The Therapy For Chronic Hepatitis C Biology
Interferon was discovered as an antiviral agent during studies on virus interference. Isaacs and Lindenmann reported in 1957 that influenza virus-infected chick cells produced a secreted factor that mediated the transfer of a virus-resistant state active against both homologous and heterologous viruses . IFNs are a multigene family of inducible cytokines [2-5]. IFNs are commonly grouped into two types . Type I IFNs are also known as viral IFNs and include IFN-α (leukocyte), IFN-β, (fibroblast) and IFN-ω. Type II IFN is also known as immune IFN (IFN- γ). The IFNs are induced by virus infection, whereas type II IFN is induced by mitogenic or antigenic stimuli. Most types of virally infected cells are capable of synthesizing IFN-α/β in cell culture.
The therapy for chronic hepatitis C has evolved steadily since alpha interferon was first approved for use in this disease more than 10 years ago. At the present time, the optimal regimen appears to be a 24- or 48-week course of the combination of pegylated alpha interferon and ribavirin.
IFN signaling involves an IFN-mediated heterodimerization of the cell surface receptor subunits, IFNAR-1 and IFNAR-2 with IFN-α/β and IFNGR-1 and IFNGR-2 with IFN-γ [3,6]. IFN binding leads to activation of overlapping pairs of Jak and Stat transcription factors by tyrosine phosphorylation. The Jak-1 and Tyk-2 kinases are activated by IFN-α/β, which leads to the phosphorylation and dimerization of the Stat-1 (p91) and Stat-2 (p113) proteins and subsequent translocation, along with IRF-9 (p48), to the nucleus. The complex of these three proteins, known as IFN-stimulated gene factor 3 (ISGF-3), trimeric complex binds to a cis-acting DNA element, designated ISRE (IFN-stimulated response element), found in IFN-α/β-inducible genes, activates the transcription of IFN-α/β-inducible genes like protein kinase PKR, 2',5'-oligoadenylate synthetase, RNase L, RNA-specific adenosine deaminase, protein Mx GTPase, major histocompatibility complex proteins and inducible nitric oxide synthase [2,7,8].
The 2',5'-Adenine (2-5A) response leading to the degradation of RNA requires two enzymes, Oligoadenylate Synthetase (OAS) and RNase L. OAS catalyzes the synthesis of oligoadenylates of the general structure ppp(A2'p)nA, commonly abbreviated 2-5A. As their name implies, they possess a 2',5'-phosphodiester bond linkage. RNase L, a latent endoribonuclease, becomes activated after binding to 2-5A oligonucleotides. The expression, regulation, and function of the OAS and the 2-5A-dependent RNase L has been characterized extensively in IFN treated and virus-infected cells [2,9,10].
Number of studies, by different groups had established that the 2-5A system functions in the antiviral effects of IFN. For example, elevated levels of 2-5A and the appearance of specific rRNA cleavage products characteristic of RNase L activation are correlated with IFN-mediated inhibition of encephalomyocarditis virus (EMCV) [11,12], vaccinia virus  and reovirus infections . Introduction of 2-5A into cells has been shown to reduce the cytopathic effects of several viruses, including vesicular stomatitis virus (VSV), EMCV, poliovirus, and Semliki Forest virus , whereas a 2-5A analog inhibitor of RNase L has reduced the anti-EMCV activity of IFN in intact cells . Further, in cell lines in which 2-5A pathway activity is defective, EMCV replication is resistant to the inhibitory effects of IFN . The cloning of cDNAs encoding 2-5A synthetase and RNase L has provided a means of definitively addressing the antiviral function of the 2-5A system in cells . Constitutive expression of the 40-kDa form of 2-5A synthetase conferred resistance to EMCV and mengovirus [19,20].
Studies have demonstrated the presence of IFN antibodies in the chronic hepatitis C patients receiving IFN therapy [21-23]. We have no information in this context from this country. We do expect the presence of IFN antibodies in chronic hepatitis C patients seen at our centre who are Non-responders. These antibodies could be interrupting the antiviral effect of IFN therapy by neutralizing them and influencing the changes in the expression levels of OAS1 gene. The study was designed to detect the presence of IFN antibodies and its effect on OAS1 expression in patients of chronic hepatitis C and correlate the data obtained with the severity of the disease.Patients and Methods:
The study includes thirty two chronic hepatitis C patients attending the medical wards and out patients departments of Maulana Azad Medical College and associated Lok Nayak Hospital, New Delhi between January 2009 and December 2009. Healthy group comprised of thirty two blood samples from voluntary donors and six liver tissue autopsy from accidentally died individuals who do not have any evidence of hepatobilliary disease. Informed consent was obtained from each patient and from relatives in case of autopsy who were included in the study.
Inclusion and Exclusion criteria:
Cases of chronic hepatitis were diagnosed as per American Association for Study of Liver Disease (AASLD) practice guideline  and included in this study. Patients who were having advanced cirrhosis, hepatocellular carcinoma were excluded. Patients with other hepatotropic virus infection, alcoholic and drug abuse were also excluded.
Study design and procedures:
The frequency of occurrence of interferon-α antibodies in chronic hepatitis C patients were compared with control group, comprising of healthy individuals who do not had any evidence of any hepatobilliary disease. Blood samples were collected in standard plain and E.D.T.A. vials from all subjects using all aseptic precautions for biochemical investigations like Heamogram, LFT, prothrombin time and KFT. Remaining serum and plasma were stored at -70OC for HCV genotyping, viral load and quantification of Anti-interferon Antibodies.
Liver biopsy was carried out in all patients of chronic hepatitis C for confirmation of the diagnosis as well as for the assessment of histological activity index. For the control group the liver tissue was obtained from autopsy of cases (as per ethical grounds) who died of accidental trauma and did not had any evidence of hepatobilliary disease. The liver tissues were collected in RNA Later (Sigma) a RNA stabilising solution and stored at -70OC for further use. Liver biopsies were subjected to histopathological examination for assessment of severity of liver disease, as well as for expression studies of OAS1 gene by RT-PCR. The antibody levels, OAS-1 gene expression levels were compared with the HCV genotype, viral load and severity of the disease. The study protocol conforms to the ethical guidelines of the 1975 declaration of Helsinki as reflected in a prior approval by the Institution Ethical Committee of Maulana Azad Medical College, New Delhi.
Serological tests were performed using commercially available ELISA kit according to instruction of the manufacturer. Antibodies for HCV were detected by 3rd generation INNOTEST HCV EIA Ab3 kit (Innogenetics NV, Ghent, Belgium).
Histological examination was performed and the severity of liver disease was assessed according to Knodell score for histologic activity index (HAI) and Ishak's fibrosis staging using sections from formalin-fixed and paraffin-embedded liver tissue samples by hematoxylin-eosin staining .
HCV RNA detection and genotyping:
HCV RNA was extracted using the TRI® reagent (Sigma) from plasma and polymerase chain reaction (PCR) was performed to detect HCV RNA, as described by KePing Qian. The genotyping was done by type specific primers in a multiplex PCR system as described by Kazuaki Chayama .
Quantification of serum Interferon-α antibody levels:
The serum concentrations of interferon antibodies in HCV infected and healthy individuals were measured using anti-IFN-alpha quantitative ELISA kit (Abnova; Taiwan). The interferon antibodies levels were determined by comparing the optical density of the samples with a standard curve containing the increasing amounts of interferon antibodies, as per guidelines of manufacturer's protocol.
Total mRNA isolation and OAS1 quantification from liver tissue biopsy:
Total mRNA from liver tissue biopsies were isolated using Qiagen RNesy Mini Kit and cDNA library was constructed. These cDNA were used for the expression analysis of 2′, 5′-OAS1 by relative quantification method with SYBR green dye in reference to a house keeping gene β-actin on the Real Time PCR machine (Corbett Research; RotorGene-3000) using 2-ΔΔCt method as explained by Livak et.al. . Six normal autopsy liver tissues were used as the calibrators for the normalization. The primer efficiency of OAS1 and β-actin was checked by plotting ï„ct values of target and referral gene serial dilutions. (Figure. 1)
Statistical significance was determined by Chi-square test with Yates correction (wherever needed) or by 2-sided Fisher exact test, Mann Whitney U test and student's t- test wherever applicable, using EpiInfo and statistical package for social science, version 10 (SPSS, Chicago, IL). The p values ≤0.05 were considered significant.Results:
Out of 32 chronic hepatitis C cases having an average age of 31.56 ± 8.97 years (range 20-51 years), 6 patients were female (18.75%) and 26 patients were males (81.25%). In 32 healthy control cases, 12 were females (37.5%) and 20 were males (62.5%) having an average age of 34.90±10 years (range 19-54 years). In chronic hepatitis group treatment naïve patients were 26 and patients treated with IFN and ribavirin were 6.
Interferon antibody levels:
The IFN-α antibodies were present in 14 (43.75%) out of 32 chronic hepatitis C patients and only 3 (9.38%) healthy individuals were carrying the antibodies out of 32 (p value = 0.002). Antibodies among the HCV patients do not show any correlation with age and sex. Eleven (42.30%) out of 26 treatment naïve patients had IFN antibody mean concentration of 6.8 ±10.01 ng/ml and 3 (50%) out of 6 treated patients had mean of 76.80±117.01 ng/ml.
The antibody levels were higher in the treated patients when compared with naïve (76.80±117.01 and 6.8±10.01 ng/ml respectively) suggesting the treatment enhances the anti-IFNα antibody production. Though the number of patients having antibodies among treated and naïve does not show any significance, the treated patients have significantly more concentrations of antibodies than treatment naïve patients (p value 0.05). (Table.1)
When we compared viral loads and IFNα antibodies in the two groups we observed a mean HCV viral load of 2.45±2.76 (log10 IU/ml) in treated and 5.57±0.66 (log10 IU/ml) in non treated patients. Treated patients significantly have lesser viral load as compared to non treated patients. Though there was no statistical significance observed in IFNα antibody concentrations but the mean concentration levels were higher in treated patients than non treated patients. Though the interferon antibodies were present, low viral loads were observed in treated patient than naïve.
OAS1 expression levels:
The IFN therapy induced increase in the OAS1 expression as an antiviral activity (76±70.15 fold) when compared with treatment naïve (19.40±28.45 fold). When antibody concentrations were compared among treatment naïve patients in association with the expression levels of OAS1, it was observed that when antibody levels decreased (0.28±0.56 ng/ml) simultaneously there was a significant increase (25.17±29.57 fold) in OAS1 gene expression. Similarly in these patients, when antibodies were increased (5.10±9.37 ng/ml) there was a decrease in OAS1 expression level (2.29±0.99 fold) (p value 0.03). Though the values are not statistically significant in treated patients the antibody presence and OAS1 expressions are showing same trend of antagonism. (Table. 2) When we correlated the OAS1 gene expression with in different genotypes, there was no significant increase or decrease in the levels, as well as presence or absence of IFNα antibodies showed no significance with in different genotypes.
Histological activity in HCV infections:
The HAI scores were correlated with different genotypes, viral load, OAS1 gene expression and IFNα antibody presence among chronic hepatitis C infective patients. We were unable to find any correlation between genotype, viral load, IFNα antibodies and HAI score. However, we observed an enhancement in the OAS1 gene expression in six cases when antibodies were absent and at the same time in six cases where antibodies were present OAS1 expression was very low among a group HAI score was 5-8. As the inflammation increases there is a decrease in trend of OAS1 gene expression. (Figure. 2)Discussion:
All humans have naturally occurring anti-interferon antibodies in their serum and it is a tempting theory that human cytokines and lymphokines are, at least partly, regulated by these auto antibodies. Ross et al. (1990) found antibodies in systemic disorders like SLE and cancer, but not before treatment . Another study by Hansen et al. (1992) had proposed binding of IgG antibodies to IFN but this binding is nonspecific and they also did not find these antibodies in normal subjects . However in the present study we found conflicting results of IFN antibody presence in 3 (9.4%) healthy subjects, 11 (42.3%) treatment naïve and 3 (50%) treated patients. In treatment naïve group the IFN secreted for viral clearance might have influenced the production of IFN antibodies as counter-regulatory mechanism.
IFN antibodies in serum were observed in a significant number of chronic hepatitis C patients when compared with normal healthy persons. We observed that the IFN antibody concentrations were higher in treated group than treatment naïve group. This data gives an inference that IFN antibodies were present in patients of chronic hepatitis C, and the IFN therapy further enhances their concentrations. Ikeda et al. (1991) postulated that though IFN antibodies were present in acute hepatitis, they do not block IFN activity . Querashi et al. in 2007 had observed that interferon antibodies were formed in a small percentage of cases receiving interferon therapy . However, in our study we observed higher concentrations of interferon antibodies levels in treated HCV patients.
Kobayashi et al. (1996) studied the affect of IFN antibody on IFN therapy in 58 chronic hepatitis C patients . They found that the response rate was not muchdifferent between patients with the antibody and those without (29% and 41%, respectively). Our study suggests that some cases of chronic hepatitis C patients who have not received INF therapy, yet harbors Anti-IFN Antibodies even before treatment and concentrations were increased in treated patients. These patients may be needed to screen for the presence of anti-IFN antibodies before initiating therapy.
The 2-prime,5-prime oligoadenylate synthetases (OASs) are interferoninduced proteins characterized by their capacity to catalyze the synthesis of 2-prime,5-prime oligomers of adenosine (2-5As). 2-5As bind to and activate RNase L, which degrades viral and cellular RNA, leading to inhibition of cellular protein synthesis and impairment of viral replication. [32,33]. Nilson et al. (1982) had demonstrated the role of 2-5A synthetase and RNAase L, in HeLa cell lines infected with Reovirus and treated with interferon . Full-sized reovirus mRNAs were detected in control infected cells, but not in interferon-treated infected cells, at 6 h post infection. Suggesting the OAS1 influence the viral RNA degradation through RNAase L cascade. In our study, we had observed that in treatment naïve patients when IFN antibody concentrations were low there were significantly higher OAS1 expressions and vice versa. Similarly in the treated patients the antibody presence and OAS1 expressions are showing inverse relationship with each other, though statistically not significant. Thus it appears that OAS1 expression was inhibited by IFN antibodies. (Figure. 3)
When we had correlated the HCV viral load with OAS1 expression, we found that, as the viral load increased there was simultaneous enhancement in the expression of OAS1, to reduce the load in treatment naïve patients (p value < 0.05) (Figure. 4). Similar observations were seen in treated patients though the inference was statistically not established due to less number of cases (Table. 2). This hypothesis needs to be confirmed in large number of patients receiving IFN and ribavirin therapy.
In one non responder patient, we had observed a very high IFNα antibody concentration (211.91 ng/ml) and very low OAS1 gene expression (2 fold) level with persistent HCV viral load, supporting the contention that pre-existing IFNα antibodies do occur and interfere in the viral clearance. Thus, this could be a matter of concern particularly for the non-responders.
We had observed that when HAI score was 0-4, i.e minimal inflammation of liver in chronic hepatitis C patients, expression of OAS 1 gene was maximum and after that as the HAI score or inflammation increased expression of OAS1 decreased. This signifies that, in the initial stage of hepatitis C, as the virus makes inroads into hepatocytes, the expression of OAS1 increased to combat HCV viral replication and reduces inflammation. However, the correlation between HAI and OAS1 expression has not been studied so far to compare our data. In our study we found that among IFN antibody carriers the levels of OAS1 expression decreased with the progression of liver disease (Figure. 2). This might be due to disruption of the 2-5A system which controls of cell growth, differentiation, and apoptosis .
Most of the reported studies from India seem to suggest a north south divide, wherein genotype 3 predominates in the north, east and west India, whereas genotype 1 is commoner in south India [35-38]. Our study also supports the already existing data in this regard from different studies. The present data had failed to show significant correlation between different genotypes of HCV and anti IFN antibodies and OAS1 gene expression.
This pilot study had shown prevalence as well as concentration of IFNα antibodies were more in chronic hepatitis C patients compared to healthy subjects. Age and sex does not affect the IFNα antibody occurrence in chronic HCV patients as well as healthy individuals. Treated patients have significantly more concentrations of antibodies than treatment naïve patients. OAS1 gene expressions were higher in treated group as compared to non treated group. IFN therapy increases the OAS1expression as an antiviral activity when compared with the naïve. The IFN antibody levels and OAS1 expressions showed a trend of antagonism. Interferon therapy was influencing the anti-IFN antibody concentration levels to rise leading to lowering of OAS1 expression levels in treated patients compared to naïve patients. No correlation exists between IFNα antibodies, OAS1 expression, genotype and viral load. The preexisting IFN antibodies might have negative regulation in the out come of the therapy. Thus screening of the patients for antibodies before initiating therapy to predict the outcome of therapy may be advisable.
However, further study with a larger sample size was required for confirmation of the possible role of anti-IFNα antibodies and their clinical significance with respect to the treatment of chronic hepatitis C patients.
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