Background/Purpose: BACKGROUND. Mixed Cryoglobulins (MCs) are cold precipitable Rheumatoid Factors (RFs) that provide a biomarker for immune complex formation, particularly associated with chronic Hepatitis C Virus (HCV) and Cryoglobulinemic Vasculitis (CryoVas) syndromes. Both the IgM and IgG components of MCs may span a spectrum of clonality, in type 2 >95% being IgMk and bearing Cross-reactive Idiotypes (CRIs) that can be revealed antigenically or by assessing specific heavy and/or light chain V-gene usage. Older studies indicated that C1q may be an integral part of MCs and that up to a third of the protein content is nonimmunoglobulin.

OBJECTIVES. We aimed to define the Immune Complexome of MCs by Mass Spectroscopy and protein sequence analysis (MALDI TOF) compared to existing data bases

Methods: METHODS MCs were isolated from the sera of patients with (a) HCV/CroVas before and after clearance of virus by Direct Acting Antivirals (DAAs), (b) persistent disease (purpura, nephritis) 2-15 years after HCV cure; (c) HIV-HCV coinfection, and (d) sjogrens or lupus patients with MCs consistently negative for HCV RNA . Isolated MCs were characterized by immunofixation and gel electrophoresis (SDS-PAGE) before being processed for MALDI-TOF. Specific gel bands identified by SDS-PAGE, including the IgM heavy chain, were excised and sequenced. In addition, serum obtained before cryoprecipitation was analyzed for the three pathways of complement activation and k/l by free and heavy-light chain immunoassays.

Results: RESULTS In a prospective clinical trial (NTC0282512) the prevalence of complement abnormalities and an expansion of k light chains was demonstrated and the short-term effects of DAA, as well as SVR 24 were analyzed to assess biomarkers concomitant with viral clearance from blood and cryoprecipitates. Depression of C4/C1q was particularly apparent among the patients with persistent cryoglobulinemia, along with IgMk clonality. The validity of MALDI-TOF was reflected in the abundance of Ig heavy and light chain sequences, IgG subclasses, k/l ratios, V-region subgroups, and MC/monoclonal RF VH1-69, VH3-30, VK3D-15 and VK3D-20 MC CRI protein sequences In addition, complement (notably C1q) co-associated with the MC Igs, as well as other proteins relevant to known phlogistic activities of MCs, binding of IgM and the aggregation of proteins in the cold.

Conclusion: CONCLUSIONS Our studies provide a platform for the analysis of MC immune complex proteomics as a laboratory biomarker relevant to CryoVas clinical syndromes

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mixed-cryoglobulin-immune-complex-proteomics-analysis-by-mass-spectroscopy/