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Mannose binding lectin as a target for neuroprotection
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Abstracts / Molecular Immunology 47 (2010) 2198–2294
significant (P < 0.001 and 0.01, respectively) following adjustment for maternal race and age. The results of this study suggest a significant link between BMI and elevated markers of complement system activation. A study is now underway to examine the contribution of these complement activation fragments to obesity in non-pregnant individuals. doi:10.1016/j.molimm.2010.05.018 103 Proteomics-based discovery of an abnormal, internally duplicated CFHR1 protein which associates with renal pathology in a Spanish family Cynthia Abarrategui-Garrido a,e , Rubén Martínez-Barricarte c,e , Alberto López-Lera b,e , Elena Fariza-Requejo a,e , Carolina Ruivo a,e , Rafael Bedoya-Pérez d , Santiago Rodríguez de Córdoba c,e , Margarita López-Trascasa b,e , Pilar Sánchez-Corral a,e a
Research Unit, Hospital Universitario La Paz, Madrid, Spain Immunology Unit, Hospital Universitario La Paz, Madrid, Spain c Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain d Pediatric Nephrology Unit, Hospital Universitario Virgen del Rocío, Sevilla, Spain e Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Spain b
Complement Factor H Related Protein 1 (CFHR1) is a structural homologue of the alternative pathway regulator factor H. Homozygous deficiency of CFHR1 and a recently described CFHR1*B allele are associated with atypical Hemolytic Uremic Syndrome, a renal pathology involving complement dysregulation. We describe an abnormal, large form of CFHR1 in a Spanish pedigree with a history of renal disease. The proband was diagnosed of Dense Deposit Disease (DDD/MPGN II) at the age of 8 years. Complement studies revealed a moderate activation of the alternative pathway. C3 convertase autoantibodies (C3NeF) were negative, and no mutations were found in Factor H, factor I and MCP. Western-blot analysis of factor H/CFHR proteins in the proband’s serum revealed an extra triplet of bands about 60 kDa. Heparin-Sepharose chromatography followed by 2D-electrophoresis showed these bands presented multiple isoforms differing in isoelectric point. Spots were picked from the gel for proteomics analysis. Peptides from the five5 SCRs domains of CFHR1 were detected in the tryptic digest of these spots, but no other protein was identified; therefore, a partial duplication of CFHR1 was suspected. MLPA analysis throughout the CFH/CFHR1–5 region revealed an extra copy of exons 3–5 of the CFHR1 gene, confirming the protein studies. The abnormal CFHR1 triplet and the extra copy of CFHR1 exons 3–5 were also observed in the patient’s mother, with a history of proteinuria, hematuria and arterial hypertension, but not in the patient’s healthy brother and maternal uncle. Samples from the patient’s grandfather, who also suffered DDD but was deceased, could not be studied. These results reveal a novel duplication within the CFHR1 gene which results in a secreted, large form of CFHR1 of likely pathological significance. The study further proves the feasibility of the proteomics-to-genomics approach for identification and characterization of clinically relevant alterations in the CFH/CFHRs region. doi:10.1016/j.molimm.2010.05.019
104 Mannose binding lectin as a target for neuroprotection F. Orsini a , E.R. Zanier a , R. Gesuete a , M. Stravalaci a , D. De Blasio a , B. Oortwijn b , M.L.M. Mannesse b , M. Gobbi a , M.G. De Simoni a a b
Mario Negri Institute, Milan, Italy Pharming Technologies B.V., Leiden, The Netherlands
Objective: We have previously shown the neuroprotective action of plasma-derived C1-inhibitor (C1-INH) (De Simoni et al., 2003, 2004; Storini et al., 2005; Longhi et al., 2008). In addition, we have recently demonstrated the surprisingly wide time-window of efficacy towards ischemic damage of recombinant human C1-INH (rhC1-INH) and its high affinity to Mannose Binding Lectin (MBL) (Gesuete et al., 2009). We have now further explored the relevance of this target to C1-INH neuroprotective properties. Methods: C57Bl/6 (WT) or MBL-A and MBL-C double knock-out (MBL−/−) mice underwent transient or permanent focal cerebral ischemia. Infarct volume, neurodegeneration and neurological deficits were evaluated following intravenous rhC1-INH or vehicle. In WT mice the colocalization in the ischemic brain tissue of rhC1-INH with MBL-A/MBL-C on cerebral vessels were performed through immunostaining and confocal microscopy. The consequences of rhC1-INH administration were analyzed by measuring the presence of circulating functional MBL/MASP-2 complexes by ELISA. Results: MBL−/− mice showed a significantly lower susceptibility to both transient and permanent ischemia. Furthermore, rhC1-INH did not exert its neuroprotective effect in MBL−/− mice while decreasing the ischemic volume by 70% in WT mice, indicating that MBL is essential for rhC1-INH protective action. In the ischemic brain of WT mice rhC1-INH colocalized with both endothelial MBL-A and MBL-C. MBL/MASP-2 activation induced by ischemia was completely prevented by rhC1-INH. Conclusions. The neuroprotective action of rhC1-INH is related to its capability to inhibit MBL. The data obtained in MBL−/− mice underline the relevance of MBL in the brain ischemic damage. We propose MBL as a novel target for stroke treatment. References De Simoni, et al., 2003. JCBFM. De Simoni, et al., 2004. Am. J. Pathol.. Gesuete, et al., 2009. Ann. Neurol.. Longhi, et al., 2008. Crit. Care Med.. Storini, et al., 2005. Neurobiol. Dis..
doi:10.1016/j.molimm.2010.05.020
Abstracts / Molecular Immunology 47 (2010) 2198–2294
significant (P < 0.001 and 0.01, respectively) following adjustment for maternal race and age. The results of this study suggest a significant link between BMI and elevated markers of complement system activation. A study is now underway to examine the contribution of these complement activation fragments to obesity in non-pregnant individuals. doi:10.1016/j.molimm.2010.05.018 103 Proteomics-based discovery of an abnormal, internally duplicated CFHR1 protein which associates with renal pathology in a Spanish family Cynthia Abarrategui-Garrido a,e , Rubén Martínez-Barricarte c,e , Alberto López-Lera b,e , Elena Fariza-Requejo a,e , Carolina Ruivo a,e , Rafael Bedoya-Pérez d , Santiago Rodríguez de Córdoba c,e , Margarita López-Trascasa b,e , Pilar Sánchez-Corral a,e a
Research Unit, Hospital Universitario La Paz, Madrid, Spain Immunology Unit, Hospital Universitario La Paz, Madrid, Spain c Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain d Pediatric Nephrology Unit, Hospital Universitario Virgen del Rocío, Sevilla, Spain e Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Spain b
Complement Factor H Related Protein 1 (CFHR1) is a structural homologue of the alternative pathway regulator factor H. Homozygous deficiency of CFHR1 and a recently described CFHR1*B allele are associated with atypical Hemolytic Uremic Syndrome, a renal pathology involving complement dysregulation. We describe an abnormal, large form of CFHR1 in a Spanish pedigree with a history of renal disease. The proband was diagnosed of Dense Deposit Disease (DDD/MPGN II) at the age of 8 years. Complement studies revealed a moderate activation of the alternative pathway. C3 convertase autoantibodies (C3NeF) were negative, and no mutations were found in Factor H, factor I and MCP. Western-blot analysis of factor H/CFHR proteins in the proband’s serum revealed an extra triplet of bands about 60 kDa. Heparin-Sepharose chromatography followed by 2D-electrophoresis showed these bands presented multiple isoforms differing in isoelectric point. Spots were picked from the gel for proteomics analysis. Peptides from the five5 SCRs domains of CFHR1 were detected in the tryptic digest of these spots, but no other protein was identified; therefore, a partial duplication of CFHR1 was suspected. MLPA analysis throughout the CFH/CFHR1–5 region revealed an extra copy of exons 3–5 of the CFHR1 gene, confirming the protein studies. The abnormal CFHR1 triplet and the extra copy of CFHR1 exons 3–5 were also observed in the patient’s mother, with a history of proteinuria, hematuria and arterial hypertension, but not in the patient’s healthy brother and maternal uncle. Samples from the patient’s grandfather, who also suffered DDD but was deceased, could not be studied. These results reveal a novel duplication within the CFHR1 gene which results in a secreted, large form of CFHR1 of likely pathological significance. The study further proves the feasibility of the proteomics-to-genomics approach for identification and characterization of clinically relevant alterations in the CFH/CFHRs region. doi:10.1016/j.molimm.2010.05.019
104 Mannose binding lectin as a target for neuroprotection F. Orsini a , E.R. Zanier a , R. Gesuete a , M. Stravalaci a , D. De Blasio a , B. Oortwijn b , M.L.M. Mannesse b , M. Gobbi a , M.G. De Simoni a a b
Mario Negri Institute, Milan, Italy Pharming Technologies B.V., Leiden, The Netherlands
Objective: We have previously shown the neuroprotective action of plasma-derived C1-inhibitor (C1-INH) (De Simoni et al., 2003, 2004; Storini et al., 2005; Longhi et al., 2008). In addition, we have recently demonstrated the surprisingly wide time-window of efficacy towards ischemic damage of recombinant human C1-INH (rhC1-INH) and its high affinity to Mannose Binding Lectin (MBL) (Gesuete et al., 2009). We have now further explored the relevance of this target to C1-INH neuroprotective properties. Methods: C57Bl/6 (WT) or MBL-A and MBL-C double knock-out (MBL−/−) mice underwent transient or permanent focal cerebral ischemia. Infarct volume, neurodegeneration and neurological deficits were evaluated following intravenous rhC1-INH or vehicle. In WT mice the colocalization in the ischemic brain tissue of rhC1-INH with MBL-A/MBL-C on cerebral vessels were performed through immunostaining and confocal microscopy. The consequences of rhC1-INH administration were analyzed by measuring the presence of circulating functional MBL/MASP-2 complexes by ELISA. Results: MBL−/− mice showed a significantly lower susceptibility to both transient and permanent ischemia. Furthermore, rhC1-INH did not exert its neuroprotective effect in MBL−/− mice while decreasing the ischemic volume by 70% in WT mice, indicating that MBL is essential for rhC1-INH protective action. In the ischemic brain of WT mice rhC1-INH colocalized with both endothelial MBL-A and MBL-C. MBL/MASP-2 activation induced by ischemia was completely prevented by rhC1-INH. Conclusions. The neuroprotective action of rhC1-INH is related to its capability to inhibit MBL. The data obtained in MBL−/− mice underline the relevance of MBL in the brain ischemic damage. We propose MBL as a novel target for stroke treatment. References De Simoni, et al., 2003. JCBFM. De Simoni, et al., 2004. Am. J. Pathol.. Gesuete, et al., 2009. Ann. Neurol.. Longhi, et al., 2008. Crit. Care Med.. Storini, et al., 2005. Neurobiol. Dis..
doi:10.1016/j.molimm.2010.05.020