© Borgis - Postępy Nauk Medycznych 1/2010, s. 10-14
*Bożena Walewska-Zielecka
Ekspresja antygenów wirusa C zapalenia wątroby i aktywność zapalna w wątrobie u osób przewlekle zakażonych HCV
HCV antigens expression and inflammatory activity in patients with chronic HCV infection
National Institute of Public Health, National Institute of Hygiene, Department of Virology
Head of Department: Bogumiła Litwińska
Streszczenie
Przewlekłe zapalenie wątroby jest istotnym czynnikiem wpływającym na globalne problemy zdrowotne. Szacuje się, że mniej niż 30% zakażonych HCV eliminuje wirusa. U pozostałych 70-80% zakażonych dochodzi do przewlekłego zapalenia wątroby, marskości i jej różnych powikłań.
Celem pracy było zbadanie częstości występowania antygenów wirusa C zapalenia wątroby (HCVAgs) i porównanie występowania ekspresji HCVAgs z całkowitą aktywnością zapalną w wątrobie u osób przewlekle zakażonych tym wirusem. Materiał badany stanowiły wycinki z biopsji wątroby 137 pacjentów przewlekle zakażonych HCV. Antygeny HCV w wątrobie wykrywano w skrawkach mrożonych metodą EnVision z zastosowaniem przeciwciał ludzkich znakowanych FITC.
HCVAgs wykryto w 67 spośród 137 badanych próbek (48,9%). Całkowita aktywność zapalna nie korelowała z ekspresją antygenów HCV. Stwierdzono jednak większą ekspresję antygenów HCV w obszarach miejscowo nasilonego odczynu zapalnego.
Rozszerzenie diagnostyki histopatologicznej o badanie immunomorfologiczne antygenów HCV w wątrobie może przyczynić się do pogłębienia wiedzy o immunopatogenezie przewlekłych zapaleń wątroby i może być wartościowym badaniem uściślającym etiologię przewlekłego zapalenia wątroby.
Summary
Chronic type C hepatitis remains a substantial burden to global health problems. It is estimated, that only up to 30% of infected persons effectively eliminate the virus. In the remaining 70-80% chronic infection develops, with threat of all consequences.
The aim of the study was to establish the expression rate of HCV antigens (HCVAgs) in liver tissue and to compare it with the degree of inflammatory reaction in the liver of patients chronically infected with HCV.
Liver biopsy specimens from 137 patients with chronic HCV infection were studied. The expression of HCV antigens was detected in frozen liver tissue sections by EnVision, DAKO with the use of FITC-labeled human antibodies to HCV antigens.
HCVAgs were detected in 67 out of 137 of the specimens examined (48.9%). Total inflammatory activity did not correlate with HCV antigens expression, however local inflammatory reaction was found as a response to abundant HCVAgs expression.
The extension of histopathological examination by immunohistochemical analysis of HCVAgs in liver tissue may contribute to broadening of the knowledge of the immunopathogenesis of chronic hepatitis and may be regarded as valuable tool for searching for chronic hepatitis etiology.
Chronic type C hepatitis remains a substantial burden to global health problems. According to recent data there are 130 million people infected with HCV, and global prevalence is 2.2% (1). Based on WHO data the prevalence of HCV in Poland is reported as 1% (2). However, Polish experts estimate HCV prevalence in Poland at 1.9%, i.e. about 730 000 persons infected with HCV (3). It is known, that only up to 30% of infected persons effectively eliminate this virus, in the remaining 70-80% patients chronic infection develops, with threat of all clinical consequences (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma) (3, 4, 5, 6).
Histopathological examination of needle biopsy specimen has been regarded as a "gold standard” in the diagnostic procedure of chronic hepatitis of different etiology. The type of liver pathology, the grade of inflammatory activity and the stage of liver fibrosis is evaluated by microscopic analysis of liver tissue (7, 8, 9). The search for tissue expression of HCV antigens seems to be a new and promising diagnostic method, but actually remains restricted mostly to research in basic sciences. Up to now HCV antigens (HCVAgs) were localised exclusively in the cytoplasm of hepatocytes, both in acute and chronic hepatitis in chimpanzees and in humans (10-19).
Inflammatory infiltrate present in a needle biopsy specimen is regarded as evidence of immunological processes in situ, in reaction to viral protein expression. Host attempts to eliminate viruses lead to damage and subsequent necrosis of hepatocytes (20-22). Damage of infected hepatocytes is a result of immune reactions aimed at eliminating infection. Therefore, necrosis of hepatocytes and mononuclear cells reaction is a key feature of this process. The intensity of the immune reaction in liver tissue depends both on the immunological status of the host and immunogenicity of HCV proteins expressed in the liver (5, 6, 23).
In this study the expression of HCVAgs in the liver of patients with chronic HCV infection was evaluated. The results were compared with inflammatory activity in the liver tissue.
Material and methods
The material studied consisted of single liver biopsy specimens and serum samples from 137 patients with chronic liver disease of HCV etiology (mean age 41; range 9-73 yrs) referred to our Department for histopathological and molecular evaluation. HCV etiology of liver disease was confirmed by clinical and serological criteria (anti-HCV and HCV RNA). No other underlying liver disease was present in these patients.
Part of each biopsy specimen (at least 12 mm length) was fixed in formalin and routinely processed to paraffin; the remaining part was divided into two fragments both rapidly frozen. One fragment, measuring approximately 5-6 mm in length was kept at -40°C until used for the detection of antigens, and the other one measuring approximately 2 mm in length was kept at -65°C for RNA extraction subsequent to homogenization.
Histopathologic diagnosis of chronic hepatitis, that included grading and staging of the process (7, 24), was established by examination of serial paraffin sections stained with hematoxylin-eosin, chromotrope 2R-aniline blue and impregnated with silver according to Gomori (25, 26). Total inflammatory activity was ascertained by histopathological activity index (tab. 1). To avoid the overestimation of fibrosis Gomori stain was applied which differentiate reticulin and collgen fibers, therefore shows the difference between reticulin fibres collapse and collagen deposits. The numerical score was presented as HAI (histopathological activity index) (7, 24).
Table 1. Histopathological evaluation and scoring system applied in this study. Modified Knodell score (Histopathological Activity Index; HAI) stratification and fibrosis evaluation (7, 24).
A. Inflammatory activity
| Range of scores of inflammation | Overall activity description |
| 1-3 | Minimal chronic hepatitis |
| 4-8 | Mild chronic hepatitis |
| 9-12 | Moderate chronic hepatitis |
| 13-18 | Severe chronic hepatitis |
B. Fibrosis score
| Fibrosis score | Description |
| S=0 | No fibrosis |
| S=1 | Mild fibrosis |
| S=2 | Moderate fibrosis |
| S=3 | Advanced fibrosis |
| S=4 | Liver cirrhosis |
HCVAgs were detected with the use of human FITC-labeled polyclonal anti-HCV antibodies (12), followed by monoclonal anti-fluorescein antibodies, and finally, by anti-mouse globulin antibodies labeled with peroxidase (EnVision system, DAKO). The specificity of the methods applied in this study was ascertained by negative staining results when primary antibody was omitted or replaced by animal or human sera that did not contain antibodies against antigens of HCV. Direct inflammatory response to viral antigens expression was evaluated during immunohistochemical examination of liver tissue.
Results
HCVAgs were detected in 67/137 cases (48.9%). These antigens were found exclusively in the cytoplasm of hepatocytes as amorphous deposits or small granules (fig. 1). Local inflammatory response, both intralobular (spotty necrosis) and periportal developed in close vicinity of hepatocytes with HCVAgs expression. Particularly numerous cells expressing HCVAgs were found in hepatocellular carcinoma occurring in liver cirrhosis caused by HCV (fig. 2).
Fig. 1. Expression of HCV Ags in numerous hepatocytes. Inflammatory mononuclear cells in direct vicinity of hepatocytes. EnVision, x200.
Fig. 2. Expression of HCVAgs in hepatocellular carcinoma. EnVision, x125.
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