RESUMO
OBJECTIVE: Kawasaki disease (KD) occurs via activation of the innate immune system. Nucleotide oligomerization domain-1 (NOD1) is a pattern recognition receptor regulating the innate immunity. We characterized histopathology of arteritis induced by FK565, a ligand for NOD1, in mice, compared with Candida albicans water-soluble fraction (CAWS)-induced model. METHODS: Vasculitis was induced by injection of FK565 or CAWS into C57BL6/J mice (n = 9 and n = 11, respectively). At 4 weeks, they were sacrificed, and plasma cytokines and chemokines were measured. RESULTS: FK565 injection induced vasculitis mainly involving bilateral coronary arteries whereas the aortic root was diffusely affected in CAWS mice. In FK565 animals, the abdominal aorta and its branching arteries also exhibited inflammation with atherosclerosis. IL-1α, IL-1ß, IL-5 and RANTES were increased in FK565 group whereas IL-6, IL-13, G-CSF, IFN-γ, and TNF-α were higher in CAWS animals (p < .05 for all variables). The total area of inflammation in FK565 mice appeared to correlate with IL-1ß levels (r = 0.71, p = .05). CONCLUSIONS: Histopathology of FK565-induced model demonstrated 'site-specific' coronary arteritis mimicking KD. This histopathological difference from CAWS model may be due to different cytokine expression profiles.
Assuntos
Adjuvantes Imunológicos/toxicidade , Candida albicans/química , Síndrome de Linfonodos Mucocutâneos/etiologia , Oligopeptídeos/toxicidade , Adjuvantes Imunológicos/farmacologia , Animais , Candida albicans/imunologia , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos/sangue , Interleucina-6/sangue , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Linfonodos Mucocutâneos/metabolismo , Síndrome de Linfonodos Mucocutâneos/patologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Oligopeptídeos/farmacologia , Ligação Proteica , Frações Subcelulares/imunologia , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: Preclinical research implementing fluorescence-based approaches is inevitable for drug discovery and technology. For example, a variety of contrast agents developed for biomedical imaging are usually evaluated in cell systems and animal models based on their conjugation to fluorescent dyes. Biodistribution studies of excised organs are often performed by macroscopic imaging, whereas the subcellular localization though vital, is often neglected or further validated by histological procedures. Available systems used to define the subcellular biodistribution of contrast agents such as intravital microscopes or ex vivo histological analysis are expensive and not affordable by the majority of researchers, or encompass tedious and time consuming steps that may modify the contrast agents and falsify the results. Thus, affordable and more reliable approaches to study the biodistribution of contrast agents are required. We developed fluorescent immunoliposomes specific for human fibroblast activation protein and murine endoglin, and used macroscopic fluorescence imaging and confocal microscopy to determine their biodistribution and subcellular localization in freshly excised mice organs at different time points post intravenous injection. RESULTS: Near infrared fluorescence macroscopic imaging revealed key differences in the biodistribution of the respective immunoliposomes at different time points post injection, which correlated to the first-pass effect as well as the binding of the probes to molecular targets within the mice organs. Thus, a higher accumulation and longer retention of the murine endoglin immunoliposomes was seen in the lungs, liver and kidneys than the FAP specific immunoliposomes. Confocal microscopy showed that tissue autofluorescence enables detection of organ morphology and cellular components within freshly excised, non-processed organs, and that fluorescent probes with absorption and emission maxima beyond the tissue autofluorescence range can be easily distinguished. Hence, the endoglin targeting immunoliposomes retained in some organs could be detected in the vascular endothelia cells of the organs. CONCLUSIONS: The underlying work represents a quick, effective and more reliable setup to validate the macroscopic and subcellular biodistribution of contrast agents in freshly excised animal organs. The approach will be highly beneficial to many researchers involved in nanodrug design or in fluorescence-based studies on disease pathogenesis.
Assuntos
Anticorpos Monoclonais/imunologia , Lipossomos/imunologia , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Frações Subcelulares/imunologia , Vísceras/imunologia , Animais , Feminino , Técnicas In Vitro , Taxa de Depuração Metabólica/imunologia , Camundongos , Camundongos Nus , Microscopia Confocal/métodos , Especificidade de Órgãos/imunologia , Distribuição Tecidual/imunologiaRESUMO
Cancer vaccines are designed to elicit an endogenous adaptive immune response that can successfully recognize and eliminate residual or recurring tumors. Such approaches can potentially overcome shortcomings of passive immunotherapies by generating long-lived therapeutic effects and immune memory while limiting systemic toxicities. A critical determinant of vaccine efficacy is efficient transport and delivery of tumor-associated antigens to professional antigen presenting cells (APCs). Plant viral nanoparticles (VNPs) with natural tropism for APCs and a high payload carrying capacity may be particularly effective vaccine carriers. The applicability of VNP platform technologies is governed by stringent structure-function relationships. We compare two distinct VNP platforms: icosahedral cowpea mosaic virus (CPMV) and filamentous potato virus X (PVX). Specifically, we evaluate in vivo capabilities of engineered VNPs delivering human epidermal growth factor receptor 2 (HER2) epitopes for therapy and prophylaxis of HER2+ malignancies. Our results corroborate the structure-function relationship where icosahedral CPMV particles showed significantly enhanced lymph node transport and retention, and greater uptake by/activation of APCs compared to filamentous PVX particles. These enhanced immune cell interactions and transport properties resulted in elevated HER2-specific antibody titers raised by CPMV- vs. PVX-based peptide vaccine. The 'synthetic virology' field is rapidly expanding with numerous platforms undergoing development and preclinical testing; our studies highlight the need for systematic studies to define rules guiding the design and rational choice of platform, in the context of peptide-vaccine display technologies.
Assuntos
Vacinas Anticâncer/imunologia , Neoplasias Experimentais/imunologia , Vírus Oncogênicos/imunologia , Vírus de Plantas/imunologia , Receptor ErbB-2/imunologia , Frações Subcelulares/imunologia , Vírion/imunologia , Imunidade Adaptativa/imunologia , Animais , Transporte Biológico Ativo/imunologia , Linhagem Celular Tumoral , Humanos , CamundongosRESUMO
The anti-ErbB2 humanized antibody trastuzumab was approved for ErbB2-positive metastatic gastric and gastro-esophageal junction cancer in 2010. Despite the effectiveness of trastuzumab, its efficacy remains variable and often modest. Thus, there is an urgent need to improve ErbB2-targeting therapy. Down-regulation of surface receptors induced by monoclonal antibody (mAb) contributes to its antitumor efficacy. Previous studies have demonstrated that if two anti-ErbB2 mAbs did not compete with each other for binding to ErbB2, the combination of them can enhance ErbB2 internalization. In the present study, we investigated ErbB2 internalization-inducing ability of non-competitive anti-ErbB2 mAb combinations and surprisingly found that most of the mAb combinations tested did not down-regulate ErbB2. Only 4 of 18 non-competitive mAb pairs efficiently induced ErbB2 internalization. Interestingly, although the non-competitive anti-ErbB2 mAbs trastuzumab and pertuzumab, either alone or in combination, were ineffective at inducing ErbB2 internalization, TPL, a bispecific antibody engineered from trastuzumab and pertuzumab, potently down-regulated the ErbB2 molecule. Importantly, TPL exhibited a far greater antitumor effect on ErbB2-overexpressing gastric cancer cell line than trastuzumab plus pertuzumab, suggesting that it may be a promising agent for the treatment of gastric cancer.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Gástricas/patologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/imunologia , Resultado do TratamentoRESUMO
Mesothelin, a cancer biomarker overexpressed in tumors of epithelial origin, is a target for nanotechnology-based diagnostic, therapeutic, and prognostic applications. The currently available anti-mesothelin antibodies present limitations, including low penetration due to large size and/or lack of in vivo stability. Single domain antibodies (sdAbs) or nanobodies (Nbs) provide powerful solutions to these specific problems. We generated a phage-display library of Nbs that were amplified from B cells of a llama that was immunized with human recombinant mesothelin. Two nanobodies (Nb A1 and Nb C6) were selected on the basis of affinity (K(D) = 15 and 30 nM, respectively). Nb A1 was further modified by adding either a cysteine to permit maleimide-based bioconjugations or a sequence for the site-specific metabolic addition of a biotin in vivo. Both systems of conjugation (thiol-maleimide and streptavidin/biotin) were used to characterize and validate Nb A1 and to functionalize nanoparticles. We showed that anti-mesothelin Nb A1 could detect native and denatured mesothelin in various diagnostic applications, including flow cytometry, western blotting, immunofluorescence, and optical imaging. In conclusion, anti-mesothelin Nbs are novel, cost-effective, small, and single domain reagents with high affinity and specificity for the tumor-associated antigen mesothelin, which can be simply bioengineered for attachment to nanoparticles or modified surfaces using multiple bioconjugation strategies. These anti-mesothelin Nbs can be useful in both conventional and nanotechnology-based diagnostic, therapeutic and prognostic biomedical applications.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Proteínas Ligadas por GPI/imunologia , Nanopartículas/uso terapêutico , Frações Subcelulares/imunologia , Anticorpos Monoclonais/genética , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Células HeLa , Humanos , Mesotelina , Nanopartículas/química , Nanopartículas/ultraestrutura , Engenharia de Proteínas/métodos , Frações Subcelulares/patologiaRESUMO
Transmembrane-4 L-six family member-1 (TM4SF1) is a small plasma membrane-associated glycoprotein that is highly and selectively expressed on the plasma membranes of tumor cells, cultured endothelial cells, and, in vivo, on tumor-associated endothelium. Immunofluorescence microscopy also demonstrated TM4SF1 in cytoplasm and, tentatively, within nuclei. With monoclonal antibody 8G4, and the finer resolution afforded by immuno-nanogold transmission electron microscopy, we now demonstrate TM4SF1 in uncoated cytoplasmic vesicles, nuclear pores and nucleoplasm. Because of its prominent surface location on tumor cells and tumor-associated endothelium, TM4SF1 has potential as a dual therapeutic target using an antibody drug conjugate (ADC) approach. For ADC to be successful, antibodies reacting with cell surface antigens must be internalized for delivery of associated toxins to intracellular targets. We now report that 8G4 is efficiently taken up into cultured endothelial cells by uncoated vesicles in a dynamin-dependent, clathrin-independent manner. It is then transported along microtubules through the cytoplasm and passes through nuclear pores into the nucleus. These findings validate TM4SF1 as an attractive candidate for cancer therapy with antibody-bound toxins that have the capacity to react with either cytoplasmic or nuclear targets in tumor cells or tumor-associated vascular endothelium.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Superfície/imunologia , Clatrina/imunologia , Dinaminas/imunologia , Células Endoteliais/imunologia , Proteínas de Neoplasias/imunologia , Frações Subcelulares/imunologia , Células Cultivadas , Células Endoteliais/ultraestrutura , HumanosRESUMO
Cardiac neonatal lupus (NL) is presumed to arise from maternal autoantibody targeting an intracellular ribonucleoprotein, Ro60, which binds noncoding Y RNA and only becomes accessible to autoantibodies during apoptosis. Despite the importance of Ro60 trafficking in the development of cardiac NL, the mechanism underlying cell surface exposure is unknown. To evaluate the influence of Y RNA on the subcellular location of Ro60 during apoptosis and activation of macrophages, stable Ro60 knockout murine fibroblasts expressing wild-type or mutated FLAG-Ro60 were assessed. FLAG3-Ro60(K170A R174A) binds Y RNA, whereas FLAG3-Ro60(H187S) does not bind Y RNA; fibroblasts expressing these constructs showed equivalent intracellular expression of Ro60. In contrast, apoptotic fibroblasts containing FLAG3-Ro60(K170A R174A) were bound by anti-Ro60, whereas FLAG3-Ro60(H187S) was not surface expressed. RNA interference of mY3 RNA in wild-type fibroblasts inhibited surface translocation of Ro60 during apoptosis, whereas depletion of mY1 RNA did not affect Ro60 exposure. Furthermore, Ro60 was not exposed following overexpression of mY1 in the mY3-depleted fibroblasts. In an in vitro model of anti-Ro60-mediated injury, Y RNA was shown to be an obligate factor for TLR-dependent activation of macrophages challenged with anti-Ro60-opsonized apoptotic fibroblasts. Murine Y3 RNA is a necessary factor to support the surface translocation of Ro60, which is pivotal to the formation of immune complexes on apoptotic cells and a TLR-dependent proinflammatory cascade. Accordingly, the Y3 RNA moiety of the Ro60 ribonucleoprotein imparts a critical role in the pathogenicity of maternal anti-Ro60 autoantibodies.
Assuntos
Cardiopatias/imunologia , Cardiopatias/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , RNA não Traduzido/metabolismo , Ribonucleoproteínas/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Autoanticorpos/metabolismo , Células Cultivadas , Criança , Técnicas de Cocultura , Cardiopatias/patologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Knockout , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/imunologia , Frações Subcelulares/imunologia , Propriedades de SuperfícieRESUMO
BACKGROUND: Toll-like receptors (TLRs) play pivotal roles in host immune responses and have been suggested to be involved in the development of many infectious diseases. In this study, the mRNA expression levels of TLR2, TLR4, and TLR9 and their relationship with periodontopathic bacteria in periodontal tissue are examined. Furthermore, the mechanism of TLR induction by Porphyromonas gingivalis is investigated in human gingival fibroblasts (HGFs). METHODS: Gingival tissue and subgingival plaque samples were collected from 19 patients with chronic periodontitis (CP) and 16 control individuals without periodontitis. Gene expression levels in the tissues and in HGFs were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The numbers of periodontopathic bacteria were determined by quantitative real-time PCR. RESULTS: The expression levels of TLR2 and TLR9 were significantly higher in the tissues of patients with CP compared to the tissues of control individuals. The mRNA levels of TLR2 and TLR9, but not TLR4, were positively correlated with the number of P. gingivalis in subgingival plaque. P. gingivalis sonicated extract, P. gingivalis lipopolysaccharide, P. gingivalis DNA, and tumor necrosis factor-α(TNF-α) could significantly upregulate the mRNA expression of TLR2 in HGFs. Furthermore, P. gingivalis-mediated TLR2 expression was suppressed by TNF-α antibody. CONCLUSIONS: This study suggests that P. gingivalis infection induces TLR2 and TLR9 upregulation in patients with CP. P. gingivalis-induced TLR2 expression in HGFs is partially dependent on TNF-α and may lead to sensitization of HGFs to bacterial components encountered in the periodontal microenvironment.
Assuntos
Porphyromonas gingivalis/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Receptor Toll-Like 9/imunologia , Adulto , Aggregatibacter actinomycetemcomitans/isolamento & purificação , Carga Bacteriana , Técnicas Bacteriológicas , Técnicas de Cultura de Células , Periodontite Crônica/imunologia , Periodontite Crônica/microbiologia , DNA Bacteriano/farmacologia , Placa Dentária/imunologia , Placa Dentária/microbiologia , Feminino , Fibroblastos/imunologia , Fibroblastos/microbiologia , Gengiva/microbiologia , Gengiva/patologia , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Perda da Inserção Periodontal/imunologia , Perda da Inserção Periodontal/microbiologia , Bolsa Periodontal/imunologia , Bolsa Periodontal/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Frações Subcelulares/imunologia , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/farmacologia , Regulação para CimaRESUMO
There is an emerging need both in pharmacology and within the biomedical industry to develop new tools to target intracellular mechanisms. The efficient delivery of functionally active proteins within cells is potentially a powerful research strategy, especially through the use of antibodies. In this work, we report on a nanovector for the efficient encapsulation and delivery of antibodies into live cells with no significant loss of cell viability or any deleterious effect on cell metabolic activity. This delivery system is based on poly[2-(methacryloyloxy)ethyl phosphorylcholine]-block-[2-(diisopropylamino)ethyl methacrylate] (PMPC-PDPA), a pH-sensitive diblock copolymer that self-assembles to form nanometer-sized vesicles, also known as polymersomes, at physiological pH. Polymersomes can successfully deliver relatively high antibody payloads within different types of live cells. We demonstrate that these antibodies can target their respective epitope showing immunolabeling of γ-tubulin, actin, Golgi protein, and the transcription factor NF-κB in live cells. Finally, we demonstrate that intracellular delivery of antibodies can control specific subcellular events, as well as modulate cell activity and proinflammatory processes.
Assuntos
Anticorpos/administração & dosagem , Fosforilcolina/análogos & derivados , Ácidos Polimetacrílicos/química , Animais , Células Cultivadas , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Células NIH 3T3 , Fosforilcolina/química , Frações Subcelulares/imunologiaRESUMO
Exosomes, 60-90-nm-sized vesicles, are produced by a large number of cell types, including tumor cells, neurons, astrocytes, hemocytes, intestinal epithelial cells, and so on. Dendritic cell (DC), the most potent professional antigen-presenting cell in the immune system, produces exosomes in the course of maturation. Mature DCs produce exosomes with the ability to elicit potent immunoactivation, resulting in tumor eradication and bacterial or virus elimination. Given the notion that exosomes are stable and easy to be modified artificially, autologous mature DC-derived exosomes have been vaccinated into patients with malignant diseases. In clinical trials utilizing exosomes as therapeutic approaches, researchers observed considerable curative effect with little side effect. However, immature or suppressive DC-derived exosomes harbor anti-inflammatory properties distinct from mature DC-derived exosomes. In murine models of autoimmune disease and transplantation, immature DC-derived exosomes reduced T cell-dependent immunoactivation, relieved clinical manifestation of autoimmune disease, and prolonged survival time of transplantation. Although the exact mechanism of how immature DC-derived exosomes function in vivo is still unclear, and there are no clinical trials regarding application of exosome vaccine into patients with autoimmune disease, we will analyze the promise of immature DC-derived exosomes as a subcellular vaccine in autoimmunity in this review.
Assuntos
Doenças Autoimunes/terapia , Células Dendríticas/citologia , Exossomos/imunologia , Vacinas , Animais , Autoimunidade , Células Dendríticas/imunologia , Humanos , Camundongos , Frações Subcelulares/imunologiaRESUMO
BACKGROUND: Schistosomiasis japonica remains a real threat to public health in China. The currently used immunodiagnostic assays are sensitive and have a certain degree of specificity, however, they all use complex crude antigens, are based on detection of schistosome-specific antibodies, and have been shown to cross-react with other parasitic diseases. Therefore, these assays cannot be used to evaluate chemotherapy efficacy. The development of highly sensitive and highly specific immunodiagnostic techniques that can monitor the decline of antibodies specific for S. japonica will be extremely valuable as part of the ongoing strategy to control schistosomiasis in endemic areas. Here we report on the identification of unique fraction antigens of soluble egg antigen (SEA) to which the antibodies disappear 7 weeks after effective treatment. Furthermore, we use these SEA fractions to develop a modified assay with both high sensitivity and specificity. METHODOLOGY/PRINCIPAL FINDINGS: SEA of S. japonicum was fractionated by electrophoresis using 7.5% SDS-PAGE under non-reducing conditions. The SEA fraction antigens to which antibodies were decreased soon after treatment were collected and used as the detection antigens to establish the FA-ELISA. Sera from patients with acute and chronic schistosomiasis infection, healthy people, and those with other parasitic diseases, were used to evaluate their sensitivity and specificity. Furthermore, sera from patients with chronic schistosomiasis infection were evaluated before and after treatment at different time points to evaluate their chemotherapeutic efficacy. CONCLUSION/SIGNIFICANCE: We demonstrated that this novel FA-ELISA provided high sensitivity and specificity, with very low cross-reactivity, and can serve as an effective tool to determine the efficacy of chemotherapy against S. japonicum.
Assuntos
Formação de Anticorpos/imunologia , Antígenos de Helmintos/imunologia , Testes Imunológicos/métodos , Óvulo/imunologia , Schistosoma japonicum/imunologia , Soro/imunologia , Soro/parasitologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Helmintos/sangue , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , Coelhos , Esquistossomose Japônica/diagnóstico , Esquistossomose Japônica/imunologia , Esquistossomose Japônica/parasitologia , Sensibilidade e Especificidade , Solubilidade , Frações Subcelulares/imunologiaRESUMO
During neuroinflammation, cytokines such as TNF-α and IFN-γ secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood-brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-γ. BEC activation with TNF-α alone or in combination with IFN-γ induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.
Assuntos
Barreira Hematoencefálica/enzimologia , Barreira Hematoencefálica/imunologia , Encéfalo/enzimologia , Encéfalo/imunologia , Citocinas/fisiologia , Endotélio Vascular/enzimologia , Endotélio Vascular/imunologia , Barreira Hematoencefálica/patologia , Encéfalo/patologia , Linhagem Celular , Endotélio Vascular/patologia , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Interferon gama/metabolismo , Microcirculação/imunologia , Receptores de Interferon/biossíntese , Receptores Tipo I de Fatores de Necrose Tumoral/biossíntese , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Frações Subcelulares/enzimologia , Frações Subcelulares/imunologia , Frações Subcelulares/patologia , Receptor de Interferon gamaRESUMO
Vesicular stomatitis virus (VSV) and Rabies Virus (RABV) are the prototypic members of the rhabdovirus family. These viruses have a particularly broad host range, and despite the availability of vaccines, RABV still causes more than 50,000 human deaths a year. Trafficking of the virion or viral particles is important at several stages of the replicative life cycle, including cellular entry, localization into the cytoplasmic inclusion bodies which primarily house the transcription and replication of the viral genome, and migration to the plasma membrane from whence the virus is released by budding. Intriguingly, specific viral proteins, VSV M and RABV P have also been shown to undergo intracellular trafficking independent of the other viral apparatus. These proteins are multifunctional, and play roles in antagonism of host processes, namely the IFN system, and as such enable viral evasion of the innate cellular antiviral response. A body of recent research has been aimed at characterizing the mechanisms by which these proteins are able to shuttle between and localize to various subcellular sites, including the nucleus, which is not required during the cytoplasmic replicative life cycle of the virus. This work has indicated that trafficking of these proteins plays a significant role in determining the ability of the viruses to replicate and cause infection, and as such, represents a viable target for development of a new generation of vaccines and prophylactic therapeutics which are required to battle these pathogens of human and agricultural significance.
Assuntos
Vírus da Raiva/imunologia , Raiva/imunologia , Frações Subcelulares/imunologia , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Humanos , Evasão da Resposta Imune , Raiva/virologia , Frações Subcelulares/virologia , Estomatite Vesicular/virologia , Proteínas Virais/imunologiaRESUMO
The adipokine adiponectin is well known to affect the function of immune cells and upregulation of CCL2 by adiponectin in monocytes/macrophages has already been reported. In the current study the effect of adiponectin on CCL2, -3, -4, and -5 and their corresponding receptors CCR1, CCR2, and CCR5 has been analyzed. Adiponectin elevates mRNA and protein of the CC chemokines in primary human monocytes. Simultaneously the surface abundance of CCR2 and CCR5 is reduced while CCR1 is not affected. Downregulation of CCR2 by adiponectin is blocked by a CCR2 antagonist although expression of the CCL2 regulated genes CCR2 and TGF-beta 1 is not altered in the adiponectin-incubated monocytes. CCL2, -3, and -5 concentrations measured in supernatants of monocytes of normal-weight (NW), overweight (OW), and type 2 diabetic (T2D) patients positively correlate with BMI and are increased in obesity and T2D. In contrast CCL4 is similarly abundant in the supernatants of all of these monocytes. The degree of adiponectin-mediated induction of the chemokines CCL3, -4, and -5 negatively correlates with their basal levels and upregulation of CCL3 and CCL5 is significantly impaired in OW and T2D cells. Serum concentrations of these chemokines are almost equal in the three groups and do not correlate with the levels in monocyte supernatants. In conclusion these data demonstrate that adiponectin stimulates release of CCL2 to CCL5 in primary human monocytes, and induction in cells of overweight probands is partly impaired. Adiponectin also lowers surface abundance of CCR2 and CCR5 and downregulation of CCR2 seems to depend on autocrine/paracrine effects of CCL2.
Assuntos
Adiponectina/farmacologia , Membrana Celular/metabolismo , Quimiocinas CC/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Receptores CCR2/metabolismo , Receptores CCR5/metabolismo , Adulto , Idoso , Peso Corporal/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/sangue , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL3/sangue , Quimiocina CCL3/genética , Quimiocina CCL3/metabolismo , Quimiocina CCL4/sangue , Quimiocina CCL4/genética , Quimiocina CCL4/metabolismo , Quimiocina CCL5/sangue , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiocinas CC/sangue , Quimiocinas CC/genética , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Monócitos/efeitos dos fármacos , Sobrepeso/sangue , Sobrepeso/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/imunologia , Regulação para Cima/efeitos dos fármacosRESUMO
Sjögren's syndrome (SS) is an autoimmune disease affecting exocrine glands, especially the salivary and lacrimal glands. Although most of the SS patients' sera have autoantibodies that can target a variety of antigens, it is not clear what determines which proteins will become autoantigens. The muscarinic receptor M3, an integral plasma membrane protein, has been proposed as a possible autoantigen in SS, and is endogenous in HeLa cells. The aim of this study was to develop a method that is able to separate and identify antigens recognised by sera from SS patients using lysates of HeLa and A-253 cells in 2D Western Blot (2DWB). The HeLa and A-253 cell lysates were fractionated in soluble and membrane-bound proteins, and the membrane-bound proteins were enriched for integral proteins. The fractions were tested using WB, confirming the presence of the main cell compartments. The rehydration solution containing ASB-14 performed better than the others in all three steps (active rehydration, focus and transfer), and efficiently separated the muscarinic receptor M3. The M3 receptor was also detected in lysates from A-253 cells. The presence of this receptor in this cell line has not been proven earlier. This work develops a suitable protocol to perform a mapping of the autoantibodies present in the sera of single SS patients, using lysates from epithelial cell lines that represent the main cell compartments as an antigen source. It is our future aim to use this protocol to perform a mapping of the antibodies present in the sera of individual SS patients.
Assuntos
Autoantígenos/imunologia , Fracionamento Celular/métodos , Proteoma/imunologia , Síndrome de Sjogren/imunologia , Autoanticorpos/imunologia , Autoantígenos/isolamento & purificação , Western Blotting/métodos , Linhagem Celular Tumoral , Hidratação/métodos , Células HeLa , Humanos , Integrinas/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/isolamento & purificação , Proteoma/isolamento & purificação , Receptor Muscarínico M3/imunologia , Frações Subcelulares/imunologiaRESUMO
Each year, during winter months, human Metapneumovirus (hMPV) is associated with epidemics of bronchiolitis resulting in the hospitalization of many infants. Bronchiolitis is an acute illness of the lower respiratory tract with a consequent inflammation of the bronchioles. The rapid onset of inflammation suggests the innate immune response may have a role to play in the pathogenesis of this hMPV infection. Since, the matrix protein is one of the most abundant proteins in the Paramyxoviridae family virion, we hypothesized that the inflammatory modulation observed in hMPV infected patients may be partly associated with the matrix protein (M-hMPV) response. By western blot analysis, we detected a soluble form of M-hMPV released from hMPV infected cell as well as from M-hMPV transfected HEK 293T cells suggesting that M-hMPV may be directly in contact with antigen presenting cells (APCs) during the course of infection. Moreover, flow cytometry and confocal microscopy allowed determining that M-hMPV was taken up by dendritic cells (moDCs) and macrophages inducing their activation. Furthermore, these moDCs enter into a maturation process inducing the secretion of a broad range of inflammatory cytokines when exposed to M-hMPV. Additionally, M-hMPV activated DCs were shown to stimulate IL-2 and IFN-γ production by allogeneic T lymphocytes. This M-hMPV-mediated activation and antigen presentation of APCs may in part explain the marked inflammatory immune response observed in pathology induced by hMPV in patients.
Assuntos
Imunidade/imunologia , Inflamação/imunologia , Metapneumovirus/imunologia , Proteínas da Matriz Viral/imunologia , Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/metabolismo , Antígenos CD1/metabolismo , Apoptose , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Endocitose , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Interferons/biossíntese , Receptores de Lipopolissacarídeos/metabolismo , Ativação Linfocitária/imunologia , Monócitos/citologia , Ligação Proteica , Frações Subcelulares/imunologia , Frações Subcelulares/virologia , Linfócitos T/imunologia , TransfecçãoRESUMO
The signaling and adaptor protein Homer3 plays a role in controlling immune homeostasis and self-reactivity. Homer3 is recruited to the immune synapse (IS) following TCR ligation, although the mechanisms regulating this subcellular localization are unknown. We show that Homer3 specifically associates with a novel ubiquitin-like domain in the IkappaB kinase (IKK) beta subunit of the IKK complex. Homer3 associates with IKKbeta in T cells and colocalizes with the IKK complex at the IS. However, Homer3 is not required for IKK activation, as NF-kappaB signaling is intact in Homer3-deficient T cells. Instead, the IKK complex recruits Homer3 to the IS following TCR engagement, and we present evidence that this association regulates actin dynamics in T cells. These findings identify a novel interaction between two major signaling proteins and reveal an unexpected NF-kappaB-independent function for the IKK complex in regulating the subcellular localization of Homer3.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas I-kappa B/metabolismo , Proteínas de Transporte/fisiologia , Linhagem Celular , Células HeLa , Proteínas de Arcabouço Homer , Humanos , Proteínas I-kappa B/fisiologia , Sinapses Imunológicas/enzimologia , Sinapses Imunológicas/imunologia , Células Jurkat , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Frações Subcelulares/imunologia , Frações Subcelulares/metabolismo , Ubiquitina/química , Ubiquitina/metabolismoAssuntos
Antígenos CD4/história , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Receptores Proteína Tirosina Quinases/história , Sequência de Aminoácidos , Animais , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Transformada , Detergentes , HIV/imunologia , HIV/patogenicidade , História do Século XX , Humanos , Ativação Linfocitária/imunologia , Camundongos , Dados de Sequência Molecular , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Frações Subcelulares/enzimologia , Frações Subcelulares/imunologia , Especificidade por Substrato/imunologia , Células U937RESUMO
Candida albicans, which represents one of the most important human pathogenic yeasts, is directly attacked by the host innate immune system upon infection. However this pathogen has developed multiple strategies to escape host immune defense. Here, we show that C. albicans secreted proteases interfere and inactivate host innate immune effector components, such as complement proteins. Secreted aspartic proteases (Saps) in the culture supernatant of C. albicans cells and also recombinant Sap1, Sap2 and Sap3 degrade host complement components C3b, C4b and C5 and also inhibit terminal complement complex (TCC) formation. This proteolytic activity is specific to the three recombinant and wild type Sap proteins. The triple knock out C. albicans strain Delta sap1-3 and also the non-pathogenic yeast S. cerevisiae lack such degrading activities. The complement inhibitory role of Sap1, Sap2 and Sap3 was confirmed in hemolysis assays with rabbit erythrocytes and normal human plasma. Secretion of complement degrading proteases provides a highly efficient complement defense response of this human pathogenic yeast that acts after the immediate acquisition of host complement regulators to the cell surface.