Skin and/or liver biopsy specimens were obtained from the following patients: 15 anti-hepatitis C virus (HCV), HCV RNA-positive patients and 3 anti-HCV, HCV RNA-negative patients with type II mixed cryoglobulinemia (MC); 7 anti-HCV, HCV RNA-positive patients with chronic active liver disease (CALD); 5 anti-HCV, HCV RNA-negative patients with noncryoglobulinemic vasculitis; and 7 anti-HCV, HCV RNA-negative patients with lichen ruber planus. A pool of murine monoclonal antibodies (MAbs) developed against c22-3, c33c, and c100-3 proteins was used to detect HCV-related antigens (Ags) by indirect immunohistochemistry. Acid electroelution (AEE) of tissue sections was performed to enhance the sensitivity of the immunohistochemical method. In anti-HCV-positive MC patients, specific HCV-related Ags were detected in the small vessels of the skin and in the cytoplasm of hepatocytes. Prior AEE of biopsy sections allowed detection of HCV Ags in 6 of 15 (40%) skin biopsy and in 9 of 14 (64.3%) liver biopsy specimens. HCV immunoreactive deposits in the skin displayed two immunohistochemical patterns: (1) coarse intraluminal material associated with dermal inflammatory infiltrates and intravascular deposition of eosinophilic hyaline material; and (2) reactivity confined to the vessel wall in the context of an apparently normal tissue. Immunoglobulin (Ig) G and IgM deposition in the skin showed immunohistochemical features comparable with those found for HCV Ag deposits.(ABSTRACT TRUNCATED AT 250 WORDS)

SUMMARYTmmunofluorescence (IF) to detect HCV antigens and non-isotopic in situ hybridization (NISH) to detect HCV RNA genome were carried out on bone marrow (BM) and peripheral blood (PB) mononuclear cells (MC) of 11 chronically HCV-infected patients. In four patients (36.4%) HCV antigens were detected in monocytes/macrophages as well as in B lymphocytes in both BMMC and PBMC. Positive T lymphocytes in BMMC were found in three of them, but only one patient showed positive T cells in PBMC. NISH invariably demonstrated minus and plus HCV RNA genomic strands either in monocytes/macrophages or B and T lymphocytes in BMMC and PBMC in the four HCV antigen-positive patients and in two further patients not expressing viral proteins in blood MC. I F signals appeared diffusely distributed within the cytoplasm, or as brilliant granules in distinct submembrane areas or else in cytoplasm membrane. Nuclei never stained. Similarly, NISH displayed HCV RNA accumulation restricted to MC cytoplasm only, nuclei being persistently negative. NISH, however, was unable to detect cell membrane signal. Infection of blood MC is a common event in naturally acquired HCV infection, since none of these patients was conditioned by immunomodulating or immunosuppressive therapies. No difference was found in terms of mean age, length of disease, anti-HCV immune response, type and severity of chronic liver damage between patients with HCV-infected MC and patients without cell infection. These results demonstrate that HCV can infect BMMC and PBMC that represent important extrahepatic sites of virus replication, and may help to explain the immunological abnormalities observed in chronic HCV carriers.Keywords chronic liver disease hepatitis C virus immunofluorescence in situ hybridization mononuclear cells INTRODUCTIONAt least 50% of HCV-infected individuals develop chronic

The high frequency of liver involvement in cryoglobulinemia is well established. Although both etiology and pathogenesis have remained so far undefined, recent studies suggest an association of mixed cryoglobulinemia with hepatitis C virus infection. To explore this hypothesis further, we assessed the prevalence of hepatitis C virus antibodies and RNA in a large group of patients, including: (1) 35 patients with cryoglobulinemia without clinical evidence of liver involvement (group 1), (2) 15 patients with symptomatic cryoglobulinemia associated with chronic liver disease (group 2) and (3) 12 patients with asymptomatic cryoglobulinemia associated with chronic liver disease (group 3). Anti-hepatitis C virus antibodies were detected by a second-generation enzyme-linked immunosorbent assay and third-generation immunoblot (SIA Prototype RIBA), whereas the polymerase chain reaction was used for the detection of viral RNA. Anti-hepatitis C virus antibodies, as detected by enzyme-linked immunosorbent assay, were demonstrated in 21 (60%) patients from group 1, 11 (73.3%) from group 2 and 10 (83.3%) from group 3. The immunoblot identified as positive 3 further patients in group 1 (giving a prevalence of 68.6%) and all patients in groups 2 and 3. Hepatitis C virus RNA was demonstrated in cryoprecipitates from 21 of 24 immunoblot-positives and from 6 of 11 immunoblot-negatives, indicating an actual active viral replication in 77.1% of group 1. This was also found in 13 (86.7%) and 10 (83.3%) cryoprecipitates of groups 2 and 3, respectively. Type II cryoglobulinemia was the prevalent form in group 1 (88.6%) and group 2 (73.3%), whereas type III was found in group 3 (58.3%) and in 26.7% of group 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Circulating immune complexes (ICs) were isolated by affinity chromatography and sucrose density gradient fractionation during acute and chronic hepatitis C virus (HCV) infection. Immunochemical and biomolecular studies showed that they basically consist of the virus component, IgG with specific anti-HCV activity and IgM bearing 17.109 epitope (IgM 17.109), an antigenic determinant common to rheumatoid factors (RFs) with WA cross-idiotype (XId). An antigen-specific IC assay was used to demonstrate IgG anti-HCV/IgM 17.109 ICs (IgG-IgM ICs) in five out of the five patients with acute and in 8 out of the 10 patients with chronic hepatitis C who mounted an IgG anti-HCV immune response. They were not detected in patients with no IgG anti-HCV response. IgG-IgM ICs appeared in step with IgG anti-HCV seroconversion and remained detectable for a long period irrespective of clinical outcome, in that they were demonstrated over a 4-year follow-up of patients with chronic hepatitis C. Their presence was unrelated to the severity and progression of liver histology. Despite similar serum levels of IgM 17.109 XId, antigen-specific IgG-IgM ICs were not found in acute and chronic hepatitis B or in acute hepatitis A. Thus, these ICs appear to be uniquely associated with HCV infection, supporting the view that IgM 17.109 XId derive from an antigen-driven response strictly related to the involved antigen. Even although they have no apparent effects on the progression of HCV-related liver disease, their presence may help to explain the immunological abnormalities and extrahepatic disorders observed in HCV infection.

We investigated by ELISA the IgE response to whole extract of the house-dust mite Dermatophagoides pteronyssinus (Dp) and to the native major allergens, Der p 1 and Der p 2, in sera from 18 adult patients (group A) with Dp-allergic asthma before (t0) and 1, 2, 3, and 4 (t1-t4) years after subcutaneous specific immunotherapy (SIT). A qualitative reduction (P = 0.05) of the IgE responses to Dp and Der p 2 was observed from t1 to t4, but a highly statistical significant decrease appeared at t3 (P < 0.01). With regard to Der p 1 IgE values, the immunotherapy induced a significant decrease (P < 0.01) at t3, but not before. In group A, the IgE responses to Der p 1 and Der p 2 were not correlated at t0 (rs = 0.31; P = 0.21) but were correlated at t3 (rs = 0.78; P = 0.001). We also examined sera from 14 adult patients (group B, same SIT schedule as group A) who were without respiratory symptoms at the end of the third year (t3) of Dp SIT. At this time (t3), there were no significant differences in Der p 1 and Der p 2 IgE levels between group A and group B.