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Complement activation and cerebral injury following ischemic stroke
Molecular Immunology 47 (2010) 2198–2294
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Abstracts
Abstracts from the XXIII International Complement Workshop
1 Complement activation and cerebral injury following ischemic stroke Andrew Elvington a , Carl Atkinson a , Liudmila Kulik b , Hong Zhu a , Jin Yu a , Mark S. Kindy a , B. Paul Morgan c , V. Michael Holers b , Stephen Tomlinson a
ilar in vitro data was obtained with a mouse brain endothelial cell line (bEnd.3). Thus, there is the potential for the direct translational development of B4/C2 mAbs and derived fragments as post-ischemic targeting agents. doi:10.1016/j.molimm.2010.05.014 10
a
Medical University of South Carolina, Charleston, SC, United States b University of Colorado, Denver, CO, United States c University of Cardiff, Cardiff, UK
Available evidence indicates that complement plays an important role in inflammation and the secondary neuronal damage that occurs after cerebral ischemia, although the complementdependent mechanisms involved are incompletely understood. We utilized complement deficient mice and, in a clinically relevant paradigm, complement inhibited mice, to investigate complement pathways involved in cerebral injury following ischemic stroke. For these studies we used a mouse model of middle cerebral artery occlusion (MCAo) (60 min) and reperfusion (24 h). We also extended previous studies on IgM-mediated activation of complement following ischemic stroke. Further supporting an important role for Ab-mediated complement activation, MBL/C1q double deficient mice were protected following MCAo and reperfusion in terms of reduced infarct, less demyelination and improved neurological function compared to wt mice. Factor B deficient mice were similarly protected, as were wt mice treated with a single dose of CR2-fH (targeted alternative pathway inhibitor) 30 min after reperfusion. On the other hand, mice deficient in either C6 or CD59 had a similar outcome to wt mice following MCAo and reperfusion. Together with data from a previous report that C1q deficient mice are not protected from ischemic stroke, these data indicate that the alternative pathway plays an important role in cerebral injury via amplification of lectin pathway activation. Further, the membrane attack complex (MAC) does not appear to play any role in cerebral injury. This is in contrast to ischemia/reperfusion injury in other organs and tissues in which the terminal complement pathway has been investigated. Finally, we previously reported that IgM mAbs specific for annexin IV (B4 mAb) or a subset of phospholipids (C2 mAb) recognize neoepitopes exposed on post-ischemic endothelium and initiate complement activation and cerebral injury after MCAo. We have now demonstrated that neoepitopes of similar specificity are expressed on human endothelial cells, since B4 and C2 mAbs bind hypoxic, but not normoxic HUVEC in vitro. Sim0161-5890/$ – see front matter doi:10.1016/j.molimm.2010.05.013
Beta-glycan linkage specificity and the role of properdin in activation of the alternative complement pathway Sarika Agarwal a , Sanjay Ram a , Haibin Huang a , Charles Spechtl a , Gary R. Ostroff b , Stuart M. Levitz a , Peter A. Rice a
A.
a
Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA b Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA Fungal cell walls are beta (b) glycans primarily composed of glucose, mannose and N-acetylglucosamine, and are considered key to immune recognition. b-Glycans recognized by the innate immune system are currently under consideration for use in antigen delivery systems and as immunomodulating agents. The aim of this study was to define how the monosaccharide composition and linkage specificity of b-glycans (b1,3 and b1,6 linked) affect alternative pathway (AP) activation, and the role that properdin (P) plays in linkage-specific b-glycan-mediated AP activation. To evaluate AP activation, we measured C3a generation and C3 deposition on glycan particles in Mg/EGTA-treated serum (AP intact; P functional) and in Mg/EGTA-treated P-depleted serum. Yeast glucan particles (YGP) that contained both b1,6 branched b1,3-D-glucans (lacking b-mannans) derived from Saccharomyces cerevisiae, scleroglucan (b1,6 branched, b1,3-D-glucan) and curdlan (single linkage b1,3D-glucan) activated AP in the absence of P; the addition of P increased AP activation 5–10-fold. Zymosan (a b-glycan particle consisting of b-glucans and b-mannans) a known AP activator and pustulan (b1,6-D-glucan only) generated C3a only in the presence of P. Neither chitin (b1,4-D-N-acetylglucosamine) nor chitosan (deacetylated chitin) activated Mg/EGTA-treated serum (AP intact; P functional). C3 deposition on particles and C5a generation paralleled C3a generation. P binding to YGP and zymosan in serum required active C3. Confocal microscopy confirmed colocalization of C3 fragments and P, suggesting that in the context of serum, P
Contents lists available at ScienceDirect
Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Abstracts
Abstracts from the XXIII International Complement Workshop
1 Complement activation and cerebral injury following ischemic stroke Andrew Elvington a , Carl Atkinson a , Liudmila Kulik b , Hong Zhu a , Jin Yu a , Mark S. Kindy a , B. Paul Morgan c , V. Michael Holers b , Stephen Tomlinson a
ilar in vitro data was obtained with a mouse brain endothelial cell line (bEnd.3). Thus, there is the potential for the direct translational development of B4/C2 mAbs and derived fragments as post-ischemic targeting agents. doi:10.1016/j.molimm.2010.05.014 10
a
Medical University of South Carolina, Charleston, SC, United States b University of Colorado, Denver, CO, United States c University of Cardiff, Cardiff, UK
Available evidence indicates that complement plays an important role in inflammation and the secondary neuronal damage that occurs after cerebral ischemia, although the complementdependent mechanisms involved are incompletely understood. We utilized complement deficient mice and, in a clinically relevant paradigm, complement inhibited mice, to investigate complement pathways involved in cerebral injury following ischemic stroke. For these studies we used a mouse model of middle cerebral artery occlusion (MCAo) (60 min) and reperfusion (24 h). We also extended previous studies on IgM-mediated activation of complement following ischemic stroke. Further supporting an important role for Ab-mediated complement activation, MBL/C1q double deficient mice were protected following MCAo and reperfusion in terms of reduced infarct, less demyelination and improved neurological function compared to wt mice. Factor B deficient mice were similarly protected, as were wt mice treated with a single dose of CR2-fH (targeted alternative pathway inhibitor) 30 min after reperfusion. On the other hand, mice deficient in either C6 or CD59 had a similar outcome to wt mice following MCAo and reperfusion. Together with data from a previous report that C1q deficient mice are not protected from ischemic stroke, these data indicate that the alternative pathway plays an important role in cerebral injury via amplification of lectin pathway activation. Further, the membrane attack complex (MAC) does not appear to play any role in cerebral injury. This is in contrast to ischemia/reperfusion injury in other organs and tissues in which the terminal complement pathway has been investigated. Finally, we previously reported that IgM mAbs specific for annexin IV (B4 mAb) or a subset of phospholipids (C2 mAb) recognize neoepitopes exposed on post-ischemic endothelium and initiate complement activation and cerebral injury after MCAo. We have now demonstrated that neoepitopes of similar specificity are expressed on human endothelial cells, since B4 and C2 mAbs bind hypoxic, but not normoxic HUVEC in vitro. Sim0161-5890/$ – see front matter doi:10.1016/j.molimm.2010.05.013
Beta-glycan linkage specificity and the role of properdin in activation of the alternative complement pathway Sarika Agarwal a , Sanjay Ram a , Haibin Huang a , Charles Spechtl a , Gary R. Ostroff b , Stuart M. Levitz a , Peter A. Rice a
A.
a
Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA b Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA Fungal cell walls are beta (b) glycans primarily composed of glucose, mannose and N-acetylglucosamine, and are considered key to immune recognition. b-Glycans recognized by the innate immune system are currently under consideration for use in antigen delivery systems and as immunomodulating agents. The aim of this study was to define how the monosaccharide composition and linkage specificity of b-glycans (b1,3 and b1,6 linked) affect alternative pathway (AP) activation, and the role that properdin (P) plays in linkage-specific b-glycan-mediated AP activation. To evaluate AP activation, we measured C3a generation and C3 deposition on glycan particles in Mg/EGTA-treated serum (AP intact; P functional) and in Mg/EGTA-treated P-depleted serum. Yeast glucan particles (YGP) that contained both b1,6 branched b1,3-D-glucans (lacking b-mannans) derived from Saccharomyces cerevisiae, scleroglucan (b1,6 branched, b1,3-D-glucan) and curdlan (single linkage b1,3D-glucan) activated AP in the absence of P; the addition of P increased AP activation 5–10-fold. Zymosan (a b-glycan particle consisting of b-glucans and b-mannans) a known AP activator and pustulan (b1,6-D-glucan only) generated C3a only in the presence of P. Neither chitin (b1,4-D-N-acetylglucosamine) nor chitosan (deacetylated chitin) activated Mg/EGTA-treated serum (AP intact; P functional). C3 deposition on particles and C5a generation paralleled C3a generation. P binding to YGP and zymosan in serum required active C3. Confocal microscopy confirmed colocalization of C3 fragments and P, suggesting that in the context of serum, P