Introduction. We have previously shown that mannose binding lectin (MBL), one of the main activators of the lectin complement pathway, is deposited on the injured tissue after cerebral ischemia and brain trauma (Orsini et al., 2012, Longhi et al., 2014). Our hypothesis is that MBL binds de-novo expressed mannose arrays on the injured endothelial cells through its carbohydrate recognition domains, leading to complement activation and brain injury. Accordingly, the reduction of MBL deposition on the endothelium results in a protective effect against brain damage. We have now established a quick ex vivo assay to assess the MBL binding properties in plasma samples, predictive of its binding to the endothelium, to be used for screening novel inhibitors of MBL deposition. Methods. An in vitro MBL binding competition assay using surface plasmon resonance (SPR) technique was initially carried out to evaluate the MBL-binding properties of a newly synthesized poly-mannosylated dendrimer, Polyman 9. To this aim, recombinant mouse MBL-C (rmMBL-C), one of the two mouse isoforms, and human MBL (rhMBL), were pre-incubated with different concentrations of Polyman 9, and flowed on a sensor chip coated with mannosylated-BSA. For the ex vivo assay, plasma from humans and naïf C57Bl/6 or MBL knock out (MBL-/-) mice were incubated for 2h on mannan-coated plates. MBL-C or hMBL binding were detected using specific primary and biotinylated secondary antibodies plus 3,3’,5,5’-tetramethylbenzidine substrate, followed by spectrophotometer reading. This assay was then used to quantify the MBL-C binding in plasma samples obtained from brain injured (controlled cortical impact, CCI) C57Bl/6 mice, intravenously injected with saline or Polyman 9 (294 μg/mouse) 10 min after injury. Results. In vitro, SPR studies showed that pre-incubation of both rmMBL-C and rhMBL with Polyman 9 reduced their binding to mannosylated-BSA with IC50= 20μM and 158μM, respectively. Using the ex vivo mannan assay we found that both MBL-C and hMBL from plasma samples bound mannan-coated plates in a concentration-dependent manner and that the pre-incubation of plasma samples with mannose reduced this binding with IC50s of 16 mM and 8.7 mM, respectively. Treatment of brain injured mice with Polyman 9 significantly reduced by 22% the ability of circulating MBL-C to bind mannans compared to saline treatment. Conclusions. We established a reliable ex vivo assay that allows to measure the binding of plasmatic MBL binding to mannans, thus mimicking in vivo condition. We confirmed that this assay allows the screening of novel MBL inhibitors in relevant in vivo settings that could be used to develop new therapeutic molecules.

A novel assay to predict mannose binding lectin deposition on the activated endothelium, a key pathogenic event in acute brain injury / D. De Blasio, F. Orsini, G. Vegliante, S. Fumagalli, L. Longhi, A. Palmioli, M. Stravalaci, M. Gobbi, A. Bernardi, M. De Simoni. ((Intervento presentato al convegno Brain Ischemia and Stroke Conference tenutosi a Roma nel 2014.

A novel assay to predict mannose binding lectin deposition on the activated endothelium, a key pathogenic event in acute brain injury

L. Longhi;A. Palmioli;A. Bernardi;
2014

Abstract

Introduction. We have previously shown that mannose binding lectin (MBL), one of the main activators of the lectin complement pathway, is deposited on the injured tissue after cerebral ischemia and brain trauma (Orsini et al., 2012, Longhi et al., 2014). Our hypothesis is that MBL binds de-novo expressed mannose arrays on the injured endothelial cells through its carbohydrate recognition domains, leading to complement activation and brain injury. Accordingly, the reduction of MBL deposition on the endothelium results in a protective effect against brain damage. We have now established a quick ex vivo assay to assess the MBL binding properties in plasma samples, predictive of its binding to the endothelium, to be used for screening novel inhibitors of MBL deposition. Methods. An in vitro MBL binding competition assay using surface plasmon resonance (SPR) technique was initially carried out to evaluate the MBL-binding properties of a newly synthesized poly-mannosylated dendrimer, Polyman 9. To this aim, recombinant mouse MBL-C (rmMBL-C), one of the two mouse isoforms, and human MBL (rhMBL), were pre-incubated with different concentrations of Polyman 9, and flowed on a sensor chip coated with mannosylated-BSA. For the ex vivo assay, plasma from humans and naïf C57Bl/6 or MBL knock out (MBL-/-) mice were incubated for 2h on mannan-coated plates. MBL-C or hMBL binding were detected using specific primary and biotinylated secondary antibodies plus 3,3’,5,5’-tetramethylbenzidine substrate, followed by spectrophotometer reading. This assay was then used to quantify the MBL-C binding in plasma samples obtained from brain injured (controlled cortical impact, CCI) C57Bl/6 mice, intravenously injected with saline or Polyman 9 (294 μg/mouse) 10 min after injury. Results. In vitro, SPR studies showed that pre-incubation of both rmMBL-C and rhMBL with Polyman 9 reduced their binding to mannosylated-BSA with IC50= 20μM and 158μM, respectively. Using the ex vivo mannan assay we found that both MBL-C and hMBL from plasma samples bound mannan-coated plates in a concentration-dependent manner and that the pre-incubation of plasma samples with mannose reduced this binding with IC50s of 16 mM and 8.7 mM, respectively. Treatment of brain injured mice with Polyman 9 significantly reduced by 22% the ability of circulating MBL-C to bind mannans compared to saline treatment. Conclusions. We established a reliable ex vivo assay that allows to measure the binding of plasmatic MBL binding to mannans, thus mimicking in vivo condition. We confirmed that this assay allows the screening of novel MBL inhibitors in relevant in vivo settings that could be used to develop new therapeutic molecules.
Settore CHIM/06 - Chimica Organica
Settore BIO/10 - Biochimica
A novel assay to predict mannose binding lectin deposition on the activated endothelium, a key pathogenic event in acute brain injury / D. De Blasio, F. Orsini, G. Vegliante, S. Fumagalli, L. Longhi, A. Palmioli, M. Stravalaci, M. Gobbi, A. Bernardi, M. De Simoni. ((Intervento presentato al convegno Brain Ischemia and Stroke Conference tenutosi a Roma nel 2014.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/251817
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