Journal of Hepatology 1998; 29: 879–886 Copyright C European Association for the Study of the Liver 1998 Printed in Denmark ¡ All rights reserved Munksgaard ¡ Copenhagen Journal of Hepatology ISSN 0168-8278 Clonality and specificity of cryoglobulins associated with HCV: pathophysiological implications Mario U. Mondelli 1 , Irene Zorzoli 2 , Antonella Cerino 1 , Agostino Cividini 1 , Morena Bissolati 1 , Laura Segagni 1 , Vittorio Perfetti 3 , Ernesto Anesi 2 , Pietro Garini 2 and Giampaolo Merlini 2 1 Laboratori di Ricerca-Area Infettivologica, Istituto di Clinica delle Malattie Infettive, and 2 Medicina Interna e Oncologia Medica, Laboratori di Ricerca-Area Biotecnologie e Tecnologie Biomediche, and 3 Unita ` di Immunologia Clinica, Area Trapianti, University of Pavia and IRCCS Policlinico San Matteo, Pavia, Italy Background/Aims: Hepatitis C virus (HCV) infection plays a central role in the pathogenesis of mixed cryo- globulinemia through molecular mechanisms which remain to be elucidated. The aim of this study was to investigate the role of antibody responses to HCV in the pathogenesis of cryoglobulinemia through char- acterization of the anti-HCV specificity and immuno- chemical characteristics of the immunoglobulins in- volved in cryoprecipitation. Methods: Sera from 50 consecutive patients with chronic HCV infection (RNA positive) were screened for the presence of cryoglobulins. The two major com- ponents of cryoprecipitates, IgM rheumatoid factors and IgG, were separated by high performance liquid chromatography and analyzed for immunochemical composition by immunoblotting and antibody speci- ficity by ELISA and immunoblotting using recombin- ant HCV proteins and synthetic peptides as antigens. Results: Cryoprecipitates were observed in 27 patients and characterized by immunofixation: 13 (48%) were classified as type II and 14 (52%) as type III. Mono- clonal immunoglobulins were detected by immuno- H  C virus (HCV) infection is frequently as- sociated with mixed cryoglobulinemia (1–3), and current hypotheses suggest that HCV may play a role in the pathogenesis of this disorder. Mixed cryoglob- ulins (MC) are immune complexes composed of rheu- Received 28 May; revised 3 August; accepted 7 August 1998 Correspondence: Mario U. Mondelli, Laboratori di Ricer- ca-Area Infettivologica, Istituto di Clinica delle Malattie Infettive, IRCCS Policlinico San Matteo and University of Pavia, via Taramelli 5, 27100 Pavia, Italy. Tel: 39 382 502 636. Fax: 39 382 526 450. E-mail: istinf/ipv36.unipv.it 879 blotting in 20 cryoprecipitates: IgM in 14 samples and IgG in 14, with a clear preponderance of IgG3 (12/ 14). Specificity studies on sera and purified IgM and IgG fractions from cryoprecipitates revealed enrich- ment in cryoglobulins, predominantly polyclonal IgG1, reactive with the HCV structural proteins, whereas specificities for nonstructural viral proteins were relatively less represented compared to whole serum. No restricted pattern of fine specificity was observed. IgG3 subclass was apparently not involved in HCV nucleoprotein binding. Conclusions: Our findings do not support a direct link between monoclonal cryoglobulins and immune re- sponse to HCV. According to the proposed pathogen- etic model, HCV infection can induce the formation of cryoprecipitable rheumatoid factors, sustain their production, and eventually lead to monoclonal B-cell expansion through several cooperative mechanisms. Key words: Clonality; Cryoglobulins; Hepatitis C vi- rus; IgG subclasses; Immune response; Peptides. matoid factor-binding IgG, which reversibly precipi- tate at low temperatures. The IgM rheumatoid factor is polyclonal in type III and monoclonal in type II, according to Brouet’s classification (4). Cryoglobulins have only been detected in a proportion of patients with HCV infection ranging from 19 to 54% (5–7), sug- gesting that either host factors may also be involved in their generation or that differences in the literature may be accounted for by the absence of standardiza- tion of methods for cryoglobulin detection. It has re- cently been proposed that the humoral immune re- sponse to HCV in cryoglobulinemic patients may differ from that observed in infected patients without cryo-