Vito Cornacchiulo

Detection of hepatitis C virus (HCV) proteins by immunofluorescence and HCV RNA genomic sequences by non-isotopic in situ hybridization in bone marrow and peripheral blood mononuclear cells of chronically HCV-infected patients

Immunofluorescence (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. IF 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.

Hepatitis C virus infection and mixed cryoglobulinemia: a striking association

SummaryThe 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) 13 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 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. Type I cryoglobulins were found only in group 1 and were negative for hepatitis C virus antibodies and RNA. Hepatitis B virus markers indicating a chronic carrier state were found in 3 patients, 2 of whom were also infected with hepatitis C. It is concluded that mixed cryoglobulinemia is very commonly associated with active hepatitis C infection. The presence of hepatitis C virions and likely virus antigen-antibody complexes in cryoprecipitates strongly suggests a causative role of this virus in the pathogenesis of tissue damage and indicates that the term “essential,” which usually defines a major proportion of types II and III cryoglobulins, should be abandoned.

In situ simultaneous detection of hepatitis C virus RNA and hepatitis C virus-related antigens in hepatocellular carcinoma

The overwhelming evidence that chronic infection with the hepatitis C virus (HCV) is an important cause of hepatocellular carcinoma (HCC) is based on epidemiologic, case‐control, and cohort studies as well as laboratory investigations. To address better the pathogenesis of HCV infection at the single‐cell level, the authors developed a specific reproducible method for the simultaneous detection of HCV specific sequences and antigens in liver tissue, using a combination of nonradioactive in situ hybridization and immunohistochemistry.

Immunochemical and biomolecular studies of circulating immune complexes isolated from patients with acute and chronic hepatitis C virus infection

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.

Circulating levels and liver tissue distribution of intercellular adhesion molecule-1 during β-interferon therapy of hepatitis C virus-associated chronic active liver disease

SummaryIntercellular adhesion molecule-1, an immunoglobulin supergene family member, is known to account for important steps in cell activation and the immune response. By a non-isotopic slot-dot immunoblotting assay, we measured circulating levels of intercellular adhesion molecule-1 in 26 patients with hepatitis C virus-associated chronic active liver disease before and after β-interferon therapy, in 6 patients with non-A, non-B acute self-limiting hepatitis and in 13 healthy subjects. Circulating intercellular adhesion molecule-1 was found in 10 of 13 (77%) normal controls at low concentrations which were not statistically different from those measured in patients with hepatitis C virus-associated chronic active liver disease responsive to β-interferon, whereas significantly higher levels were found in unresponsive patients. Higher serum intercellular adhesion molecule-1 levels were found in 4 of 10 (40%) β-interferon-responsive patients compared with 13 of 16 (18%) unresponsive patients. Intercellular adhesion molecule-1 levels persisted after discontinuation of β-interferon treatment and did not correlate with hepatocytolysis (as indicated by alanine aminotransferase serum activity) either in chronic active liver disease or acute hepatitis. However, a good correlation was found between intercellular adhesion molecule-1 and its expression on liver cells, thus emphasizing that induced circulating levels may reflect the state of activation at the sites of the inflammatory process. These data strongly support the view that intercellular adhesion molecule-1 plays an important role in liver cell damage in hepatitis C virus-associated acute and chronic liver disease, and that its circulating levels may be a good prognostic parameter of responsiveness to β-interferon therapy.

HCV-Induced Cryoglobulinemic Vasculitis: Pathogenesis and Therapeutic Implications

Leucocytoclastic vasculitis is the dominant lesion of mixed cryoglobulinemia (MC). The high prevalence of antibodies to hepatitis C virus (HCV) in association with the higher concentration of HCV RNA genomic sequences in the cryoglobulins and analysis of the composition of immune complexes (ICs) suggest a close relation between MC and HCV infection and strongly supports the view that this virus plays a key role in causing vascular damage. Our data indicate that endothelial cells are fully susceptible to infection of HCV, and support the contention that they serve as sufficient targets for the binding of HCV proteins expressed on the cell surface to serum immunoglobulins. The in situ demonstration of IgM RF WA cross-idiotype (XId) adds further evidence that RF of the WA group participates in the development of vasculitis, and probably stabilizes the binding of ICs. Lymphomonocytes may be crucial in the infection of endothelial cells by acting as a circulating viral reservoir. The efficacy of interferon in the treatment of MC, has been shown in more than 50% of patients and includes improvement of cutaneous vasculitis and renal function. However, almost 80% of responders have a clinical and biochemical relapse after treatment withdrawal.