RESUMO
Aspergillus fumigatus is an opportunistic fungal pathogen responsible for a spectrum of clinical manifestations. Dendritic cells recognize pathogen-associated molecular patterns of Aspergillus via two main receptor families, Toll-like receptors (TLRs) and C-type lectin receptors (CLR). Here, the importance of TLR and CLR signaling in the regulation of T-helper cell type 2 (Th2) responses was analyzed using a mouse model based on the transfer of bone marrow-derived dendritic cells (BMDCs) pulsed with A. fumigatus conidia. BMDCs were generated from mice deficient in either MyD88 or MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1). Both the MyD88 and MALT1 signaling pathway in BMDCs contributed to the production of inflammatory cytokines induced by A. fumigatus conidia. Mice sensitized with MyD88-/- BMDCs pulsed in vitro with A. fumigatus conidia showed an exacerbated allergic inflammation, with stronger eosinophil recruitment in the BAL and higher Th2 cytokine production compared with mice sensitized with wild-type or MALT1-/- BMDCs. This exacerbation was not observed when MyD88-/- BMDCs were pulsed with Cladosporium sphaerospermum, a nonpathogenic mold. A lack of TLR2 signaling recapitulated the exacerbation of the A. fumigatus Th2 response observed in the absence of MyD88 signaling, whereas TLR2 agonist dampened the response induced with A. fumigatus and C. sphaerospermum conidia. IL-10 production by BMDCs in response to A. fumigatus was dependent on the expression of TLR2 and MyD88. IL-10-/- BMDCs exacerbated, whereas MyD88-/- BMDCs supplemented with exogenous IL-10 decreased the allergic pulmonary inflammation. These results indicate that TLR2/MyD88-specific recognition of PAMPs from A. fumigatus conidia can upregulate IL-10 production and downregulate lung eosinophilia and the development of a Th2 response.
Assuntos
Aspergillus fumigatus/imunologia , Células Dendríticas/imunologia , Inflamação/imunologia , Pulmão/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Aspergilose/imunologia , Asma/imunologia , Células Cultivadas , Cladosporium/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Lectinas Tipo C/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Eosinofilia Pulmonar/imunologia , Células Th2/imunologiaRESUMO
Agonists of Toll-like receptors (TLRs) are potent activators of the innate immune system and hold promise as vaccine adjuvant and for anticancer immunotherapy. Unfortunately, in soluble form they readily enter systemic circulation and cause systemic inflammatory toxicity. Here we demonstrate that by covalent ligation of a small-molecule imidazoquinoline-based TLR7/8 agonist to 50-nm-sized degradable polymeric nanogels the potency of the agonist to activate TLR7/8 in in vitro cultured dendritic cells is largely retained. Importantly, imidazoquinoline-ligated nanogels focused the in vivo immune activation on the draining lymph nodes while dramatically reducing systemic inflammation. Mechanistic studies revealed a prevalent passive diffusion of the nanogels to the draining lymph node. Moreover, immunization studies in mice have shown that relative to soluble TLR7/8 agonist, imidazoquinoline-ligated nanogels induce superior antibody and T-cell responses against a tuberculosis antigen. This approach opens possibilities to enhance the therapeutic benefit of small-molecule TLR agonist for a variety of applications.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Imidazóis/administração & dosagem , Nanoestruturas/administração & dosagem , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Bactérias/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Géis , Concentração de Íons de Hidrogênio , Imidazóis/química , Imidazóis/farmacologia , Imunização , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Glicoproteínas de Membrana/agonistas , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Linfócitos T/imunologia , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistasRESUMO
The differentiation of macrophages into lipid-filled foam cells is a hallmark of the lung granuloma that forms in patients with active tuberculosis (TB). Mycolic acids (MAs), the abundant lipid virulence factors in the cell wall of Mycobacterium tuberculosis (Mtb), can induce this foam phenotype possibly as a way to perturb host cell lipid homeostasis to support the infection. It is not exactly clear how MAs allow differentiation of foam cells during Mtb infection. Here we investigated how chemically synthetic MAs, each with a defined stereochemistry similar to natural Mtb-associated mycolates, influence cell foamy phenotype and mycobacterial proliferation in murine host macrophages. Using light and laser-scanning-confocal microscopy, we assessed the influence of MA structure first on the induction of granuloma cell types, second on intracellular cholesterol accumulation, and finally on mycobacterial growth. While methoxy-MAs (mMAs) effected multi-vacuolar giant cell formation, keto-MAs (kMAs) induced abundant intracellular lipid droplets that were packed with esterified cholesterol. Macrophages from mice treated with kMA were permissive to mycobacterial growth, whereas cells from mMA treatment were not. This suggests a separate yet key involvement of oxygenated MAs in manipulating host cell lipid homeostasis to establish the state of TB.
Assuntos
Colesterol/metabolismo , Granuloma/tratamento farmacológico , Macrófagos/metabolismo , Ácidos Micólicos/administração & dosagem , Tuberculose/tratamento farmacológico , Animais , Diferenciação Celular/efeitos dos fármacos , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Granuloma/metabolismo , Granuloma/patologia , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/patogenicidade , Ácidos Micólicos/síntese química , Tuberculose/metabolismo , Tuberculose/microbiologia , Tuberculose/patologiaRESUMO
Mycolic acids (MAs) are highly hydrophobic long-chain α-alkyl ß-hydroxy fatty acids present in the cell wall of Mycobacterium tuberculosis (Mtb) as a complex mixture of molecules with a common general structure but with variable functional groups in the meromycolate chain. In this study, we addressed the relationship between the MA molecular structure and their contribution to the development of T-cell immune responses. Hereto, we used the model antigen ovalbumin and single synthetic MAs, differing in oxygenation class and cis versus trans proximal cyclopropane configuration, as immune stimulatory agents. Subcutaneous delivery of liposome-formulated MAs with a proximal cis cyclopropane elicited antigen-specific Th1 and cytotoxic T-cell immune responses, whereas intratracheal immunization elicited pulmonary Th17 responses. These immune stimulatory activities depended not only on the cis versus trans proximal cyclopropane configuration but also on the MA oxygenation class. Our study thus shows that both the presence and nature of the functional groups in the meromycolate chain affect the immune stimulatory adjuvant activity of Mtb mycolates and suggests that Mtb bacilli may impact on the host protective immune response by modulating the cis versus trans stereochemistry of its mycolates as well as by altering the oxygenation class of the meromycolate functional group.
Assuntos
Adjuvantes Imunológicos , Mycobacterium tuberculosis/imunologia , Ácidos Micólicos/imunologia , Tuberculose/imunologia , Animais , Citocinas/biossíntese , Feminino , Imunização , Imunoglobulina G/imunologia , Fatores Imunológicos , Imunomodulação , Injeção Intratimpânica , Injeções Subcutâneas , Interferon gama/biossíntese , Lipossomos/química , Ativação Linfocitária , Camundongos , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/administração & dosagem , Ácidos Micólicos/química , Fosfatidilcolinas/química , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/administração & dosagem , Vacinas contra a Tuberculose/imunologiaRESUMO
DNA vaccination holds great promise for the prevention and treatment of cancer and infectious diseases. However, the clinical ability of DNA vaccines is still controversial due to the limited immune response initially observed in humans. We hypothesized that electroporation of a plasmid encoding the HIV-1 Gag viral capsid protein would enhance cancer DNA vaccine potency. DNA electroporation used to deliver plasmids in vivo, induced type I interferons, thereby supporting the activation of innate immunity. The coadministration of ovalbumin (OVA) and HIV-1 Gag encoding plasmids modulated the adaptive immune response. This strategy favored antigen-specific Th1 immunity, delayed B16F10-OVA tumor growth and improved mouse survival in both prophylactic and therapeutic vaccination approaches. Similarly, a prophylactic DNA immunization against the melanoma-associated antigen gp100 was enhanced by the codelivery of the HIV-1 Gag plasmid. The adjuvant effect was not driven by the formation of HIV-1 Gag virus-like particles. This work highlights the ability of both electroporation and the HIV-1 Gag plasmid to stimulate innate immunity for enhancing cancer DNA vaccine immunogenicity and demonstrates interesting tracks for the design of new translational genetic adjuvants to overcome the current limitations of DNA vaccines in humans.
Assuntos
Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Plasmídeos/genética , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Animais , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Humanos , Interferon Tipo I/biossíntese , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Plasmídeos/administração & dosagem , Modelos de Riscos Proporcionais , Células Th1/imunologia , Células Th1/metabolismo , Transfecção , Resultado do Tratamento , Carga Tumoral , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/ultraestruturaRESUMO
Given their high potential to evoke cytolytic T cell responses, tumor antigen-encoding messenger RNA (mRNA) vaccines are now being intensively explored as therapeutic cancer vaccines. mRNA vaccines clearly benefit from wrapping the mRNA into nano-sized carriers such as lipoplexes that protect the mRNA from degradation and increase its uptake by dendritic cells in vivo. Nevertheless, the early innate host factors that regulate the induction of cytolytic T cells to mRNA lipoplex vaccines have remained unresolved. Here, we demonstrate that mRNA lipoplexes induce a potent type I interferon (IFN) response upon subcutaneous, intradermal and intranodal injection. Regardless of the route of immunization applied, these type I IFNs interfered with the generation of potent cytolytic T cell responses. Most importantly, blocking type I IFN signaling at the site of immunization through the use of an IFNAR blocking antibody greatly enhanced the prophylactic and therapeutic antitumor efficacy of mRNA lipoplexes in the highly aggressive B16 melanoma model. As type I IFN induction appears to be inherent to the mRNA itself rather than to unique properties of the mRNA lipoplex formulation, preventing type I IFN induction and/or IFNAR signaling at the site of immunization might constitute a widely applicable strategy to improve the potency of mRNA vaccination.
Assuntos
Vacinas Anticâncer/administração & dosagem , Interferon Tipo I/metabolismo , Melanoma Experimental/tratamento farmacológico , RNA Mensageiro/administração & dosagem , Linfócitos T Citotóxicos/metabolismo , Animais , Anticorpos/administração & dosagem , Vacinas Anticâncer/imunologia , Humanos , Injeções Intradérmicas , Injeções Subcutâneas , Lipossomos , Melanoma Experimental/imunologia , Camundongos , RNA Mensageiro/imunologia , Receptor de Interferon alfa e beta/antagonistas & inibidores , Resultado do TratamentoRESUMO
BACKGROUND: Human respiratory syncytial virus (RSV) is a frequent cause of asthma exacerbations, yet the susceptibility of asthmatic patients to RSV is poorly understood. OBJECTIVE: We sought to address the contribution of resident alveolar macrophages (rAMs) to susceptibility to RSV infection in mice that recovered from allergic airway eosinophilia. METHODS: Mice were infected with RSV virus after clearance of allergic airway inflammation (AAI). The contribution of post-AAI rAMs was studied in vivo by means of clodronate liposome-mediated depletion, adoptive transfer, and treatment with recombinant cytokines before RSV infection. RESULTS: After clearing the allergic bronchial inflammation, post-AAI mice had bronchial hyperreactivity and increased inflammatory cell influx when infected with RSV compared with nonallergic mice, whereas viral clearance was comparable in both mouse groups. Post-AAI rAMs were necessary and sufficient for mediating these proinflammatory effects. In post-AAI mice the residing CD11c(hi) autofluorescent rAM population did not upregulate the terminal differentiation marker sialic acid-binding immunoglobulin-like lectin F and overproduced TNF and IL-6 through increased nuclear factor κB nuclear translocation. In line with these results, post-AAI lungs had reduced levels of the rAM maturation cytokine GM-CSF. Intratracheal administration of GM-CSF induced final rAM maturation in post-AAI mice and prevented the increased susceptibility to RSV-induced hyperreactivity and inflammation. CONCLUSION: Defective production of GM-CSF leads to insufficient post-AAI rAM maturation in mice that recovered from an AAI, causing increased susceptibility to RSV-induced immunopathology. Promoting the differentiation of post-AAI rAMs might be a therapeutic option for preventing RSV-induced exacerbations in human asthmatic patients.
Assuntos
Asma/complicações , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Infecções por Vírus Respiratório Sincicial/complicações , Vírus Sincicial Respiratório Humano , Transferência Adotiva , Alérgenos/imunologia , Animais , Asma/imunologia , Asma/metabolismo , Asma/patologia , Asma/terapia , Líquido da Lavagem Broncoalveolar , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Imunofenotipagem , Mediadores da Inflamação/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , NF-kappa B/metabolismo , Fenótipo , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Infecções por Vírus Respiratório Sincicial/patologiaRESUMO
We report on the design of glycosylated nanogels via core-cross-linking of amphiphilic non-water-soluble block copolymers composed of an acetylated glycosylated block and a pentafluorophenyl (PFP) activated ester block prepared by reversible addition-fragmentation (RAFT) polymerization. Self-assembly, pH-sensitive core-cross-linking, and removal of remaining PFP esters and protecting groups are achieved in one pot and yield fully hydrated sub-100 nm nanogels. Using cell subsets that exhibit high and low expression of the mannose receptor (MR) under conditions that suppress active endocytosis, we show that mannosylated but not galactosylated nanogels can efficiently target the MR that is expressed on the cell surface of primary dendritic cells (DCs). These nanogels hold promise for immunological applications involving DCs and macrophage subsets.
Assuntos
Células Dendríticas/citologia , Células Dendríticas/metabolismo , Manose/química , Polietilenoglicóis/química , Polietilenoimina/química , Polímeros/química , Animais , Células Cultivadas , Concentração de Íons de Hidrogênio , Lectinas Tipo C/metabolismo , Manose/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Nanogéis , Polimerização , Receptores de Superfície Celular/metabolismoRESUMO
The induction of antigen-specific adaptive immunity exclusively occurs in lymphoid organs. As a consequence, the efficacy by which vaccines reach these tissues strongly affects the efficacy of the vaccine. Here, we report the design of polymer hydrogel nanoparticles that efficiently target multiple immune cell subsets in the draining lymph nodes. Nanoparticles are fabricated by infiltrating mesoporous silica particles (ca. 200â nm) with poly(methacrylic acid) followed by disulfide-based crosslinking and template removal. PEGylation of these nanoparticles does not affect their cellular association inâ vitro, but dramatically improves their lymphatic drainage inâ vivo. The functional relevance of these observations is further illustrated by the increased priming of antigen-specific Tâ cells. Our findings highlight the potential of engineered hydrogel nanoparticles for the lymphatic delivery of antigens and immune-modulating compounds.
Assuntos
Hidrogéis , Linfonodos/metabolismo , Nanopartículas , Polímeros/química , Vacinas/administração & dosagem , Animais , Antígenos CD/imunologia , Linfonodos/imunologia , Camundongos , Microscopia Eletrônica de TransmissãoRESUMO
The use of DNA and viral vector-based vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to the lack of their genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study, we evaluated the potency of antigen-encoding mRNA complexed with the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOTAP/DOPE ) as a novel vaccination approach. We demonstrate that subcutaneous immunization of mice with mRNA encoding the HIV-1 antigen Gag complexed with DOTAP/DOPE elicits antigen-specific, functional T cell responses resulting in specific killing of Gag peptide-pulsed cells and the induction of humoral responses. In addition, we show that DOTAP/DOPE complexed antigen-encoding mRNA displays immune-activating properties characterized by secretion of type I interferon (IFN) and the recruitment of proinflammatory monocytes to the draining lymph nodes. Finally, we demonstrate that type I IFN inhibit the expression of DOTAP/DOPE complexed antigen-encoding mRNA and the subsequent induction of antigen-specific immune responses. These results are of high relevance as they will stimulate the design and development of improved mRNA-based vaccination approaches.
Assuntos
Antígenos/imunologia , Imunidade Celular/efeitos dos fármacos , Interferon Tipo I/farmacologia , RNA Mensageiro/imunologia , Animais , Camundongos , RNA Mensageiro/administração & dosagemRESUMO
Resident alveolar macrophages (rAMs) residing in the bronchoalveolar lumen of the airways play an important role in limiting excessive inflammatory responses in the respiratory tract. High phagocytic activity along with hyporesponsiveness to inflammatory insults and lack of autonomous IFN-ß production are crucial assets in this regulatory function. Using a mouse model of asthma, we analyzed the fate of rAMs both during and after allergic bronchial inflammation. Although nearly indistinguishable phenotypically from naïve rAMs, postinflammation rAMs exhibited a strongly reduced basal phagocytic capacity, accompanied by a markedly increased inflammatory reactivity to Toll-like receptors TLR-3 (poly I:C), TLR-4 [lipopolysaccharide (LPS)], and TLR-7 (imiquimod). Importantly, after inflammation, rAMs exhibited a switch from an IFN-ß-defective to an IFN-ß-competent phenotype, thus indicating the occurrence of a new, inflammatory-released rAM population in the postallergic lung. Analysis of rAM turnover revealed a rapid disappearance of naïve rAMs after the onset of inflammation. This inflammation-induced rAM turnover is critical for the development of the hyperinflammatory rAM phenotype observed after clearance of bronchial inflammation. These data document a novel mechanism of innate imprinting in which noninfectious bronchial inflammation causes alveolar macrophages to acquire a highly modified innate reactivity. The resulting increase in secretion of inflammatory mediators on TLR stimulation implies a role for this phenomenon of innate imprinting in the increased sensitivity of postallergic lungs to inflammatory insults.
Assuntos
Asma/imunologia , Bronquite/imunologia , Macrófagos Alveolares/imunologia , Animais , Asma/fisiopatologia , Hiper-Reatividade Brônquica/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/biossíntese , Progressão da Doença , Feminino , Mediadores da Inflamação/imunologia , Interferon beta/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Fagocitose/imunologia , Fenótipo , Receptores Toll-Like/imunologiaRESUMO
Mycolic acids (MAs) occur in the cell wall of Mycobacterium tuberculosis as variable mixtures of different classes and chain lengths. Here, we address the relationship between the structure and its inflammatory function of this virulence factor using single synthetic MA isomers, differing in oxygenation class and cis- versus α-methyl-trans proximal cyclopropane orientation. Analysis of bronchoalveolar inflammation, lung histopathology and alveolar macrophage transcription revealed a strong dependence on these meromycolic chemistries of mouse pulmonary inflammation in response to intratracheal treatments with MAs. Whereas α-MA was inert, oxygenated methoxy- and keto-MA with cis-cyclopropane stereochemistry elicited solid to mild inflammatory responses respectively. In trans-cyclopropane orientation, methoxy-MA partially lost its inflammatory activity and keto-MA exerted anti-inflammatory alternative activation of alveolar macrophages and counteracted cis-methoxy-MA induced airway inflammation. The differential innate immune activities of MAs demonstrated here, dependent on oxygenation class and cis versus α-methyl-trans cyclopropane chemistry, identify a novel means for M. tuberculosis to steer host immune responses during infection.
Assuntos
Mycobacterium tuberculosis/química , Ácidos Micólicos/química , Ácidos Micólicos/imunologia , Fatores de Virulência/química , Fatores de Virulência/imunologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Contagem de Células , Feminino , Expressão Gênica/genética , Imunidade Inata/imunologia , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Lipossomos , Pulmão/imunologia , Pulmão/patologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Mycobacterium tuberculosis/imunologia , Ácidos Micólicos/administração & dosagem , Ácidos Micólicos/farmacologia , Neutrófilos/imunologia , Neutrófilos/patologia , Estereoisomerismo , Fatores de Virulência/administração & dosagem , Fatores de Virulência/farmacologiaRESUMO
BACKGROUND: mRNA levels of members of the Vascular Endothelial Growth Factor family (VEGF-A, -B, -C, -D, Placental Growth Factor/PlGF) have been investigated as tissue-based markers of colon cancer. These studies, which used specimens obtained by surgical resection or colonoscopic biopsy, yielded contradictory results. We studied the effect of the sampling method on the marker accuracy of VEGF family members. METHODS: Comparative RT-qPCR analysis was performed on healthy colon and colon carcinoma samples obtained by biopsy (n = 38) or resection (n = 39) to measure mRNA expression levels of individual VEGF family members. mRNA levels of genes encoding the eicosanoid enzymes cyclooxygenase 2 (COX2) and 5-lipoxygenase (5-LOX) and of genes encoding the hypoxia markers glucose transporter 1 (GLUT-1) and carbonic anhydrase IX (CAIX) were included as markers for cellular stress and hypoxia. RESULTS: Expression levels of COX2, 5-LOX, GLUT-1 and CAIX revealed the occurrence in healthy colon resection samples of hypoxic cellular stress and a concurrent increment of basal expression levels of VEGF family members. This increment abolished differential expression of VEGF-B and VEGF-C in matched carcinoma resection samples and created a surgery-induced underexpression of VEGF-D. VEGF-A and PlGF showed strong overexpression in carcinoma samples regardless of the sampling method. CONCLUSIONS: Sampling-induced hypoxia in resection samples but not in biopsy samples affects the marker-reliability of VEGF family members. Therefore, biopsy samples provide a more accurate report on VEGF family mRNA levels. Furthermore, this limited expression analysis proposes VEGF-A and PlGF as reliable, sampling procedure insensitive mRNA-markers for molecular diagnosis of colon cancer.
Assuntos
Carcinoma/metabolismo , Neoplasias do Colo/metabolismo , Manejo de Espécimes/métodos , Fator A de Crescimento do Endotélio Vascular/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Área Sob a Curva , Biomarcadores Tumorais/análise , Biópsia , Carcinoma/patologia , Carcinoma/cirurgia , Hipóxia Celular/fisiologia , Neoplasias do Colo/patologia , Neoplasias do Colo/cirurgia , Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Because of their large surface area, the lungs appear an attractive route for noninvasive vaccine delivery, harboring the potential to induce local mucosal immune responses in addition to systemic immunity. To evoke adaptive immunity, Ags require the addition of adjuvants that not only enhance the strength of the immune response but also determine the type of response elicited. In this study, we evaluate the adjuvant characteristics of polyelectrolyte microcapsules (PEMs) consisting of the biopolymers dextran-sulfate and poly-L-arginine. PEMs form an entirely new class of microcapsules that are generated by the sequential adsorption of oppositely charged polymers (polyelectrolytes) onto a sacrificial colloidal template, which is subsequently dissolved leaving a hollow microcapsule surrounded by a thin shell. Following intratracheal instillation, PEMs were not only efficiently taken up by APCs but also enhanced their activation status. Pulmonary adaptive immune responses were characterized by the induction of a strongly Th17-polarized response. When compared with a mixture of soluble Ag with empty microcapsules, Ag encapsulation significantly enhanced the strength of this local mucosal response. Given their unique property to selectively generate Th17-polarized immune responses, PEMs may become of significant interest in the development of effective vaccines against fungal and bacterial species.
Assuntos
Implantes Absorvíveis , Adjuvantes Imunológicos/administração & dosagem , Cápsulas/administração & dosagem , Interleucina-17/imunologia , Pulmão/imunologia , Vacinação/métodos , Adjuvantes Imunológicos/síntese química , Animais , Células Apresentadoras de Antígenos/imunologia , Antígenos/administração & dosagem , Antígenos/imunologia , Lavagem Broncoalveolar , Cápsulas/síntese química , Sulfato de Dextrana/imunologia , Eletrólitos/imunologia , Feminino , Citometria de Fluxo , Imunidade nas Mucosas/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
By targeting dendritic cells, polymeric carriers in the nano to lower micron range constitute very interesting tools for antigen delivery. In this critical review, we review how new immunological insights can be exploited to design new carriers allowing one to tune immune responses and to further increase vaccine potency (137 references).
Assuntos
Antígenos/administração & dosagem , Portadores de Fármacos/química , Polímeros/química , Antígenos/imunologia , Células Dendríticas/imunologia , Humanos , Nanotecnologia , Vacinas/imunologiaRESUMO
Immunizing: to evoke highly potent immune responses against recombinant antigens, hollow capsules consisting of layers of dextran sulphate and poly-L-arginine that encapsulate the antigen ovalbumin (orange circles) were coated with immune-activating CpG-containing oligonucleotides (green). These capsules were readily internalized by dendritic cells and showed activity in further immunization experiments.
Assuntos
Cápsulas/química , Eletrólitos/química , Vacinas Sintéticas/imunologia , Animais , Células Dendríticas/imunologia , Sulfato de Dextrana/química , Interferon gama/metabolismo , Camundongos , Oligodesoxirribonucleotídeos/química , Ovalbumina/genética , Ovalbumina/imunologia , Ovalbumina/metabolismo , Peptídeos/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Propriedades de Superfície , Vacinas Sintéticas/químicaRESUMO
Contrary to the T-helper (Th)-2 bias and eosinophil-dominated bronchial inflammation encountered in most asthmatic subjects, other patients may exhibit neutrophil-predominant asthma subphenotypes, along with Th-1 and Th-17 cells. However, the etiology of many neutrophil-dominated asthma subphenotypes remains ill-understood, in part due to a lack of appropriate experimental models. To better understand the distinct immune-pathological features of eosinophilic vs. neutrophilic asthma types, we developed an ovalbumin (OVA)-based mouse model of neutrophil-dominated allergic pulmonary inflammation. Consequently, we probed for particular inflammatory signatures and checkpoints underlying the immune pathology in this new model, as well as in a conventional, eosinophil-dominated asthma model. Briefly, mice were OVA sensitized using either aluminum hydroxide (alum) or complete Freund's adjuvants, followed by OVA aerosol challenge. T-cell, granulocyte, and inflammatory mediator profiles were determined, along with alveolar macrophage genomewide transcriptome profiling. In contrast to the Th-2-dominated phenotype provoked by alum, OVA/ complete Freund's adjuvants adjuvant-based sensitization, followed by allergen challenge, elicited a pulmonary inflammation that was poorly controlled by dexamethasone, and in which Th-1 and Th-17 cells additionally participated. Analysis of the overall pulmonary and alveolar macrophage inflammatory mediator profiles revealed remarkable similarities between both models. Nevertheless, we observed pronounced differences in the IL-12/IFN-γ axis and its control by IL-18 and IL-18 binding protein, but also in macrophage arachidonic acid metabolism and expression of T-cell instructive ligands. These differential signatures, superimposed onto a generic inflammatory signature, denote distinctive inflammatory checkpoints potentially involved in orchestrating neutrophil-dominated asthma.
Assuntos
Eosinófilos/imunologia , Neutrófilos/imunologia , Pneumonia/imunologia , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/imunologia , Modelos Animais de Doenças , Feminino , Adjuvante de Freund/imunologia , Perfilação da Expressão Gênica , Mediadores da Inflamação/análise , Interleucina-12/imunologia , Interleucina-18/imunologia , Pulmão/imunologia , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologiaRESUMO
BACKGROUND: Research involving gene expression profiling and clinical applications, such as diagnostics and prognostics, often require a DNA array platform that is flexibly customisable and cost-effective, but at the same time is highly sensitive and capable of accurately and reproducibly quantifying the transcriptional expression of a vast number of genes over the whole transcriptome dynamic range using low amounts of RNA sample. Hereto, a set of easy-to-implement practical optimisations to the design of cDNA-based nylon macroarrays as well as sample (33)P-labeling, hybridisation protocols and phosphor screen image processing were analysed for macroarray performance. RESULTS: The here proposed custom macroarray platform had an absolute sensitivity as low as 50,000 transcripts and a linear range of over 5 log-orders. Its quality of identifying differentially expressed genes was at least comparable to commercially available microchips. Interestingly, the quantitative accuracy was found to correlate significantly with corresponding reversed transcriptase - quantitative PCR values, the gold standard gene expression measure (Pearson's correlation test p < 0.0001). Furthermore, the assay has low cost and input RNA requirements (0.5 µg and less) and has a sound reproducibility. CONCLUSIONS: Results presented here, demonstrate for the first time that self-made cDNA-based nylon macroarrays can produce highly reliable gene expression data with high sensitivity and covering the entire mammalian dynamic range of mRNA abundances. Starting off from minimal amounts of unamplified total RNA per sample, a reasonable amount of samples can be assayed simultaneously for the quantitative expression of hundreds of genes in an easily customisable and cost-effective manner.
Assuntos
Perfilação da Expressão Gênica/métodos , Macrófagos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Mensageiro/análise , Transcriptoma , Primers do DNA , Perfilação da Expressão Gênica/instrumentação , Humanos , Nylons/química , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , RNA Mensageiro/biossíntese , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
Alternatively activated macrophages (AAMs), triggered by interleukin-4 (IL-4) and IL-13, play a modulating role during Th2 cytokine-driven pathologies, but their molecular armament remains poorly characterized. Here, we established E-cadherin (Cdh1) as a selective marker for IL-4/IL-13-exposed mouse and human macrophages, which is STAT6-dependently induced during polarized Th2 responses associated with Taenia crassiceps helminth infections or allergic airway inflammation. The IL-4-dependent, arginase-1/ornithine decarboxylase-mediated production of polyamines is important for maximal Cdh1 induction, unveiling a novel mechanism for IL-4-dependent gene transcription. At the macrophage surface, E-cadherin forms a functional complex with the catenins that accumulates at sites of cell contact. Macrophage-specific deletion of the Cdh1 gene illustrates the implication of E-cadherin in IL-4-driven macrophage fusion and heterotypic interactions with CD103(+) and KLRG1(+) T cells. This study identifies the E-cadherin/catenin complex as a discriminative, partly polyamine-regulated feature of IL-4/IL-13-exposed alternatively activated macrophages that contributes to homotypic and heterotypic cellular interactions.
Assuntos
Caderinas/imunologia , Interleucina-4/imunologia , Macrófagos/imunologia , Poliaminas/imunologia , Transdução de Sinais/imunologia , alfa Catenina/imunologia , Animais , Asma/imunologia , Western Blotting , Caderinas/metabolismo , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Hipersensibilidade/imunologia , Imunoprecipitação , Interleucina-13/imunologia , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Teníase/imunologia , alfa Catenina/metabolismoRESUMO
PURPOSE: To evaluate if lyophilization can be used to obtain a dry formulation of polyelectrolyte microcapsules, which have emerged as a new class of microparticles for the encapsulation and delivery of biomacromolecules. METHODS: Microcapsules composed of dextran sulfate and poly-L-arginine were obtained by coating CaCO(3) microparticles by means of the layer-by-layer technique. Microcapsules were lyophilized using different stabilizers; intactness was checked by CLSM and SEM. Horseradish peroxidase was encapsulated as model enzyme and retained activity after freeze-drying was determined using a fluorescence assay. Ovalbumin was encapsulated as model antigen; immunogenicity after lyophilization was evaluated in vitro by a T-cell proliferation assay and in vivo by measuring the antibody titer in mice. RESULTS: The results clearly demonstrate the necessity of using polyols in the formulation to prevent rupture of the microcapsules and to preserve the activity of encapsulated enzymes. Lyophilized microcapsules appeared as a promising adjuvant for antigen delivery, as both in vitro as in vivo assays showed higher immune activation compared to free antigen. CONCLUSIONS: Lyophilization is a promising strategy towards improved stability of protein-loaded microcapsules.