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Treatment of experimental C3 Glomerulopathy by human complement factor H produced in glycosylation-optimized Physcomitrella patens
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Abstracts / Molecular Immunology 89 (2017) 115–120
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Complement profile and autoantibodies against adipocytes on acquired lipodystrophies
Treatment of experimental C3 Glomerulopathy by human complement factor H produced in glycosylation-optimized Physcomitrella patens
Fernando Corvillo 1,∗ , Verónica Aparicio 2 , Sofía Garrido 1 , María Paz de Miguel 2 , Margarita López-Trascasa 1 1
Immunology Unit, La Paz University Hospital, IdiPAZ, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER U754), Madrid, Spain 2 Cell Engineering Laboratory, La Paz University Hospital Research Institute, IdiPAZ, Madrid, Spain
Background: Lipodystrophies are a heterogeneous group of extremely rare disorders characterized by a selective deficiency of adipose tissue (AT) in absence of nutritional deprivation or catabolic state. Acquired partial lipodystrophy (APL, also called Barraquer-Simons syndrome) is the most commonly extrarenal manifestation of C3 glomerulopathy. Between 80 and 90% of APL patients have complement alternative pathway (AP) abnormalities, associated frequently to the presence of C3 nephritic factor (C3NeF). On the other hand, in acquired generalized lipodystrophy (AGL) data relating to complement alterations are poor. In this study we analyze the complement profile (C3 and C4 levels) and autoantibodies related with AP deregulation. Moreover, we focus our investigation to study the possible autoimmune origin of both acquired lipodystrophies. Materials and methods: We have a cohort of 8 patients with APL and 5 with AGL, collected along all the country. C3 and C4 levels were measured using nephelometry and autoantibodies, including C3NeF, against complement proteins were screened using ELISA techniques. Autoantibodies against adipocyte were analyzed by using immunofluorescence and western blot assays. Results and conclusions: Complement abnormalities were found exclusively in APL patients. Low C3 levels were found in 75% of patients, and as novelty decreased C4 levels were present in 37.5% of patients. Although the acquired factor more prevalent was C3NeF (62.5%), we can highlight the presence of antibodies against proteins that conforms the C3 convertase, as C3 (12.5%), factor B (37.5%) and properdin (25%). Moreover, anti-factor H antibodies were detected in two patients (25%). The high prevalence of autoantibodies against AP proteins could be explain the complement abnormalities in APL patients. Regarding the presence of antibodies against AT, we found IgG reactivity around the adipocyte in patients with both diseases, and we confirmed the presence of autoantibodies by western blot using AT extracts. The blots results showed a complex pattern of bands, but clearly different between both types. Our data confirms the existence of autoantibodies against adipocytes proteins, but further studies are necessary to identify the antigen and to characterize the effect of these autoantibodies in adipocyte viability. http://dx.doi.org/10.1016/j.molimm.2017.06.049
Karsten Häffner 1,∗ , Juliana Parsons 2 , Lennard L. Bohlender 2 , Sebastian Hoernstein 2 , Holger Niederkrüger 2 , Benjamin Fode 3 , Andreas Busch 3 , Nicola Krieghoff 3 , Jonas Koch 3 , Andreas Schaaf 3 , Thomas Frischmuth 3 , Peter F. Zipfel 4 , Martin Pohl 1 , Ralf Reski 2 , Eva L. Decker 2 , Stefan Michelfelder 1 1 Department of General Pediatrics, Adolescent Medicine and Neonatology, University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany 2 Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany 3 Greenovation Biotech GmbH, Freiburg, Germany 4 Leibnitz Institute for Natural Product Research and Infection Biology, Friedrich Schiller University, Jena, Germany
Background: Genetic defects in complement proteins, mostly in glycoprotein factor H (FH), can cause renal diseases like atypical hemolytic uremic syndrome (aHUS) or C3 glomerulopathies (C3G). Therapeutic options, specifically targeting AP activation, are highly desirable but attempts to produce biologically active recombinant FH have until now mostly failed to demonstrate therapeutic value. We reported on the ability of Physcomitrella patens to produce this complex and highly glycosylated protein (moss-FH). Moss-FH displays a predominantly di-antennary complex-type glycosylation pattern (GnGn) and showed full AP regulatory activity as well as the ability to protect host cells from complement-mediated lysis. Here we show the in vivo effects of moss-FH using FH-deficient (FH−/−) mice, a mouse model for experimental C3G. Materials and methods: Moss-FH was expressed and purified from transgenic moss lines devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans via two-step affinity chromatography. Moss-FH or human serum-derived FH (hFH) was injected into the tail vein of FH−/− mice at 50 g/per gram body weight and monitored over five days. Serum C3 and FH levels were quantified by ELISA and western blotting. Immunostaining of C3/C3d was performed on renal cryosections. For quantification of glomerular C3 staining, mean fluorescence intensity of 27 glomeruli per mouse were determined. Results and conclusions: Intravenous administration of mossFH in FH−/− mice significantly reduced abnormal C3 depositions comparable to hFH over five days. No change in glomerular C3d staining was detectable. Moss-FH was found in serum until 6 h after injection and serum C3 levels were increased from 2 to 72 h (peak level ∼46% of wild-type after 12 h). In contrast, hFH was detectable until day 5 and lead to a more sustained increase in serum C3 levels from 2 to 120 h (peak level ∼68% of wild-type after 24 h). Thus, we consider moss-FH as a promising pharmaceutical product for patients with complement dysregulation but further modification of terminal sialylation may be beneficial to increase serum half-life of this moss-derived biopharmaceutical. http://dx.doi.org/10.1016/j.molimm.2017.06.050
Abstracts / Molecular Immunology 89 (2017) 115–120
011
012
Complement profile and autoantibodies against adipocytes on acquired lipodystrophies
Treatment of experimental C3 Glomerulopathy by human complement factor H produced in glycosylation-optimized Physcomitrella patens
Fernando Corvillo 1,∗ , Verónica Aparicio 2 , Sofía Garrido 1 , María Paz de Miguel 2 , Margarita López-Trascasa 1 1
Immunology Unit, La Paz University Hospital, IdiPAZ, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER U754), Madrid, Spain 2 Cell Engineering Laboratory, La Paz University Hospital Research Institute, IdiPAZ, Madrid, Spain
Background: Lipodystrophies are a heterogeneous group of extremely rare disorders characterized by a selective deficiency of adipose tissue (AT) in absence of nutritional deprivation or catabolic state. Acquired partial lipodystrophy (APL, also called Barraquer-Simons syndrome) is the most commonly extrarenal manifestation of C3 glomerulopathy. Between 80 and 90% of APL patients have complement alternative pathway (AP) abnormalities, associated frequently to the presence of C3 nephritic factor (C3NeF). On the other hand, in acquired generalized lipodystrophy (AGL) data relating to complement alterations are poor. In this study we analyze the complement profile (C3 and C4 levels) and autoantibodies related with AP deregulation. Moreover, we focus our investigation to study the possible autoimmune origin of both acquired lipodystrophies. Materials and methods: We have a cohort of 8 patients with APL and 5 with AGL, collected along all the country. C3 and C4 levels were measured using nephelometry and autoantibodies, including C3NeF, against complement proteins were screened using ELISA techniques. Autoantibodies against adipocyte were analyzed by using immunofluorescence and western blot assays. Results and conclusions: Complement abnormalities were found exclusively in APL patients. Low C3 levels were found in 75% of patients, and as novelty decreased C4 levels were present in 37.5% of patients. Although the acquired factor more prevalent was C3NeF (62.5%), we can highlight the presence of antibodies against proteins that conforms the C3 convertase, as C3 (12.5%), factor B (37.5%) and properdin (25%). Moreover, anti-factor H antibodies were detected in two patients (25%). The high prevalence of autoantibodies against AP proteins could be explain the complement abnormalities in APL patients. Regarding the presence of antibodies against AT, we found IgG reactivity around the adipocyte in patients with both diseases, and we confirmed the presence of autoantibodies by western blot using AT extracts. The blots results showed a complex pattern of bands, but clearly different between both types. Our data confirms the existence of autoantibodies against adipocytes proteins, but further studies are necessary to identify the antigen and to characterize the effect of these autoantibodies in adipocyte viability. http://dx.doi.org/10.1016/j.molimm.2017.06.049
Karsten Häffner 1,∗ , Juliana Parsons 2 , Lennard L. Bohlender 2 , Sebastian Hoernstein 2 , Holger Niederkrüger 2 , Benjamin Fode 3 , Andreas Busch 3 , Nicola Krieghoff 3 , Jonas Koch 3 , Andreas Schaaf 3 , Thomas Frischmuth 3 , Peter F. Zipfel 4 , Martin Pohl 1 , Ralf Reski 2 , Eva L. Decker 2 , Stefan Michelfelder 1 1 Department of General Pediatrics, Adolescent Medicine and Neonatology, University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany 2 Plant Biotechnology, Faculty of Biology, University of Freiburg, Freiburg, Germany 3 Greenovation Biotech GmbH, Freiburg, Germany 4 Leibnitz Institute for Natural Product Research and Infection Biology, Friedrich Schiller University, Jena, Germany
Background: Genetic defects in complement proteins, mostly in glycoprotein factor H (FH), can cause renal diseases like atypical hemolytic uremic syndrome (aHUS) or C3 glomerulopathies (C3G). Therapeutic options, specifically targeting AP activation, are highly desirable but attempts to produce biologically active recombinant FH have until now mostly failed to demonstrate therapeutic value. We reported on the ability of Physcomitrella patens to produce this complex and highly glycosylated protein (moss-FH). Moss-FH displays a predominantly di-antennary complex-type glycosylation pattern (GnGn) and showed full AP regulatory activity as well as the ability to protect host cells from complement-mediated lysis. Here we show the in vivo effects of moss-FH using FH-deficient (FH−/−) mice, a mouse model for experimental C3G. Materials and methods: Moss-FH was expressed and purified from transgenic moss lines devoid of potentially immunogenic plant-specific sugar residues on protein N-glycans via two-step affinity chromatography. Moss-FH or human serum-derived FH (hFH) was injected into the tail vein of FH−/− mice at 50 g/per gram body weight and monitored over five days. Serum C3 and FH levels were quantified by ELISA and western blotting. Immunostaining of C3/C3d was performed on renal cryosections. For quantification of glomerular C3 staining, mean fluorescence intensity of 27 glomeruli per mouse were determined. Results and conclusions: Intravenous administration of mossFH in FH−/− mice significantly reduced abnormal C3 depositions comparable to hFH over five days. No change in glomerular C3d staining was detectable. Moss-FH was found in serum until 6 h after injection and serum C3 levels were increased from 2 to 72 h (peak level ∼46% of wild-type after 12 h). In contrast, hFH was detectable until day 5 and lead to a more sustained increase in serum C3 levels from 2 to 120 h (peak level ∼68% of wild-type after 24 h). Thus, we consider moss-FH as a promising pharmaceutical product for patients with complement dysregulation but further modification of terminal sialylation may be beneficial to increase serum half-life of this moss-derived biopharmaceutical. http://dx.doi.org/10.1016/j.molimm.2017.06.050