiltrative helper, cytotoxic, and regulatory T lymphocytes as well as apoptotic cells were increased in tumors from immunized rats compared with controls. These findings make microvesicle-based vaccination a promising immunotherapeutic approach against glioblastoma.


Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Vacinas Anticâncer/imunologia , Micropartículas Derivadas de Células/imunologia , Glioblastoma/imunologia , Glioblastoma/terapia , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/efeitos da radiação , Modelos Animais de Doenças , Glioblastoma/mortalidade , Glioblastoma/patologia , Imunidade , Imunização , Ratos , Resultado do Tratamento , Carga Tumoral/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...