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Assay for the pattern-recognition molecule CL-L1
1140
Abstracts / Immunobiology 217 (2012) 1129–1222
31 Anticomplementary activity of horse IgG and F(ab )2 antivenoms Carla Cristina Squaiella-Baptistão 1 , Jose Roberto Marcelino 2 , Luiz Eduardo Ribeiro da Cunha 3 , José María Gutiérrez 4 , Denise V. Tambourgi 1 1
Laboratório de Imunoquímica, Instituto Butantan, São Paulo, SP, Brazil 2 Sec¸ão de Processamento de Plasmas Hiperimunes, Instituto Butantan, São Paulo, SP, Brazil 3 Instituto Vital Brazil, Niterói, Rio de Janeiro, Brazil 4 Instituto Clodomiro Picado, Universidad de Costa Rica, San José, Costa Rica Envenomation by poisonous animals is a tropical neglected disease according to the World Health Organization. The only effective treatment is the antivenom therapy. Antivenoms are in the WHO List of Essential Medicines and should be part of any primary health care package where snakebite accidents occur. Intact heterologous antibodies can activate the complement system (C) via Fc, leading to early adverse reactions, which justify the production of F(ab )2 antivenoms. However, studies have indicated that these fragments can also activate C, leading to such reactions. Herein, we aimed to evaluate the anticomplementary activity of antivenoms (horse antibodies against snakes, spiders, scorpions or caterpillar venoms) produced by Butantan, Vital Brazil and Clodomiro Picado Institutes. These antivenoms have different composition, with the presence or not of Fc portion, and different protein contents. Samples of the antivenoms were incubated with normal human serum and the activation of the three C-pathways was analyzed. Only the classical pathway was activated by some antivenoms from the three Institutes, indicating that anticomplementary activity is not only associated with the presence of the IgG-Fc portion, since F(ab )2 antivenoms also activated the classical pathway. The C-activation/consumption was positively correlated with the amount of heterologous protein only for the antivenom from Vital Brazil Institute. SDS-PAGE showed the presence of several contaminant proteins in most antivenoms, and protein aggregates were detected in those from Clodomiro Picado Institute. Western Blot analysis showed that some antivenoms from Butantan Institute contained some intact IgG heavy chains, indicating that the cleavage of the antibodies for the production of F(ab )2 fragments was not complete. Our results suggest that several factors, such as composition, contaminant proteins and aggregates, are contributing for the C-activation/consumption by the antivenoms. Support: FAPESP, INCTTOX, CNPq. http://dx.doi.org/10.1016/j.imbio.2012.08.032 32 Assay for the pattern-recognition molecule CL-L1 Esben Axelgaard, Lisbeth Jensen, Steffen Thiel, Jens Chr. Jensenius Department of Biomedicine, Aarhus University, Aarhus, Denmark Pattern-recognition molecules of the innate immune system have been an area of intense research the last decade. The pattern recognition molecules belonging to the group of collectins are C-type lectins showing diverse binding patterns towards carbohydrate antigen structures on microorganisms and endogenous non-self structures in a calcium-dependent manner. The collectins are made up of polypeptides consisting of an N-terminal cross-linking region (CLR), where Cys–Cys disulphide bonds allow formation of higher oligomeric structures, a collagen-like region
with repeated Gly-Xaa-Yaa motifs, an alpha-helical neck region that induces trimerization of the polypeptide chains through the formation of a coiled-coil, and finally a C-terminal carbohydrate recognition domain (CRD), which facilitates the binding to ligands upon recognition. Structural basis and biological activity of collectins, like mannan-binding lectin (MBL), surfactant protein A (SP-A) and surfactant protein D (SP-D), have been quite extensively studied, and much is now know about these collectins. Recently, however, three further collectins, collectin liver 1 (CLL1, also termed collectin-10), collectin kidney 1 (CL-K1, also termed CL-11 and collectin-11) and collectin placenta 1 (CL-P1 also termed collectin-12) were discovered by the Wakamiya group. CL-K1 shows binding activity towards carbohydrate structures and association with MBL-associated serine proteases (MASPs), indicating a possible role in innate immunity. The structure and function of CLL1 remain largely unknown. We present a sandwich-type TRIFMA assay specific for the measurement of CL-L1 in solutions and analyze the physiological concentrations of this protein in normal and acute-phase conditions. http://dx.doi.org/10.1016/j.imbio.2012.08.033 33 Autoregulation of thromboinflammation on biomaterials and cells by a novel therapeutic coating technique Yuji Teramura 1 , Per H. Nilsson 2 , Kristina N. Ekdahl 2 , Peetra U. Magnusson 1 , Hongchang Qu 3 , Daniel Ricklin 3 , Jaan Hong 1 , John D. Lambris 3 , Bo Nilsson 1 1 Department of Immunology, Genetics and Pathology, Uppsala University, Sweden 2 School of Natural Sciences, Linnaeus University, Kalmar, Sweden 3 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Activation of the coagulation and complement systems provide the main recognition and effector mechanisms in the multiple biological responses triggered when biomaterials, therapeutic cells and whole organs come into contact with whole blood. Activation of these cascade systems can result in serious side effects of the biomaterial or cells. In this study, we created a self-regulatory surface to attenuate coagulation and complement activation on both cell and material surfaces by targeting ADP-dependent platelet aggregation/activation and the complement alternative pathway. The ADP-degrading enzyme apyrase and a factor H-binding peptide were co-immobilized onto cell and material surfaces using a blood compatible PEG linker. We demonstrated in human plasma/wholeblood models that platelet activation/coagulation can be regulated simultaneously with complement activation, by co-immobilizing a factor H-binding peptide and apyrase on either material (glass) or cell (rabbit erythrocytes, porcine aortic endothelial cells) surfaces when exposed to human plasma or whole blood. Factor H was effectively recruited by the factor H-binding peptide from blood plasma to both types of surfaces and was able to inhibit complement activation. Apyrase was shown to degrade ADP, thereby suppressing platelet and coagulation activation under the same conditions. In summary, we have inhibited thromboinflammation by using a multicomponent approach, which created a hybrid surface that makes the surface blood compatible and which promises to reduce incompatibility reactions in treatments involving biomaterials and in transplantation of cells and whole organs. http://dx.doi.org/10.1016/j.imbio.2012.08.034
Abstracts / Immunobiology 217 (2012) 1129–1222
31 Anticomplementary activity of horse IgG and F(ab )2 antivenoms Carla Cristina Squaiella-Baptistão 1 , Jose Roberto Marcelino 2 , Luiz Eduardo Ribeiro da Cunha 3 , José María Gutiérrez 4 , Denise V. Tambourgi 1 1
Laboratório de Imunoquímica, Instituto Butantan, São Paulo, SP, Brazil 2 Sec¸ão de Processamento de Plasmas Hiperimunes, Instituto Butantan, São Paulo, SP, Brazil 3 Instituto Vital Brazil, Niterói, Rio de Janeiro, Brazil 4 Instituto Clodomiro Picado, Universidad de Costa Rica, San José, Costa Rica Envenomation by poisonous animals is a tropical neglected disease according to the World Health Organization. The only effective treatment is the antivenom therapy. Antivenoms are in the WHO List of Essential Medicines and should be part of any primary health care package where snakebite accidents occur. Intact heterologous antibodies can activate the complement system (C) via Fc, leading to early adverse reactions, which justify the production of F(ab )2 antivenoms. However, studies have indicated that these fragments can also activate C, leading to such reactions. Herein, we aimed to evaluate the anticomplementary activity of antivenoms (horse antibodies against snakes, spiders, scorpions or caterpillar venoms) produced by Butantan, Vital Brazil and Clodomiro Picado Institutes. These antivenoms have different composition, with the presence or not of Fc portion, and different protein contents. Samples of the antivenoms were incubated with normal human serum and the activation of the three C-pathways was analyzed. Only the classical pathway was activated by some antivenoms from the three Institutes, indicating that anticomplementary activity is not only associated with the presence of the IgG-Fc portion, since F(ab )2 antivenoms also activated the classical pathway. The C-activation/consumption was positively correlated with the amount of heterologous protein only for the antivenom from Vital Brazil Institute. SDS-PAGE showed the presence of several contaminant proteins in most antivenoms, and protein aggregates were detected in those from Clodomiro Picado Institute. Western Blot analysis showed that some antivenoms from Butantan Institute contained some intact IgG heavy chains, indicating that the cleavage of the antibodies for the production of F(ab )2 fragments was not complete. Our results suggest that several factors, such as composition, contaminant proteins and aggregates, are contributing for the C-activation/consumption by the antivenoms. Support: FAPESP, INCTTOX, CNPq. http://dx.doi.org/10.1016/j.imbio.2012.08.032 32 Assay for the pattern-recognition molecule CL-L1 Esben Axelgaard, Lisbeth Jensen, Steffen Thiel, Jens Chr. Jensenius Department of Biomedicine, Aarhus University, Aarhus, Denmark Pattern-recognition molecules of the innate immune system have been an area of intense research the last decade. The pattern recognition molecules belonging to the group of collectins are C-type lectins showing diverse binding patterns towards carbohydrate antigen structures on microorganisms and endogenous non-self structures in a calcium-dependent manner. The collectins are made up of polypeptides consisting of an N-terminal cross-linking region (CLR), where Cys–Cys disulphide bonds allow formation of higher oligomeric structures, a collagen-like region
with repeated Gly-Xaa-Yaa motifs, an alpha-helical neck region that induces trimerization of the polypeptide chains through the formation of a coiled-coil, and finally a C-terminal carbohydrate recognition domain (CRD), which facilitates the binding to ligands upon recognition. Structural basis and biological activity of collectins, like mannan-binding lectin (MBL), surfactant protein A (SP-A) and surfactant protein D (SP-D), have been quite extensively studied, and much is now know about these collectins. Recently, however, three further collectins, collectin liver 1 (CLL1, also termed collectin-10), collectin kidney 1 (CL-K1, also termed CL-11 and collectin-11) and collectin placenta 1 (CL-P1 also termed collectin-12) were discovered by the Wakamiya group. CL-K1 shows binding activity towards carbohydrate structures and association with MBL-associated serine proteases (MASPs), indicating a possible role in innate immunity. The structure and function of CLL1 remain largely unknown. We present a sandwich-type TRIFMA assay specific for the measurement of CL-L1 in solutions and analyze the physiological concentrations of this protein in normal and acute-phase conditions. http://dx.doi.org/10.1016/j.imbio.2012.08.033 33 Autoregulation of thromboinflammation on biomaterials and cells by a novel therapeutic coating technique Yuji Teramura 1 , Per H. Nilsson 2 , Kristina N. Ekdahl 2 , Peetra U. Magnusson 1 , Hongchang Qu 3 , Daniel Ricklin 3 , Jaan Hong 1 , John D. Lambris 3 , Bo Nilsson 1 1 Department of Immunology, Genetics and Pathology, Uppsala University, Sweden 2 School of Natural Sciences, Linnaeus University, Kalmar, Sweden 3 Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Activation of the coagulation and complement systems provide the main recognition and effector mechanisms in the multiple biological responses triggered when biomaterials, therapeutic cells and whole organs come into contact with whole blood. Activation of these cascade systems can result in serious side effects of the biomaterial or cells. In this study, we created a self-regulatory surface to attenuate coagulation and complement activation on both cell and material surfaces by targeting ADP-dependent platelet aggregation/activation and the complement alternative pathway. The ADP-degrading enzyme apyrase and a factor H-binding peptide were co-immobilized onto cell and material surfaces using a blood compatible PEG linker. We demonstrated in human plasma/wholeblood models that platelet activation/coagulation can be regulated simultaneously with complement activation, by co-immobilizing a factor H-binding peptide and apyrase on either material (glass) or cell (rabbit erythrocytes, porcine aortic endothelial cells) surfaces when exposed to human plasma or whole blood. Factor H was effectively recruited by the factor H-binding peptide from blood plasma to both types of surfaces and was able to inhibit complement activation. Apyrase was shown to degrade ADP, thereby suppressing platelet and coagulation activation under the same conditions. In summary, we have inhibited thromboinflammation by using a multicomponent approach, which created a hybrid surface that makes the surface blood compatible and which promises to reduce incompatibility reactions in treatments involving biomaterials and in transplantation of cells and whole organs. http://dx.doi.org/10.1016/j.imbio.2012.08.034