Detection of HCV Components and Pathological Reactions in Apoptotic Hepatocytes from Chronically HCV-infected Patients

Abstract

&#13; Analysis of Hepatitis C Virus (HCV)-infected hepatocytes at the cellular level may contribute to elucidate the mechanisms of HCV pathogenesis. In this work, the presence of HCV components and pathological reactions in apoptotic hepatocytes from chronic HCV-infected patients were studied by electron microscopy and confocal microscopy. Eight samples of liver biopsies from patients with chronic hepatitis C were studied by laser scanning confocal microscopy, Transmission Electron Microscopy (TEM) and Immunoelectron Microscopy (IEM). Data provide evidence for apoptosis of hepatocytes from HCV-infected liver biopsies during chronic HCV infection. Confirmation of this process was based on the morphological data by TEM including cell shrinkage; chromatin condensation; formation of apoptotic bodies; phagocytosis by neighbouring cells; and the presence of DNA fragmentation by TUNEL assay and caspase 3 activation. Interestingly, Hepatitis C core protein (HCcAg) was specifically immunolabeled in the rough endoplasmic reticulum, mitochondria as well as in the nucleus of apoptotic hepatocytes. In addition, E1 was specifically immunostained in the cytoplasm and in the mitochondria of some hepatocytes. The presence of Crystalloid Bodies (CB) similar to those observed in recombinant<i> P. pastoris</i> expressing HCcAg was observed in the cytoplasm of some hepatocytes. Immunogold labelling showed that HCcAg co-localized with these CB. In addition, structures forming a paracristalline array and particles with a diameter of 50 nm appeared in the mitochondria of some apoptotic hepatocytes. Moreover, unstructured large aggregates containing HCcAg similar to those detected at late stages of HCcAg expression in recombinant <i>P. pastoris</i> cells were frequently observed in damaged hepatocytes. Of note, these aggregates were specifically immunostained with anti-HCcAg. Data suggest the possibility for a direct role of these HCV-related structures as well as HCcAg and E1 in apoptosis and pathogenicity

Ultrastructural Evidences of Hepatitis B Infection in Human Liver Biopsies Disclose Complex Assembly and Morphogenesis Pathways for Hepatitis B Virus

“…On the other hand, HBsAg was noted in the cytoplasm as well as on tubular structures [7,10] . It is interesting to note that the HBV genome has been detected in hepatocytes from these liver biopsies using an in situ hybridization assay [20] followed by confocal microscopy (Falcon et al, unpublished results). Thus, data support the infection of hepatocytes by HBV in these samples.…”

Interaction of a C-terminal Truncated Hepatitis C Virus Core Protein with Plasmid DNA Vaccine Leads toin vitro Assembly of Heterogeneous Virus-like Particles

“…For immunofluorescence staining (IF) analysis, samples were immediately fixed with 4% paraformaldehyde in PBS at 4 ºC and then mounted on gelatine-coated glass slides and stored for 2 days at -200 ºC. Then samples were treated as previously described [20] and incubated overnight at 4 ºC with with anti-HCcAg.120 IgG antibodies. Specific reactions were detected as described elsewhere [20] .…”

“…Then samples were treated as previously described [20] and incubated overnight at 4 ºC with with anti-HCcAg.120 IgG antibodies. Specific reactions were detected as described elsewhere [20] . Stained samples were coverslipped in Vectashield mountaing medium (Vector Laboratories, Inc. Burlingame, CA., USA), sealed with nail polish and viewed on an immunofluorescence microscope.…”

Evidences of Apoptosis in Hepatitis C Virus-Infected Hepatocytes and Peripheral Blood Monocuclear Cells in the Absence of Liver Injury

“…For controls, an antisense biotin-labeled probe for rat prolactin mRNA (5'-biotin-ACATATCTGTATACAGGGTAG-3', position 63-82) was used. These probes were synthesized and purified using conditions previously described [13] .…”