Reactivity of an Anti NS3-HCV-Induced Autoantibody in HCV-Positive Gastric Tissue by 2D Gel Immunoblot.

Abstract

In a previous study we demonstrated an IgM cross reactivity against Fc region of human IgG and HCV-NS3 antigen in HCV-induced type II mixed cryoglobulinemia (MC). We identified NS3 and Fc epitopes recognized by these IgM by epitope excision approach. HLA determination confirms their ability to present these epitopes. The same cross reactivity has been observed with a monoclonal antibody, (mAb B3-18), obtained after immunization of a mouse with one of the identified NS3 epitopes. Previous, observations indicate that HCV may localize in several tissues besides the liver (such as gastric mucosa) possibly inducing some of the extra hepatic manifestations of chronic HCV infection. The aim of this study is to characterize the mAb B3-18 reactivity by an immuno-proteomic approach. Such novel information may be of interest to identify new potential antigens implicated in HCV pathogenesis useful to design new molecules for diagnostic or therapeutic approaches. Proteins were extracted from 12 gastric biopsies of patients with gastric disturb and HCV-infection (one of them with a concomitant malt lymphoma of the stomach) and from 3 patients with gastric disturb but without HCV infection as control. Proteins were analysed by 2D gel electrophoresis and mAb B3-18 immunoblot. Proteins recognized by mAb B3-18 were identified by MALDI-TOF mass spectrometry. Differential protein expression levels between patients and controls were detected by 2D-DIGE. Samples were also tested by immunohistochemistry with the mAb B3-18, by VDJ-BCR and VDJ-TCR genscan analysis and by KIR/ligands haplotypes. MAb B3-18 is functional both in 2D gel electrophoresis and in immunohistochemistry. Two specific proteins were identified by B3-18 immunoblot in HCV-infected patients: 18 kDa antrum mucosa protein (AMP 18) and TFIZ1. Both proteins function as growth factors at least partly responsible for maintaining a functional gastric epithelium by seeking evidence of epithelial cell mitogenic activity. They are expressed and secreted in normal gastric mucosa but down-regulated in gastric cancer. These proteins, as well as some others, also showed an up-regulation in gastric mucosa from HCV-infected patients. Mab B3–18 reactivity in immunohistochemistry was restricted to gastric tissue surrounding the NHL lesion. An oligo/monoclonal B cell pattern was found in 3 patients. The characterization of KIR/ligand is in course. The use of a monoclonal antibody miming IgM autoantibodies from HCV+ patients with type II MC and a concomitant immunocytoma, is useful to identify autoantigens that could sustain HCV-related B cell proliferations and HCV infection-associated antigens.