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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2011  |  Volume : 54  |  Issue : 2  |  Page : 355-358
Relation of hepatitis B core antigen expression with histological activity, serum HBeAg, and HBV DNA levels


1 Department of Pathology, Izmir Ataturk Researh and Training Hospital, Izmir, Turkey
2 Department of Gastroenterology, Izmir Ataturk Researh and Training Hospital, Izmir, Turkey

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Date of Web Publication27-May-2011
 

   Abstract 

Background: Hepatitis B virus (HBV) is a potentially life-threatening liver infection which may progress to liver failure and cirrhosis. Intrahepatic expression patterns of viral antigens detected by immunohistochemistry may have prognostic implications in disease process. Aim: In this study, we aimed to investigate the relationship between the HBV core antigen (HBcAg) expression and histological activity index (HAI), fibrosis, serum hepatitis B e-antigen (HBeAg) status and HBV DNA levels in patients with chronic HBV infection. Materials and Methods: A total of 114 liver biopsies from patients with chronic HBV infection were included in the study. Immunohistochemical expression of HBcAg and its relation with HAI, fibrosis, serum alanine aminotransferase (ALT) levels, HBeAg status and HBV DNA levels were assessed. Results: The presence of nuclear expression of HBcAg did not show any correlations with ALT levels, HAI and fibrosis score. When the groups were categorized according to the HBeAg status, nuclear HBcAg expression was found to be high in HBeAg positive patients. However, HBcAg nuclear expression showed significant correlations with HBV DNA levels and fibrosis scores in HBeAg negative but not HBeAg positive patients. HBV DNA levels were also significantly associated with HAI and fibrosis scores in HBeAg negative patients. Conclusions: Significant differences found between HBeAg positive and negative patients suggest that HBeAg negative disease is different from HBeAg positive disease, and also point outs that in HBeAg negative disease, patients with nuclear HBcAg expression and increased levels of HBV DNA levels are at a higher risk of developing progressive liver disease.

Keywords: Chronic hepatitis, hepatitis B, hepatitis B core antigen, immunohistochemistry

How to cite this article:
Sari A, Dere Y, Pakoz B, Calli A, Unal B, Tunakan M. Relation of hepatitis B core antigen expression with histological activity, serum HBeAg, and HBV DNA levels. Indian J Pathol Microbiol 2011;54:355-8

How to cite this URL:
Sari A, Dere Y, Pakoz B, Calli A, Unal B, Tunakan M. Relation of hepatitis B core antigen expression with histological activity, serum HBeAg, and HBV DNA levels. Indian J Pathol Microbiol [serial online] 2011 [cited 2019 Dec 16];54:355-8. Available from: http://www.ijpmonline.org/text.asp?2011/54/2/355/79972



   Introduction Top


Chronic hepatitis B virus (HBV) infection is a major health problem which may progress to liver failure, cirrhosis and hepatocellular carcinoma (HCC). [1] The outcome of the chronic HBV infection depends on both viral and host-specific factors and it is important to predict which chronic HBV patients will progress to cirrhosis or HCC. [2] The association of serum HBV DNA levels, an indicator of HBV replication, and the development of chronic HBV related comorbidities has been pointed out by several studies. [3] On the other hand, intra-hepatic expression and localization patterns of viral antigens detected by immunohistochemistry are thought to serve additional benefit regarding viral replication.[4] Hepatocyte expressions and localizations of HBV core antigen (HBcAg) and HBV surface antigen (HBsAg) were studied previously and inconsistent findings were found regarding their relations with viral replication parameters. [5],[6],[7],[8],[9] As there are limited numbers of reports and the data are inconsistent, we aimed to study immunohistochemical expression of HBcAg and its relationships between histological activity, fibrosis scores, hepatitis B e-antigen (HBeAg) and HBV DNA levels.


   Materials and Methods Top


Liver biopsies from a total of 114 patients with chronic HBV infection who were followed up regularly between 2005 and 2009 were included in the study. All were HBsAg positive, and antibody against HCV (anti-HCV), antibody against hepatitis D virus (anti-HDV), autoimmune markers and antibody against human immunodeficiency virus (anti-HIV) were negative. None of the patients received antiviral or immunosuppressive therapy. All patients' HBeAg data, HBV DNA titer and alanine aminotransferase (ALT) levels were collected from the hospital records. HBV DNA levels and ALT levels used in this study were determined within 1 week before the liver biopsy.

HBeAg, anti-HBe, anti-HCV, anti-HDV and anti-HIV were tested by enzyme immunoassay. Serum HBV DNA levels were determined by polymerase chain reaction (PCR). Liver biopsy specimens were fixed in 10% formaldehyde, and after routine tissue processing, all biopsies were stained with hematoxylin-eosin, reticulum and Masson's trichrome. Histopathologic findings were assessed and scored according to Ishak's scoring system [10] by a pathologist who was unaware of the HBV DNA levels and the virological test results.

Immunohistochemical staining for HBcAg was performed by using streptavidin-biotin-peroxidase technique. Paraffin-embedded tissue samples were cut into 5-μm thick sections and placed onto poly-l-lysine-coated slides for immunostaining with HBcAg (rabbit anti-HBcAg, Neomarkers, 1413R810A, Fremont, CA, USA). Paraffin sections were incubated at 60°C overnight. Then, the paraffin was removed by washing with xylene followed by absolute ethanol. Endogenous peroxidase activity was inactivated by 3% hydrogen peroxide in methanol for 30 minutes. The slides were heated in ethylenediaminetetraacetic acid (EDTA), pH 6.0, in a pressure cooker oven for 20 minutes for antigen retrieval, allowed to cool at room temperature, and washed in distilled water and phosphate-buffered saline (PBS). Diaminobenzidine was used as the chromogen. The slides were counterstained with Harris's hematoxylin and dehydrated through graded alcohols to xylene and mounted.

Any positive immunoreactivity in the nuclei of hepatocytes was accepted as positive for HBcAg expression [Figure 1] and negative if there was no nuclear immunoreactivity in the nuclei.
Figure 1: Immunohistochemical expression of HBcAg in nuclei of hepatocytes, IHC, x400

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Statistical Analysis

Data were presented as median with minimum and maximum values. Comparison between groups of continuous variables was performed by using the Student's t-test. Differences between categorical variables were analyzed with Chi-square test. Relationships between variables were analyzed using Pearson's correlation coefficients. The statistical analysis was carried out using Statistical Package of Social Science (SPSS), version 13.0 (SPSS Inc., Chicago, IL, USA). A double-tailed P value of <0.05 was considered as statistically significant.


   Results Top


There were 114 patients (76 M/38 F) [mean age 41.5 (20-84) years] in the study group. Twenty-five patients (22%) were positive for HBeAg. There were 29 patients who had nuclear HBcAg expression. The clinical and laboratory data of the cases are listed in [Table 1].
Table 1: Demographic and laboratory characteristics of the study group

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Comparison of HBeAg(+) vs. HBeAg(−) Subjects

HBeAg positive patients were significantly younger than the HBeAg negative subjects [29 (22-84) vs. 44 (20-79) years; P < 0.05]. Sex distributions and ALT levels were not different between the groups (P > 0.05). HBV DNA levels were significantly higher in the HBeAg positive patients than the HBeAg negatives {100,000 (10,000-500,000) vs. 5000 (2-900,000) IU/mL, ´10 3 , respectively; P < 0.05}.

Nuclear staining of HBcAg was significantly pronounced in the HBeAg positive patients (72% in HBeAg positive patients vs. 12% in HBeAg negatives; P < 0.001) [Table 2].
Table 2: Comparison of HBeAg(+) vs. HBeAg(−) subjects

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Hepatitis activity index (HAI) and fibrosis scores were not different between HBeAg positive vs. negative group (P > 0.05) [Table 2]. [Table 2] represents the data regarding HBeAg positive and negative patient groups.

Relationship of the Nuclear Expressions of HBcAg with Histological Activity, Fibrosis, and HBV DNA Levels

There was no relation of the presence of nuclear expression of HBcAg with ALT levels, HAI, and fibrosis scores (P > 0.05). However, nuclear HBcAg expression correlated positively with HBV DNA levels (P < 0.05; r = 0.4).

When the groups were categorized according to the HBeAg status, HBcAg nuclear expression showed significant correlations with HBV DNA levels and fibrosis scores (P < 0.05; r = 0.35 and 0.3, respectively) in HBeAg negative patients. HBV DNA levels were also significantly associated with HAI and fibrosis scores (P < 0.05; r = 0.5 and 0.4, respectively) in HBeAg negative patients.

Relationships Between Hepatic Activity Index, Fibrosis and Other Parameters

There were significant correlations between HAI, fibrosis, HBV DNA levels and ALT concentrations (P < 0.05; r = 0.76, 0.38, and 0.23, respectively). Similarly, fibrosis correlated with HAI and HBV DNA levels (P < 0.05; r = 0.76 and 0.32, respectively). However, we could not observe correlations between HAI, fibrosis, nuclear expression of HBcAg and the presence of HBeAg. [Table 3] presents the correlations between HAI, fibrosis and other parameters.
Table 3: The correlation coefficients between HAI, fibrosis and other parameters

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   Discussion Top


Intrahepatic distributions of HBV related antigens are of interest and are currently being studied in chronic HBV infection. The presence of HBcAg usually indicates active viral replication and the staining patterns of HBcAg may also show a relation with HBV DNA and/or histological activity. [8],[11] HBcAg may be present in the cytoplasm, nucleus or both.

The localization of the core antigen may reflect the behavior of the disease and it may also predict the response to the antiviral treatments. [12],[13],[14] In general, cytoplasmic expression of HBcAg reflects a low level of viral replication but higher hepatocellular damage, while nuclear expression of the core antigen reflects a high level of viremia but lower hepatocyte injury. [5],[6],[8],[12],[15],[16],[17] The reason why the level of HBV DNA in serum correlates with expression of HBcAg in the hepatocyte nucleus rather than the cytoplasmic expression is unclear. In prior studies, it is reported that the replication of HBV is cell cycle regulated; in quiescent cells, HBcAg is located in the nucleus but in proliferating cells it is located in the cytoplasm. [5],[18] Upon infection with HBV, the virus enters a cell and is transported to the nucleus with concomitant uncoating. During this process, viral DNA is converted to covalently closed circular (CCC) DNA in the nucleus. It is proposed that the possible function of the transport of the HBcAg protein from the cytoplasm to the nucleus is to amplify to pool of CCC DNA. [18] Therefore, subcellular localization of core antigen is correlated with the level of viral replication in chronic HBV infection. [12],[18] In accordance with these observations, we found that the nuclear expression of HBcAg correlated with serum HBV DNA titers, but we did not find any relation with histological activity. In our study, background and nonspecific cytoplasmic staining found in many biopsies rendered the reliable interpretation of cytoplasmic HBcAg challenging. Therefore, we could not be sure whether the cytoplasmic positivity reflects the true cytoplasmic positivity or a nonspesific background staining. So, we only evaluated the nuclear expression of HBcAg staining.

HBeAg is generally considered to be a marker of HBV replication and infectivity. The presence of HBeAg is usually associated with high levels of HBV DNA in serum and higher rates of transmission of HBV infection from carriers to healthy subjects. [19] In our study, when HBeAg positive and negative groups were compared, patients were found to be significantly younger in HBeAg positive group, suggesting early stages of disease, which is in concordance with previous studies. [12],[20] We also observed that serum HBV DNA levels and nuclear expression of HBcAg were also significantly higher in HBeAg positive group. Similar findings were reported by other studies, supporting the belief that nuclear expression of core antigen characterizes immune tolerance phase with high level of viremia. [12],[20] There are inconsistent data regarding the relation of HBcAg expression with HBV DNA and histological activity. [11],[12],[21] Disparate results involving the quantitative expression and topographical distribution of HBcAg have been attributed to the different anti-HBc antibodies used. [22] Furthermore, requirement of a method which was capable of detecting low concentrations of cytoplasmic HBcAg had been proposed. [22] Nonspecific cytoplasmic HBcAg expression found in many liver biopsies made up the limitation of our study to investigate the role of cytoplasmic HBcAg staining in chronic HBV disease. Ramakrishna et al. found that the nuclear expression and cytoplasmic expression of HBcAg was related with disease activity in HBeAg negative and HBeAg positive diseases, respectively. [12] Kim et al. [11] reported a positive correlation between cytoplasmic HBcAg expression and histological activity in both HBeAg positive and negative patients.

In the current study, serum levels of HBV DNA showed a positive correlation with HAI, fibrosis and nuclear expression of HBcAg in HBeAg negative subjects but not in HBeAg positive subjects. When the whole study group was taken into account, we observed significant but weak correlations between HBV DNA levels, HAI, and fibrosis which may probably be a result of HBeAg negative subjects group. Increased nuclear HBcAg expression also showed positive relation with fibrosis scores in HBeAg negative patients and not with HBeAg positive and the whole study group patients. These findings are in agreement with Ramarkishna's study [12] which suggests that HBeAg negative disease is different from HBeAg positive disease, and also confirms that in HBeAg negative disease, patients with increased levels of HBV DNA levels had increased risk of progressive liver disease.

In summary, we did not find any correlation between the presence of nuclear expression of HBcAg and ALT levels, HAI and fibrosis score. When the groups were classified according to the HBeAg status, we found that HBeAg positive patients presented at a younger age had higher HBV DNA levels and increased nuclear HBcAg expression. HBcAg nuclear expression showed significant correlations with HBV DNA levels and fibrosis scores in HBeAg negative patients, while HBV DNA levels were also significantly associated with HAI and fibrosis scores in HBeAg negative patients but not in HBeAg positive patients. These results imply that HBeAg positive and negative disease have biological differences, and in HBeAg negative disease, patients with nuclear HBcAg expression and increased levels of HBV DNA levels are at a higher risk of developing progressive liver disease.

 
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Correspondence Address:
Aysegul Sari
Department of Pathology, Ataturk Caddesi No. 184, Kat 3, Daire 6, Firat Apt, PK 35210, Alsancak, Izmir
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.79972

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