| 1. |
- Gerogianni, Alexandra, et al.
(författare)
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Functional evaluation of complement factor I variants by immunoassays and SDS-PAGE
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Factor I (FI) is an essential regulator of the complement system. Together with co-factors, FI degrades C3b, which inhibits further complement activation. Genetic mutations in FI are associated with pathological conditions like age-related macular degeneration and atypical hemolytic uremic syndome. Here, we evaluated eight recombinant FI genetic variants found in patients. We assessed FI's co-factor activity in the presence of two co-factors; Factor H and soluble CR1. Different analytical assays were employed; SDS-PAGE to evaluate the degradation of C3b, ELISA to measure the generation of fluid phase iC3b and the degradation of surface-bound C3b using a novel Luminex bead-based assay. We demonstrate that mutations in the FIMAC and SP domains of FI led to significantly reduced protease activity, whereas the two analyzed mutations in the LDLRA2 domain did not result in any profound changes in FI's function. The different assays employed displayed a strong positive correlation, but differences in the activity of the genetic variants Ile55Phe and Gly261Asp could only be observed by combining different methods and co-factors for evaluating FI activity. In conclusion, our results provide a new perspective regarding available diagnostic tools for assessing the impact of mutations in FI.
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| 2. |
- Michels, Marloes A.H.M., et al.
(författare)
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Overactivity of alternative pathway convertases in patients with complement-mediated renal diseases
- 2018
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9:APR
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Tidskriftsartikel (refereegranskat)abstract
- Overactivation of the alternative pathway of the complement system is associated with the renal diseases atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G). C3 nephritic factors (C3NeF) play an important role in C3G pathogenesis by stabilizing the key enzymatic complex of complement, the C3 convertase. However, the reliability of assays detecting these autoantibodies is limited. Therefore, in this study, we validated and optimized a prototype hemolytic method for robust detection and characterization of factors causing convertase overactivity in large patient cohorts. The assay assesses convertase activity directly in the physiological milieu of serum and therefore is not restricted to detection of stabilizing autoantibodies such as C3NeF but may also reveal genetic variants resulting in prolonged convertase activity. We first defined clear cutoff values based on convertase activity in healthy controls. Next, we evaluated 27 C3G patient samples and found 16 positive for prolonged convertase activity, indicating the presence of factors influencing convertase stability. In three patients, the overactive convertase profile was persistent over disease course while in another patient the increased stability normalized in remission. In all these four patients, the convertase-stabilizing activity resided in the purified immunoglobulin (Ig) fraction, demonstrating the autoantibody nature. By contrast, the Igs of a familial aHUS patient carrying the complement factor B mutation p.Lys323Glu did not reveal convertase stabilization. However, in serum prolonged convertase activity was observed and segregated with the mutation in both affected and unaffected family members. In conclusion, we present a robust and reliable method for the detection, characterization, and evaluation over time of factors prolonging convertase activity (C3NeF or certain mutations) in patient cohorts. This assay may provide new insights in disease pathogenesis and may contribute to the development of more personalized treatment strategies.
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| 3. |
- Van Den Heuvel, Lambertus, et al.
(författare)
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Genetic predisposition to infection in a case of atypical hemolytic uremic syndrome
- 2018
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Ingår i: Journal of Human Genetics. - : Springer Science and Business Media LLC. - 1434-5161 .- 1435-232X. ; 63:1, s. 93-96
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Tidskriftsartikel (refereegranskat)abstract
- Most cases of hemolytic uremic syndrome (HUS) are caused by infection with enterohemorrhagic Escherichia coli (EHEC). Genetic defects causing uncontrolled complement activation are associated with the more severe atypical HUS (aHUS). Non-EHEC infections can trigger the disease, however, complement defects predisposing to such infections have not yet been studied. We describe a 2-month-old patient infected with different Gram-negative bacterial species resulting in aHUS. Serum analysis revealed slow complement activation kinetics. Rare variant R229C was found in complement inhibitor vitronectin. Recombinant mutated vitronectin showed enhanced complement inhibition in vitro and may have been a predisposing factor for infection. Our work indicates that genetic changes in aHUS can not only result in uncontrolled complement activation but also increase vulnerability to infections contributing to aHUS.
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| 4. |
- Assink, Karin, et al.
(författare)
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Mutation analysis and clinical implications of von Willebrand factor-cleaving protease deficiency.
- 2003
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Ingår i: Kidney International. - : Elsevier BV. - 1523-1755 .- 0085-2538. ; 63:6, s. 1995-1999
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Tidskriftsartikel (refereegranskat)abstract
- Background. The pentad of thrombocytopenia, hemolytic anemia, mild renal dysfunction, neurologic signs, and fever, classically characterizes the syndrome of thrombotic thrombocytopenic purpura (TTP). TTP usually occurs in adults as an acquired form but a congenital form in children has also been described. In the latter case, the initial presentation is often with neonatal jaundice and thrombocytopenia. The disorder may subsequently take a relapsing course. Deficiency of a recently identified novel metalloprotease, the von Willebrand factor (vWF)-cleaving protease, originating from mutations in the ADAMTS13 gene plays a major role in the development of TTP. Methods. Blood for DNA analysis was collected from six unrelated TTP families, consisting of nine patients from four different countries, and was screened for mutations in the ADAMTS 13 gene. This gene spans 29 exons encompassing ~37 kb. Conventional techniques of DNA extraction, polymerase chain reaction (PCR), and direct cycle sequencing were used. Results. Eight novel ADAMTS 13 mutations are presented. Half of the total number of mutant ADAMTS13 alleles are amino acid substitutions. The disease-causing mutations are spread over the gene. The pathogenicity of the individual mutations is based upon their predicted effect on the ADAMTS13 protein and segregation in family members. Although most of the patients (seven out of nine) had symptoms during the neonatal period, they were in a remarkably good condition. Only one of the nine patients had a decreased glomerular filtration rate (GFR) with proteinuria and hematuria. Another patient had epileptic seizures. Conclusion. We confirm that deficiency of ADAMTS13 is a molecular mechanism responsible for familial TTP. An early diagnosis allows prophylactic treatment with fresh plasma infusions.
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| 5. |
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| 6. |
- Blom, Anna, et al.
(författare)
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A novel method for direct measurement of complement convertases activity in human serum.
- 2014
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Ingår i: Clinical and Experimental Immunology. - : Oxford University Press (OUP). - 0009-9104 .- 1365-2249. ; 178:1, s. 142-153
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Tidskriftsartikel (refereegranskat)abstract
- Complement convertases are enzymatic complexes that play a central role in sustaining and amplification of the complement cascade. Impairment of complement function directly or indirectly leads to pathologic conditions including higher infection rate, kidney diseases, autoimmune- or neurodegenerative diseases and ischemia-reperfusion injury. An assay for direct measurement of activity of the convertases in patient sera is not available. Existing assays testing convertase function are based on purified complement components and thus, convertase formation occurs under non-physiological conditions. We designed a new assay, in which C5 blocking compounds enabled separation of the complement cascade into two phases: the first ending at the stage of C5 convertases and the second ending with membrane attack complex formation. Use of rabbit erythrocytes or antibody-sensitized sheep erythrocytes as the platforms for convertase formation enabled easy readout based on measurement of hemolysis. Thus, properties of patient sera could be studied directly regarding convertase activity and membrane attack complex formation. Another advantage of this assay was the possibility to screen for host factors such as C3 nephritic factor and other anti-complement autoantibodies, or gain-of-function mutations, which prolong half-live of complement convertases. Herein, we present proof of concept, detailed description and validation of this novel assay.
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| 7. |
- Nilsson, Per H., 1980-, et al.
(författare)
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Eculizumab-C5 complexes express a C5a neoepitope in vivo : Consequences for interpretation of patient complement analyses
- 2017
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Ingår i: Molecular Immunology. - : Elsevier. - 0161-5890 .- 1872-9142. ; 89, s. 111-114
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Tidskriftsartikel (refereegranskat)abstract
- The complement system has obtained renewed clinical focus due to increasing number of patients treated with eculizumab, a monoclonal antibody inhibiting cleavage of C5 into C5a and C5b. The FDA approved indications are paroxysmal nocturnal haemoglobinuria and atypical haemolytic uremic syndrome, but many other diseases are candidates for complement inhibition. It has been postulated that eculizumab does not inhibit C5a formation in vivo, in contrast to what would be expected since it blocks C5 cleavage. We recently revealed that this finding was due to a false positive reaction in a C5a assay. In the present study, we identified expression of a neoepitope which was exposed on C5 after binding to eculizumab in vivo. By size exclusion chromatography of patient serum obtained before and after infusion of eculizumab, we document that the neoepitope was exposed in the fractions containing the eculizumab-C5 complexes, being positive in this actual C5a assay and negative in others. Furthermore, we confirmed that it was the eculizumab-C5 complexes that were detected in the C5a assay by adding an anti-IgG4 antibody as detection antibody. Competitive inhibition by anti-C5 antibodies localized the epitope to the C5a moiety of C5. Finally, acidification of C5, known to alter C5 conformation, induced a neoepitope reacting identical to the one we explored, in the C5a assays. These data are important for interpretation of complement analyses in patients treated with eculizumab.
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| 8. |
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