RESUMO
Tumour necrosis factor (TNF) is a cytokine belonging to a family of trimeric proteins; it has been shown to be a key mediator in autoimmune diseases such as rheumatoid arthritis and Crohn's disease. While TNF is the target of several successful biologic drugs, attempts to design small molecule therapies directed to this cytokine have not led to approved products. Here we report the discovery of potent small molecule inhibitors of TNF that stabilise an asymmetrical form of the soluble TNF trimer, compromising signalling and inhibiting the functions of TNF in vitro and in vivo. This discovery paves the way for a class of small molecule drugs capable of modulating TNF function by stabilising a naturally sampled, receptor-incompetent conformation of TNF. Furthermore, this approach may prove to be a more general mechanism for inhibiting protein-protein interactions.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Multimerização Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anti-Inflamatórios/uso terapêutico , Artrite Experimental/imunologia , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Masculino , Camundongos , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Estabilidade Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína/efeitos dos fármacos , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Transdução de Sinais/imunologia , Relação Estrutura-Atividade , Resultado do Tratamento , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/isolamento & purificação , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/ultraestruturaRESUMO
Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNFα). TNFα forms a stable homotrimer with unique surface-exposed sites for Adalimumab, Infliximab, and TNF receptor binding. Here, we report the structures of Adalimumab-TNFα and Infliximab-TNFα complexes modeled from negative stain EM and cryo-EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2, and 3:2 complexes of Adalimumab-TNFα and Infliximab-TNFα. The 2:2 complex structures of Adalimumab-TNFα and Infliximab-TNFα show diamond-shaped profiles and the 2D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNFα. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab-TNFα or Infliximab-TNFα complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cryo-EM analysis of 3:2 Adalimumab-TNFα complex generated a low-resolution density consistent with a TNFα trimer bound with three Fab domains from three individual antibody molecules, while each antibody molecule binds to two molecules of TNFα trimer. The Fc domains are not visible in the reconstruction. These results show the two mAbs form structurally distinct complexes with TNFα.
Assuntos
Adalimumab , Infliximab , Fator de Necrose Tumoral alfa , Adalimumab/química , Adalimumab/metabolismo , Adalimumab/ultraestrutura , Humanos , Infliximab/química , Infliximab/metabolismo , Infliximab/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Ligação Proteica , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/ultraestruturaRESUMO
This study examined the ultrastructural changes in the pulmonary mechanical barriers in a rat model of severe acute pancreatitis (SAP)-associated acute lung injury (ALI). Animals were randomized into the SAP group (n = 60) and the control group (n = 60). SAP was induced by retrograde injection of 5% taurocholic acid into the biliopancreatic duct. The morphological abnormalities assessed by histology and the lung wet/dry weight ratio and the ultrastructural abnormalities assessed by transmission electron microscope and scanning electron microscope examinations plus lanthanum nitrate tracing were compared between the two groups at 6, 12, and 24 h post-SAP induction (n = 10/group/time point). The SAP group had significantly greater extravascular effusion than the control group at each time point as assessed by the lung wet/dry weight ratio (p < .001). The severity of the tissue damage increased in the lung and pancreas over time in the SAP group (all p < .001). In the SAP group, ultrastructural damages to the endothelial, epithelial, and pleural barriers were apparent and the damages to the endothelial barrier were detected earlier than the other two barriers, suggesting its fundamental role in preventing the further development of SAP-associated ALI. Moreover, the ultrastructural abnormalities were detected earlier than symptoms and morphological changes. The ultrastructural damages in the endothelial, epithelial, and pleural barriers occurred in the early stage of SAP. The endothelial barrier is likely to be the first line to prevent the further development in this rat model of SAP-associated ALI.
Assuntos
Lesão Pulmonar Aguda/patologia , Lesão Pulmonar/patologia , Pulmão/ultraestrutura , Pancreatite/patologia , Fator de Necrose Tumoral alfa/ultraestrutura , Lesão Pulmonar Aguda/diagnóstico , Animais , Modelos Animais de Doenças , Pulmão/patologia , Masculino , Microscopia Eletrônica de Transmissão/métodos , Ratos Sprague-DawleyRESUMO
It is suggested that cholesterol efflux mediated by ATP binding cassette transporter A1 (ABCA1) plays an important role in anti-atherogenesis. However, the effects of inflammatory cytokines on ABCA1 expression and cholesterol accumulation in foam cells are little known. This study investigates the effects of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells. ABCA1mRNA and protein levels were determined by RT-PCR and Western blot, respectively. The total cholesterol content in THP-1 macrophage-derived foam cells was detected by the zymochemistry method. Results revealed that TNF-alpha could increase cholesterol content by down-regulating ABCA1 expression in a time-dependent manner in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. In addition IL-10 time-dependently decreased cholesterol accumulation by up-regulating ABCA1 expression and inhibited the down-regulation of ABCA1 by TNF-alpha in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Células Espumosas/metabolismo , Interleucina-10/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Linhagem Celular , Colesterol/metabolismo , Regulação para Baixo , Células Espumosas/citologia , Humanos , Lipoproteínas LDL/metabolismo , Monócitos/citologia , Monócitos/metabolismo , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/ultraestrutura , Regulação para CimaRESUMO
Activated macrophages secrete an array of proinflammatory cytokines, including tumor necrosis factor-alpha (TNFalpha) and interleukin 6 (IL-6), that are temporally secreted for sequential roles in inflammation. We have previously characterized aspects of the intracellular trafficking of membrane-bound TNFalpha and its delivery to the cell surface at the site of phagocytic cups for secretion (Murray, R.Z., J.G. Kay, D.G. Sangermani, and J.L. Stow. 2005. Science. 310:1492-1495). The trafficking pathway and surface delivery of IL-6, a soluble cytokine, were studied here using approaches such as live cell imaging of fluorescently tagged IL-6 and immunoelectron microscopy. Newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers either as the sole labeled cargo or together with TNFalpha, utilizing specific soluble NSF attachment protein receptor (SNARE) proteins to fuse with the recycling endosome. Within recycling endosomes, we demonstrate the compartmentalization of cargo proteins, wherein IL-6 is dynamically segregated from TNFalpha and from surface recycling transferrin. Thereafter, these cytokines are independently secreted, with TNFalpha delivered to phagocytic cups but not IL-6. Therefore, the recycling endosome has a central role in orchestrating the differential secretion of cytokines during inflammation.
Assuntos
Endossomos/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , DNA Complementar/genética , Eletroporação , Endossomos/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Interleucina-6/análise , Macrófagos/ultraestrutura , Camundongos , Microscopia de Fluorescência , RNA Interferente Pequeno/farmacologia , Rodaminas/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/ultraestruturaRESUMO
BACKGROUND: The heart undergoes repair and initiates protective mechanisms via ventricular unloading. We examined the presence of 2 markers in pre-unloaded and post-unloaded human cardiac tissue that are important indicators of cardiac failure, tumor necrosis factor-alpha and inducible nitric oxide synthase. We also measured 2 nuclear transcription factors, NFkappaB50 and NFkappaB65, comparing quantities and localizations to determine if mechanical unloading reduced their presence, as these markers are also thought to be indicators of impending heart failure. Amounts and localizations in patients that had been diagnosed with either ischemic or non-ischemic cardiomyopathy were compared after mechanical unloading with a left ventricular assist device. To establish that unloading had been achieved, levels of atrial natriuretic protein were determined. METHODS: Core biopsies were harvested at assist device implantation and removal. Fluorescence deconvolution microscopy image reconstructions of fluorescence probes were correlated with data obtained by western Blot and electrobility shift assays. RESULTS: Statistically significant differences in localization and amounts of tumor necrosis factor and nitric oxide synthase were seen between pre- and post-assist device samples. Amounts of tumor necrosis factor and nitric oxide synthase in ischemic tissue were increased at the time of assist device removal, but decreased in dilated or idiomyopathic samples. Ventricular unloading resulted in reduced levels of natriuretic protein, with the greatest reduction being seen in ischemic tissue. Both NFkappaB50 and NFkappaB65 increased in ischemic tissue, but only NFkappaB50 in non-ischemic samples. CONCLUSIONS: Changes in localization of the factors and altered levels of cytokine and nitric oxide synthase indicate that the heart switches to a "protective and repair" mode, and mechanical unloading allows this transition to occur. Observed changes were dependent on the etiology of the disease.
Assuntos
Fator Natriurético Atrial/ultraestrutura , Cardiomiopatia Dilatada/metabolismo , Coração Auxiliar , Isquemia Miocárdica/metabolismo , Miocárdio/ultraestrutura , Óxido Nítrico Sintase/ultraestrutura , Fator de Necrose Tumoral alfa/ultraestrutura , Fator Natriurético Atrial/metabolismo , Biomarcadores/metabolismo , Biópsia , Western Blotting , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/terapia , Remoção de Dispositivo , Eletroforese , Ventrículos do Coração/metabolismo , Ventrículos do Coração/ultraestrutura , Humanos , Microscopia de Fluorescência , Isquemia Miocárdica/patologia , Isquemia Miocárdica/terapia , Miocárdio/metabolismo , Óxido Nítrico Sintase/metabolismo , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/metabolismoRESUMO
TALL-1/BAFF/BLyS was recently identified as a member of the tumor necrosis factor (TNF) ligand family. The crystal structure of the functional soluble TALL-1 (sTALL-1) has been determined at 3.0 A. sTALL-1 forms a virus-like assembly with 200 A diameter in the crystals, containing 60 sTALL-1 monomers. The cluster formation is mediated by a "flap" region of the sTALL-1 monomer. The virus-like assembly was also detected in solution using gel filtration and electron microscopy. Deletion of the flap region disrupted the formation of the virus-like assembly. The mutant sTALL-1 still bound its receptor but could not activate NF-kappaB and did not stimulate B lymphocyte proliferation. Finally, we found the virus-like cluster of sTALL-1 exists in physiological condition. We propose that this virus-like assembly of sTALL-1 is the functional unit for TALL-1 in vivo.
Assuntos
Proteínas de Membrana/química , Fator de Necrose Tumoral alfa/química , Sequência de Aminoácidos , Fator Ativador de Células B , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Receptores do Fator de Necrose Tumoral/metabolismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/ultraestrutura , Montagem de VírusRESUMO
In this study, the prevention of rat cytomegalovirus (RCMV) infection-enhanced experimental obliterative bronchiolitis in rat tracheal allografts was investigated. RCMV infection markedly enhanced cell proliferation and histological changes of obliterative bronchiolitis, a form of chronic rejection after lung transplantation. These alterations were linked to increased interleukin (IL)-2 and tumor necrosis factor-alpha (TNF-alpha) immunoreactivity, and reduction of IL-10 expression. In recipient rats with acute RCMV infection, prophylaxis with either ganciclovir (DHPG) or hyperimmune serum (HIS) totally prevented RCMV infection-enhanced tracheal occlusion. DHPG treatment initiated during acute RCMV infection also reduced lesion development but markedly less than DHPG prophylaxis. Treatment of acute RCMV infection with HIS alone or in combination with DHPG had no significant effect on tracheal occlusion. Inhibition of the transcription of cytokines by high doses of cyclosporine A significantly reduced RCMV infection-enhanced tracheal obliteration. In rats with chronic RCMV infection, obliterative alterations were prevented by DHPG prophylaxis initiated at the time of transplantation. Prophylaxis either with DHPG or HIS did not affect the amount of infectious RCMV recovered from host salivary glands, nor were there differences seen in RCMV major immediate early DNA expression in tracheal allografts between different antiviral drug regimens. Immunohistochemical analysis of allografts revealed that inhibition of tracheal occlusion by antiviral prophylaxis was associated with a reduction in the number of ED1(+) macrophages and cells staining for Th1 cytokines and TNF-alpha, while immune modulation by cyclosporine A up-regulated IL-10 production. In conclusion, the results of the present study suggest that the CMV infection-enhanced chronic rejection develops independently of viral load but requires both immune activation and simultaneous CMV gene expression beyond immediate early genes.
Assuntos
Antivirais/uso terapêutico , Bronquiolite Obliterante/prevenção & controle , Bronquiolite Obliterante/virologia , Infecções por Citomegalovirus/complicações , Modelos Animais de Doenças , Ganciclovir/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Rejeição de Enxerto/virologia , Soros Imunes/administração & dosagem , Transplante de Pulmão/efeitos adversos , Pré-Medicação/métodos , Doença Aguda , Animais , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Doença Crônica , Citomegalovirus/genética , Avaliação Pré-Clínica de Medicamentos , Regulação Viral da Expressão Gênica , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Imuno-Histoquímica , Macrófagos Alveolares/ultraestrutura , Masculino , Ratos , Ratos Endogâmicos , Células Th1/ultraestrutura , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/ultraestrutura , Carga ViralRESUMO
Peripheral blood eosinophils from two normal donors and two patients with the hypereosinophilic syndrome (HES) were analyzed with a post-embedding immunogold method to detect the substructural location of tumor necrosis factor-alpha (TNF-alpha). In eosinophils of HES patients, TNF-alpha was localized to the matrix compartment of 64% of the specific secondary granules. Other structures in the HES eosinophils were unlabeled. No TNF-alpha was detected in eosinophils of normal donors. These studies document the first ultrastructural subcellular localization of any cytokine within the major population of secretory granules in human eosinophils and support other lines of evidence indicating that the expression of TNF-alpha may be greater in the eosinophils of HES patients than in those of normal donors.
Assuntos
Grânulos Citoplasmáticos/ultraestrutura , Eosinófilos/ultraestrutura , Matriz Extracelular/ultraestrutura , Síndrome Hipereosinofílica/patologia , Microscopia Imunoeletrônica , Fator de Necrose Tumoral alfa/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Eosinófilos/metabolismo , Matriz Extracelular/metabolismo , Humanos , Síndrome Hipereosinofílica/metabolismo , Imuno-Histoquímica , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Comprovado experimentalmente o efeito anti-tumoral do Staphylococcus aureus sobre um sarcoma transplantado em ratos(S-E 100).Ratos adultos endocriados e inoculados s.c.com S-E 100 receberam via s.c. uma suspensäo de 8*10elevado a sexta S.aureus mortos por tindalizaçäo.Grupos experimentais:1)S-E 100+S.aureus 7 dias antes:2)S-E 100+S.aureus simultaneamente;3)S-E 100+S.aureus 7 dias depois;4)S-E 100 (grupo de controle).A incidência do S-E 100 näo modificou de forma significativa nos grupos experimentais.As medidas efetuadas nos dias 7, 14, 21, 28 depois de inoculado o tumor evidenciaram que os animais desafiados, prévia e simultaneamente com S.aureus apresentavam tumores com um tamanho significativamente menor que o grupo de controle.Nos animais que pertenciam aos grupos 1 e 2, o período de sobrevivência foi significativamente superior ao grupo controle, mas a porcentagem de mortalidade näo foi diferente entre os grupos.O estudo histológico do tumor evidenciou uma maior superfície de tecido necrosado, sete(7) dias antes do desafio tumoral com o microorganismo, e nos ratos inoculados simultaneamente, em relaçäo aos animais que pertenciam ao grupo de controle.Conclusäo:S.aureus quando é inoculado antes(vacina)ou em forma simultânea ao desafio com sarcoma E-100 exerce uma inibiçäo do crescimento do tumor e também induz um aumento no processo de necrose do tumor além de aumentar o período de sobrevida dos animais.