RESUMO
While vaccination against HIV-1 has been so far unsuccessful, recently broadly neutralizing antibodies (bNAbs) against HIV-1 envelope glycoprotein were shown to induce long-term suppression in the absence of antiretroviral therapy in patients with antibody-sensitive viral reservoirs. The requirement of neutralizing antibodies indicates that the antibody mediated removal (clearance) of HIV-1 in itself is not efficient enough in these immune compromised patients. Here we present a novel, alternative approach that is independent of a functional immune system to clear HIV-1, by capturing the virus and redirecting it to non-target cells where it is internalized and degraded. We use bispecific antibodies with domains derived from small single chain Llama antibodies (VHHs). These bind with one domain to HIV-1 envelope proteins and with the other domain direct the virus to cells expressing epidermal growth factor receptor (EGFR), a receptor that is ubiquitously expressed in the body. We show that HIV envelope proteins, virus-like particles and HIV-1 viruses (representing HIV-1 subtypes A, B and C) are efficiently recruited to EGFR, internalized and degraded in the lysosomal pathway at low nM concentrations of bispecific VHHs. This directed degradation in non-target cells may provide a clearance platform for the removal of viruses and other unwanted agents from the circulation, including toxins, and may thus provide a novel method for curing.
Assuntos
Camelídeos Americanos , Infecções por HIV , Soropositividade para HIV , HIV-1 , Anticorpos de Cadeia Única , Animais , Anticorpos Neutralizantes , Receptores ErbB , Anticorpos Anti-HIV , HumanosRESUMO
Extracellular vesicles (EVs) are nanoparticles which are released by cells from all three domains of life: Archaea, Bacteria and Eukarya. They can mediate cell-cell communication by transferring cargoes such as proteins and nucleic acids between cells. EVs receive great interest in both academia and industry as they have the potential to be natural drug carriers or vaccine candidates. However, limitations to their clinical translation exist as efficient isolation, loading, labelling and surface-engineering methods are lacking. In this article, we investigate a 'post-insertion' approach, which is commonly used in the functionalization of liposomes in the pharmaceutical field, on two different EV types: mammalian cell-derived EVs and bacteria-derived EVs. We aimed to find an easy and flexible approach to functionalize EVs, thereby improving the labelling, isolation, and surface-engineering.
Assuntos
Bactérias/química , Membrana Externa Bacteriana/química , Vesículas Extracelulares/química , Imuno-Histoquímica/métodos , Animais , Membrana Externa Bacteriana/ultraestrutura , Western Blotting/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida/métodos , Vesículas Extracelulares/ultraestrutura , Citometria de Fluxo/métodos , Células HEK293 , Humanos , Camundongos , Microscopia Eletrônica de Transmissão/métodos , Propriedades de SuperfícieAssuntos
Betacoronavirus , Bradicinina/metabolismo , Infecções por Coronavirus/complicações , Pandemias , Pneumonia Viral/complicações , Edema Pulmonar/etiologia , Angiotensina II/fisiologia , Enzima de Conversão de Angiotensina 2 , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Animais , Bradicinina/análogos & derivados , COVID-19 , Embrião de Galinha , Infecções por Coronavirus/metabolismo , Modelos Animais de Doenças , Humanos , Cininogênios/metabolismo , Camundongos , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/metabolismo , Sistema Renina-Angiotensina/fisiologia , SARS-CoV-2RESUMO
Factor XII (FXII) zymogen activation requires cleavage after arginine 353 located in the activation loop. This cleavage can be executed by activated FXII (autoactivation), plasma kallikrein (PKa), or plasmin. Previous studies proposed that the activation loop of FXII is shielded to regulate FXII activation and subsequent contact activation. In this study, we aimed to elucidate this mechanism by expressing and characterizing seven consecutive N-terminally truncated FXII variants as well as full-length wild-type (WT) FXII. As soon as the fibronectin type II domain is lacking (FXII Δ1-71), FXII cleavage products appear on Western blot. These fragments display spontaneous amidolytic activity, indicating that FXII without the fibronectin type II domain is susceptible to autoactivation. Additionally, truncated FXII Δ1-71 is more easily activated by PKa or plasmin than full-length WT FXII. To exclude a contribution of autoactivation, we expressed active-site incapacitated FXII truncation variants (S544A). FXII S544A Δ1-71 is highly susceptible to cleavage by PKa, indicating exposure of the activation loop. In surface binding experiments, we found that the fibronectin type II domain is non-essential for binding to kaolin or polyphosphate, whereas the following epidermal growth factor-like domain is indispensable. Binding of full-length FXII S544A to kaolin or polyphosphate increases its susceptibility to cleavage by PKa. Moreover, the activation of full-length WT FXII by PKa increases approximately threefold in the presence of kaolin. Deletion of the fibronectin type II domain eliminates this effect. Combined, these findings suggest that the fibronectin type II domain shields the activation loop of FXII, ensuring zymogen quiescence.
Assuntos
Precursores Enzimáticos/química , Fator XII/química , Fibrinolisina/química , Fibronectinas/química , Calicreínas/química , Animais , Sítios de Ligação , Coagulação Sanguínea , Bradicinina/química , Domínio Catalítico , Bovinos , Fator XIIa/química , Fibronectinas/sangue , Células HEK293 , Humanos , Calicreínas/sangue , Caulim/química , Polifosfatos/química , Ligação Proteica , Domínios ProteicosRESUMO
Heparin is a widely used anticoagulant which inhibits factor Xa and thrombin through potentiation of antithrombin. We recently identified that the nucleic acid stain SYTOX reacts with platelet polyphosphate due to molecular similarities, some of which are shared by heparin. We attempted to study heparin in flowing blood by live-cell fluorescence microscopy, using SYTOX for heparin visualisation. Immunostaining was performed with monoclonal antibodies directed against various heparin-binding proteins. In addition, we studied modulation of heparin activity in coagulation assays, as well its effects on fibrin formation under flow in recalcified whole blood. We found that SYTOX-positive polymers appear in heparinised blood under flow. These polymers typically associate with platelet aggregates and their length (reversibly) increases with shear rate. Immunostaining revealed that of the heparin-binding proteins assessed, they only contain histones. In coagulation assays and flow studies on fibrin formation, we found that addition of exogenous histones reverses the anticoagulant effects of heparin. Furthermore, the polymers do not appear in the presence of DNase I, heparinase I/III, or the heparin antidote protamine. These findings suggest that heparin forms polymeric complexes with cell-free DNA in whole blood through a currently unidentified mechanism.
Assuntos
Anticoagulantes/sangue , Ácidos Nucleicos Livres/sangue , Heparina/sangue , Histonas/sangue , Plaquetas/metabolismo , Humanos , Compostos Orgânicos , Polímeros/metabolismo , Resistência ao CisalhamentoRESUMO
ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system. We next investigated how plasmin affects both VWF and ADAMTS13 under static conditions in plasma by western blotting. We found that globular VWF is largely protected from plasmin cleavage. However, ADAMTS13 is rapidly cleaved under these conditions, suggesting inactivation. Surprisingly, we observed that plasmin enhances ADAMTS13 activity in a modified two-stage FRETS-VWF73 assay that protects FRETS-VWF73 substrate from degradation. In direct binding studies under the same conditions, we found that plasmin generates multiple C-terminally truncated forms of ADAMTS13 with VWF-binding capacity. In an effort to seek evidence for this mechanism in vivo, we analysed plasma from patients with systemic amyloidosis, which is hallmarked by a hyperfibrinolytic state. We found that their plasma contained increased levels of C-terminally truncated forms of ADAMTS13, which correlated with their hyperfibrinolytic state.We propose that truncation of ADAMTS13 by plasmin abolishes intramolecular self-association, which improves interaction with unfolded VWF.
Assuntos
Proteína ADAMTS13/química , Amiloidose/imunologia , Fibrinolisina/química , Proteínas Recombinantes/química , Fator de von Willebrand/química , Proteína ADAMTS13/sangue , Motivos de Aminoácidos , Amiloidose/sangue , Sítios de Ligação , Fibrinólise , Humanos , Immunoblotting , Peptídeos/química , Ligação Proteica , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes/sangueRESUMO
Increased procoagulant activity in the alveolar compartment and uncontrolled inflammation are hallmarks of the acute respiratory distress syndrome (ARDS). Here, we investigated whether the contact phase system of coagulation is activated and may regulate inflammatory responses in human lungs. Components of the contact phase system were characterized in bronchoalveolar lavage fluids (BALF) from 54 ARDS patients and 43 controls, and their impact on cytokine/chemokine expression in human precision cut lung slices (PCLS) was assessed by a PCR array. Activation of the contact system, associated with high levels of coagulation factor XIIa (Hageman factor, FXIIa), plasma kallikrein and bradykinin, occurred rapidly in ARDS lungs after the onset of the disease and virtually normalized within one week from time of diagnosis. FXII levels in BALF were higher in ARDS non-survivors than survivors and were positively correlated with tumor necrosis factor (TNF)-α concentration. FXII induced the production and release of interleukin (IL)-8, IL-1ß, IL-6, leukemia inhibitory factor (LIF), CXCL5 and TNF-α in human PCLS in a kallikrein-kinin-independent manner. In conclusion, accumulation of FXII in ARDS lungs may contribute to the release of pro-inflammatory mediators and is associated with clinical outcome. FXII inhibition may thus offer a novel and promising therapeutic approach to antagonize overwhelming inflammatory responses in ARDS lungs without interfering with vital haemostasis.
Assuntos
Coagulação Sanguínea , Citocinas/metabolismo , Fator XII/metabolismo , Mediadores da Inflamação/metabolismo , Pulmão/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Adulto , Líquido da Lavagem Broncoalveolar/química , Citocinas/genética , Feminino , Humanos , Pulmão/imunologia , Masculino , Pessoa de Meia-Idade , Pneumonia/sangue , Pneumonia/genética , Pneumonia/imunologia , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/imunologia , Estudos Retrospectivos , Transdução de Sinais , Adulto JovemRESUMO
Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/metabolismo , Polifosfatos/metabolismo , Plaquetas/química , Plaquetas/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Fator XII/metabolismo , Humanos , Nanopartículas/química , Polifosfatos/farmacologiaRESUMO
RATIONALE: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. OBJECTIVES: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. METHODS: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. CONCLUSIONS: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.
Assuntos
Proteína Inibidora do Complemento C1/farmacologia , Histonas/metabolismo , Lesão Pulmonar/prevenção & controle , Síndrome do Desconforto Respiratório/fisiopatologia , Animais , Líquido da Lavagem Broncoalveolar , Proteína Inibidora do Complemento C1/metabolismo , Modelos Animais de Doenças , Humanos , Pulmão/metabolismo , Pulmão/fisiopatologia , Lesão Pulmonar/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting translational failure. Experimentally, MI is induced artificially and the effect of the experimental procedures may influence outcome and thus clinical applicability. The aim of this study was to investigate if invasive surgery, as in the common open chest MI model affects IS and cardiac function. Twenty female landrace pigs were subjected to MI by transluminal balloon occlusion. In 10 of 20 pigs, balloon occlusion was preceded by invasive surgery (medial sternotomy). After 72 hrs, pigs were subjected to echocardiography and Evans blue/triphenyl tetrazoliumchloride double staining to determine IS and area at risk. Quantification of IS showed a significant IS reduction in the open chest group compared to the closed chest group (IS versus area at risk: 50.9 ± 5.4% versus 69.9 ± 3.4%, P = 0.007). End systolic LV volume and LV ejection fraction measured by echocardiography at follow-up differed significantly between both groups (51 ± 5 ml versus 65 ± 3 ml, P = 0.033; 47.5 ± 2.6% versus 38.8 ± 1.2%, P = 0.005). The inflammatory response in the damaged myocardium did not differ between groups. This study indicates that invasive surgery reduces IS and preserves cardiac function in a porcine MI model. Future studies need to elucidate the effect of infarct induction technique on the efficacy of pharmacological therapies in large animal cardioprotection studies.