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Standardisation of the factor H autoantibody assay
Abstracts / Molecular Immunology 56 (2013) 240–316
P CDT 15 Soluble CR1 and its potential as a therapy for C3 glomerulopathy C. Nester 1,∗ , Y. Yang 2 , D. Holanda 3 , H. Marsh 4 , R. Hammond 4 , L. Thomas 4 , S. Sethi 5 , R.J.H. Smith 1 1 University of Iowa, Molecular Otolaryngology and Renal Research Laboratories, Interdepartmental PhD Program in Genetics, Rare Renal Disease Clinic, Departments of Pediatrics and Internal Medicine, Iowa City, United States 2 University of Iowa, Molecular Otolaryngology and Renal Research Laboratories, Iowa City, United States 3 University of Iowa, Department of Pathology, Iowa City, United States 4 Celldex Therapeutics, Needham, United States 5 Mayo Clinic, Department of Laboratory Medicine and Pathology, Rochester, United States
Introduction: Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are two widely recognized subtypes of C3 glomerulopathy (C3G). Both diseases are characterized by fluidphase dysregulation of the alternative complement pathway that leads to deposition of complement proteins in the renal glomerulus. Disease triggers are unknown and targeted treatments are lacking. Objectives: We studied soluble CR1, a potent regulator of the C3 and C5 convertases and the only regulator of complement that serves as a cofactor of factor I to cleave iC3b to C3c and C3dg, to test whether it could restore complement regulation in C3 glomerulopathy. Materials and methods: We studied soluble CR1 in DDD patient sera, in a mouse model of C3GN, and in a DDD patient with endstage renal failure. Results: In DDD patient sera, soluble CR1 prevents dysregulation of the alternative pathway C3 convertase even in the presence of C3 nephritic factors. In mice deficient in complement factor H and transgenic for human CR1, soluble CR1 stops alternative pathway activation, normalizes serum C3 levels, and clears C3 from glomerular basement membranes. In a patient with end-stage renal failure, short term administration of soluble CR1 normalized activity of the terminal complement pathway. Conclusions: These data support the use of sCR1 in a carefully selected patient population to evaluate its efficacy as a specific therapy for C3G. A trial to evaluate the safety and activity of sCR1 in C3 Glomerulopathy is now registered with ClinicalTrials.gov and is open to patient recruitment.
293
The screening of all atypical haemolytic uraemic syndrome (aHUS) patients for factor H autoantibodies is regarded as best practice. Objective: Currently, there is no recommended assay or a consensus on the reporting of factor H autoantibody titres. On behalf of the European Working Party on Complement Genetics in Renal Disease, this study set out to provide a recommended protocol to all laboratories which establish factor H autoantibody testing. Methods: Three European complement laboratories with expertise in the field of autoantibody testing systematically evaluated several ELISA methods used for the detection factor H autoantibodies on various previously tested aHUS patient samples. Results: All methods tested adequately detect high titre samples and where generally robust in the detection of factor H autoantibodies in challenging samples. The importance of individual sample background subtraction in these ELISA tests was established. The use of a relative or arbitrary unit index with a common positive and negative serum allowed for consistent comparison of findings from different test centres, even when using different ELISA methods. This study strongly recommends that a standard arbitrary unit scale based on a titration curve from a common positive anti-serum be adopted across centres to allow future establishment of the relative importance of particular titres of factor H autoantibodies in aHUS. Conclusion: When all elements were taken into consideration, this study recommends one specific method (the Paris protocol) for the detection and reporting of factor H autoantibodies to be used when setting up a factor H autoantibody screen. As well as being robust, it was found to be the most user-friendly and economical of the ELISAs tested. Systematic assay for the presence of factor H autoantibodies in patients using the Paris protocol will provide the longitudinal analysis needed to fully establish the importance of factor H autoantibodies in disease. http://dx.doi.org/10.1016/j.molimm.2013.05.151 P CDT 17 Skewing of SAG mediated therapy for a predominant Th1 during Visceral Leishmaniasis on triggering CD2 epitope S. Sinha 1,∗ , S. Bimal 2 , S. Sundaram 1 1
University of Allahabad, Centre for Biotechnology, Allahabad, India Rajendra Memorial Research Institute of Medical Sciences, Division of Immunology, Patna, India 2
http://dx.doi.org/10.1016/j.molimm.2013.05.150 P CDT 16 Standardisation of the factor H autoantibody assay R. Watson 1 , S. Lindner 2 , P. Bordereau 3 , E.-M. Hunze 1 , F. Tak 1 , S. Ngo 2 , P. Zipfel 2,4 , C. Skerka 2 , M.-A. Dragon-Durey 3 , K.J. Marchbank 1,∗ 1 Newcastle University, Institute of Cellular Medicine, Newcastleupon-tyne, United Kingdom 2 Leibniz Institute for Natural Product Research and Infection Biology, Dept. of Infection Biology, Jena, Germany 3 INSERM, Unite Mixte de Recherche en sante, Paris, France 4 Friedrich Schiller University, Jena, Germany
Introduction: Atypical haemolytic uraemic syndrome (aHUS) is a rare kidney disease defined by complement dysregulation, either due to mutations of complement activator or regulatory genes. Furthermore, autoantibodies to several members of the complement system can deregulate complement and contribute to aHUS.
Background: Visceral Leishmaniasis is a macrophage associated disorder which is linked with a profound decrease in the immunotherapeutic potential of the infected subjects leading to a marked reduction in the CD4 linked Th1 protective immune response. We have previously reported down regulation of CD2 co receptor on the surface of CD4 cells in patients suffering from Visceral Leishmaniasis. Stimulation of CD2 epitope with antiCD2 antibody has led to a remarkable increase in the Protein kinase C alpha mediated phosphorylation on CD2 co receptor on CD4T cells, induction of IFN-␥ led Th1 dominated immune response, a substantial increase in the lymphoblast population and this response remained Th1 dominated even in the presence of Th2 predominant conditions signified with rIL4. Methodology/principal findings: In the present part of the study we have tried to evaluate the use of CD2 antibody as an immunotherapeutic agent along with SAG in ensuring treatment of BALB/c mice induced with experimental Visceral Leishmaniasis. It has been found in the present set of studies that stimulation of CD2 co receptor along with along with therapeutic dose of SAG
P CDT 15 Soluble CR1 and its potential as a therapy for C3 glomerulopathy C. Nester 1,∗ , Y. Yang 2 , D. Holanda 3 , H. Marsh 4 , R. Hammond 4 , L. Thomas 4 , S. Sethi 5 , R.J.H. Smith 1 1 University of Iowa, Molecular Otolaryngology and Renal Research Laboratories, Interdepartmental PhD Program in Genetics, Rare Renal Disease Clinic, Departments of Pediatrics and Internal Medicine, Iowa City, United States 2 University of Iowa, Molecular Otolaryngology and Renal Research Laboratories, Iowa City, United States 3 University of Iowa, Department of Pathology, Iowa City, United States 4 Celldex Therapeutics, Needham, United States 5 Mayo Clinic, Department of Laboratory Medicine and Pathology, Rochester, United States
Introduction: Dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) are two widely recognized subtypes of C3 glomerulopathy (C3G). Both diseases are characterized by fluidphase dysregulation of the alternative complement pathway that leads to deposition of complement proteins in the renal glomerulus. Disease triggers are unknown and targeted treatments are lacking. Objectives: We studied soluble CR1, a potent regulator of the C3 and C5 convertases and the only regulator of complement that serves as a cofactor of factor I to cleave iC3b to C3c and C3dg, to test whether it could restore complement regulation in C3 glomerulopathy. Materials and methods: We studied soluble CR1 in DDD patient sera, in a mouse model of C3GN, and in a DDD patient with endstage renal failure. Results: In DDD patient sera, soluble CR1 prevents dysregulation of the alternative pathway C3 convertase even in the presence of C3 nephritic factors. In mice deficient in complement factor H and transgenic for human CR1, soluble CR1 stops alternative pathway activation, normalizes serum C3 levels, and clears C3 from glomerular basement membranes. In a patient with end-stage renal failure, short term administration of soluble CR1 normalized activity of the terminal complement pathway. Conclusions: These data support the use of sCR1 in a carefully selected patient population to evaluate its efficacy as a specific therapy for C3G. A trial to evaluate the safety and activity of sCR1 in C3 Glomerulopathy is now registered with ClinicalTrials.gov and is open to patient recruitment.
293
The screening of all atypical haemolytic uraemic syndrome (aHUS) patients for factor H autoantibodies is regarded as best practice. Objective: Currently, there is no recommended assay or a consensus on the reporting of factor H autoantibody titres. On behalf of the European Working Party on Complement Genetics in Renal Disease, this study set out to provide a recommended protocol to all laboratories which establish factor H autoantibody testing. Methods: Three European complement laboratories with expertise in the field of autoantibody testing systematically evaluated several ELISA methods used for the detection factor H autoantibodies on various previously tested aHUS patient samples. Results: All methods tested adequately detect high titre samples and where generally robust in the detection of factor H autoantibodies in challenging samples. The importance of individual sample background subtraction in these ELISA tests was established. The use of a relative or arbitrary unit index with a common positive and negative serum allowed for consistent comparison of findings from different test centres, even when using different ELISA methods. This study strongly recommends that a standard arbitrary unit scale based on a titration curve from a common positive anti-serum be adopted across centres to allow future establishment of the relative importance of particular titres of factor H autoantibodies in aHUS. Conclusion: When all elements were taken into consideration, this study recommends one specific method (the Paris protocol) for the detection and reporting of factor H autoantibodies to be used when setting up a factor H autoantibody screen. As well as being robust, it was found to be the most user-friendly and economical of the ELISAs tested. Systematic assay for the presence of factor H autoantibodies in patients using the Paris protocol will provide the longitudinal analysis needed to fully establish the importance of factor H autoantibodies in disease. http://dx.doi.org/10.1016/j.molimm.2013.05.151 P CDT 17 Skewing of SAG mediated therapy for a predominant Th1 during Visceral Leishmaniasis on triggering CD2 epitope S. Sinha 1,∗ , S. Bimal 2 , S. Sundaram 1 1
University of Allahabad, Centre for Biotechnology, Allahabad, India Rajendra Memorial Research Institute of Medical Sciences, Division of Immunology, Patna, India 2
http://dx.doi.org/10.1016/j.molimm.2013.05.150 P CDT 16 Standardisation of the factor H autoantibody assay R. Watson 1 , S. Lindner 2 , P. Bordereau 3 , E.-M. Hunze 1 , F. Tak 1 , S. Ngo 2 , P. Zipfel 2,4 , C. Skerka 2 , M.-A. Dragon-Durey 3 , K.J. Marchbank 1,∗ 1 Newcastle University, Institute of Cellular Medicine, Newcastleupon-tyne, United Kingdom 2 Leibniz Institute for Natural Product Research and Infection Biology, Dept. of Infection Biology, Jena, Germany 3 INSERM, Unite Mixte de Recherche en sante, Paris, France 4 Friedrich Schiller University, Jena, Germany
Introduction: Atypical haemolytic uraemic syndrome (aHUS) is a rare kidney disease defined by complement dysregulation, either due to mutations of complement activator or regulatory genes. Furthermore, autoantibodies to several members of the complement system can deregulate complement and contribute to aHUS.
Background: Visceral Leishmaniasis is a macrophage associated disorder which is linked with a profound decrease in the immunotherapeutic potential of the infected subjects leading to a marked reduction in the CD4 linked Th1 protective immune response. We have previously reported down regulation of CD2 co receptor on the surface of CD4 cells in patients suffering from Visceral Leishmaniasis. Stimulation of CD2 epitope with antiCD2 antibody has led to a remarkable increase in the Protein kinase C alpha mediated phosphorylation on CD2 co receptor on CD4T cells, induction of IFN-␥ led Th1 dominated immune response, a substantial increase in the lymphoblast population and this response remained Th1 dominated even in the presence of Th2 predominant conditions signified with rIL4. Methodology/principal findings: In the present part of the study we have tried to evaluate the use of CD2 antibody as an immunotherapeutic agent along with SAG in ensuring treatment of BALB/c mice induced with experimental Visceral Leishmaniasis. It has been found in the present set of studies that stimulation of CD2 co receptor along with along with therapeutic dose of SAG