RESUMEN
Thrombin activation of C5 connects thrombosis to inflammation. Complement research in whole blood ex vivo necessitates anticoagulation, which potentially interferes with the inflammatory modulation by thrombin. We challenged the concept of thrombin as an activator of native C5 by analyzing complement activation and C5 cleavage in human whole blood anticoagulated with Gly-Pro-Arg-Pro (GPRP), a peptide targeting fibrin polymerization downstream of thrombin, allowing complete endogenous thrombin generation. GPRP dose-dependently inhibited coagulation but allowed for platelet activation in accordance with thrombin generation. Spontaneous and bacterial-induced complement activation by Escherichia coli and Staphylococcus aureus, analyzed at the level of C3 and C5, were similar in blood anticoagulated with GPRP and the thrombin inhibitor lepirudin. In the GPRP model, endogenous thrombin, even at supra-physiologic concentrations, did not cleave native C5, despite efficiently cleaving commercially sourced purified C5 protein, both in buffer and when added to C5-deficient serum. In normal serum, only exogenously added, commercially sourced C5 was cleaved, whereas the native plasma C5 remained intact. Crucially, affinity-purified C5, eluted under mild conditions using an MgCl2 solution, was not cleaved by thrombin. Acidification of plasma to pH ≤ 6.8 by hydrochloric or lactic acid induced a C5 antigenic change, nonreversible by pH neutralization, that permitted cleavage by thrombin. Circular dichroism on purified C5 confirmed the structural change during acidification. Thus, we propose that pH-induced conformational change allows thrombin-mediated cleavage of C5 and that, contrary to previous reports, thrombin does not cleave plasma C5 in its native form, suggesting that thrombin cleavage of C5 may be restricted to certain pathophysiological conditions.
Asunto(s)
Complemento C5 , Trombina , Coagulación Sanguínea , Activación de Complemento , Fibrina , HumanosRESUMEN
Dengue results in substantial human morbidity and significant socio-economic impacts, but a specific dengue therapeutic is not available. The currently available dengue vaccine has low efficacy and high rate of adverse effects, necessitating different strategies for the development of a safer and more efficient vaccine against dengue virus. We describe here a hybrid combination of different-sized gold nanoparticles (AuNPs) and domain III of envelope glycoprotein derived from serotype 2 of dengue virus (EDIII) as dengue subunit vaccine. The efficacy of the EDIII-functionalized AuNPs (AuNP-E) to induce neutralizing antibody in BALB/c mice is evaluated. Obtained results show that AuNP-E induced a high level of antibody which mediates serotype-specific neutralization of dengue virus. More importantly, the level of antibody is dependent on both the size of AuNPs and the concentration of AuNP-E, implicating the possibility to modulate it through adjusting these parameters. These results represent an important step towards the development of tetravalent AuNP-based subunit dengue vaccine. STATEMENT OF SIGNIFICANCE: This research presents a novel subunit vaccine against dengue virus using a hybrid comprising gold nanoparticles (AuNPs) and domain III of envelop protein (EDIII). We proved the neutralizing activity of anti-EDIII antibody induced in immunized mice on Dengue virus serotype 2 in an AuNP core size and concentration dependent manner. The hybrid concept behind this work could also be adopted for the development of a tetravalent vaccine against four serotypes of Dengue virus.
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Virus del Dengue/inmunología , Oro/química , Nanopartículas del Metal/química , Pruebas de Neutralización , Tamaño de la Partícula , Vacunas de Subunidad/inmunología , Animales , Anticuerpos Neutralizantes/metabolismo , Proliferación Celular , Activación de Complemento , Femenino , Sueros Inmunes , Inmunización , Nanopartículas del Metal/ultraestructura , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos , Células RAW 264.7 , Bazo/citología , Linfocitos T/inmunología , Distribución Tisular , Proteínas Virales/química , Proteínas Virales/inmunologíaRESUMEN
Gold nanoparticles (AuNPs) are widely used in biomedical applications, but much less is known about their immunological properties, particularly their interaction with the complement system, a key component of innate immunity serving as an indicator of their biocompatibility. Using a library of different-sized AuNPs (10, 20, 40, and 80 nm) passivated with polyethylene glycol (PEG) of different molecular weight ( Mw = 1, 2, 5, and 10 kDa), we demonstrated that citrate-capped AuNPs activated the whole complement system in a size-dependent manner, characterized by the formation of the end-point activation product, SC5b-9, in human serum. Although PEGylation of AuNPs mitigated, but did not abolish, the activation level, complement activation by PEGylated AuNPs was independent of both the core size of AuNPs and the molecular weight of PEG. The cellular uptake of both citrate-capped and PEGylated AuNPs by human U937 promonocytic cells which expresses complement receptors were highly correlated to the level of complement activation. Taken together, our results provided new insights on the innate complement activation by PEGylated AuNPs that are widely considered to be inert biocompatible nanomaterials.
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Materiales Biocompatibles Revestidos/efectos adversos , Activación de Complemento , Oro/efectos adversos , Nanopartículas del Metal/efectos adversos , Polietilenglicoles/efectos adversos , Ácido Cítrico/efectos adversos , Ácido Cítrico/química , Ácido Cítrico/inmunología , Ácido Cítrico/farmacocinética , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/farmacocinética , Activación de Complemento/efectos de los fármacos , Oro/química , Oro/inmunología , Oro/farmacocinética , Humanos , Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Tamaño de la Partícula , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Células U937RESUMEN
Gold nanoparticles passivated by polyelectrolyte ligands are widely used to confer stability and biofunctionality. While nanoparticles and polyelectrolytes have been reported as activators, their ability to activate the complement system as hybrid polyelectrolyte-coated nanoparticles is poorly characterized. Here, we found that gold nanoparticles passivated by common polyelectrolytes activated the system differently. The surface area of AuNPs appeared to be a major determinant of complement activation level as it determined the amount of adsorbed polyelectrolytes. Although a moderate negative correlation between AuNP surface hydrophilicity and their activation level was observed, the surface charge and functional group of polyelectrolyte ligands also influenced the final complement activation level.
RESUMEN
[This corrects the article DOI: 10.1039/C7RA13325A.].
RESUMEN
The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs in vitro, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.
Asunto(s)
Activación de Complemento/efectos de los fármacos , Proteínas del Sistema Complemento/farmacología , Oro/farmacología , Nanoestructuras , Adsorción , Complemento C1q/farmacología , Complemento C3b/farmacología , Humanos , Macrófagos/metabolismo , Nanomedicina , Células U937RESUMEN
A simple, rapid, and signal-on fluorescent assay was developed for activity analysis of DNA methyltransferase and for screening of its inhibitors based on a methylation-resistant endonuclease and SYBR Green I.
Asunto(s)
Metilación de ADN , Metilasas de Modificación del ADN/metabolismo , Endonucleasas/metabolismoRESUMEN
A highly sensitive and rapid PCR-free telomerase activity assay has been developed that uses SYBR Green intercalation into the G-quadruplex structures in the presence of K(+).