AUTOIMMUNITY IN HEPATITIS C VIRUS INFECTION
M J Dhorda, S R Kankonkar, A L Kirpalani, D N Amarapurkar
Medical Research Centre of Bombay Hospital, Mumbai, India.
Hepatitis 'C' virus (HCV) is a positive strand RNA virus classified as a separate genus within the family Flaviviridae, though this classification may be changed. Its genome consists of about 9400 bases that code for a polyprotein of approximately 3000 amino acids.1 This polyprotein is cleaved after translation by cellular or viral proteases to give the structural proteins that have a presumed enzymatic role in viral replication. HCV is known to have 11 distinct genotypes, with over seventy subtypes, each associated with a varying degree of severity of disease, prognosis and response to interferon therapy.
Typical features of HCV infection include :
-The acute phase of the infection is often subclinical; many infected individuals remain asymptomatic for years.
-A high proportion of infected individuals develop a chronic infection, which often progresses to cirrhosis and hepatocellular carcinoma (HCC).,
-There is a high prevalence of immune disorders such as cryoglobulinaemia and autoimmune disease amongst HCV patients.
HCV is known to be implicated autoimmune hepatitis type 2B (AIH-2b),, with autoantibodies produced against cytochrome P450 IID6 in hepatocytes. However, there is some evidence for the involvement of HCV in AIH-1 as well.,
The present study seeks to study the prevalence of markers of autoimmunity, such as Anti-nuclear antibody (ANA), anti-mitochondrial antibody (AMA), anti-smooth muscle antibody (ASMA) and anti-liver-kidney-microsomal antigen antibody (ALKM-1) in patients infected with HCV.
PATIENTS AND METHODS
The patients selected for this study were referred to a secondary care centre. Infection with hepatitis C virus was confirmed in each case by HCV RNA detection by PCR. 8-10 ml of blood was collected by venepuncture; after separation of the serum from whole blood, it was aliquotted and stored at -70o C until further use. In all, 60 patients were tested for ALKM-1 (35 males, 25 females). Thirty five patients (23 males, 12 females) were tested for ANA and ASMA, and 30 for AMA (20 males, 10 females). The age of these patients ranged from 8 to 64 years; the period of infection, as calculated from presumed date of first exposure, ranged from 10 months to 27 years. 23/60, 17/35 and 17/30 patients in the study were also suffering from chronic renal failure (CRF).
ANA, AMA and ASMA were tested for using the indirect immunofluorescence technique with kits from incstar, USA. The kits included slides with the appropriate tissue fixed on them, appropriate controls, fluorescein isothiocyanate (FITC)-conjugated anti-human IgG antibody, counterstain and mounting medium as well as ready wash buffer that needed to be dissolved in one liter of water. The procedure as outlined in the accompanying manual were followed for each of the kits. Briefly, each serum sample was diluted with the provided phosphate buffered saline (PBS) to a screening dilution as recommended in the instruction manual (1:40 for ANA and ASMA,1:20 for AMA). A drop (30-40 mL) of the diluted sample was applied to individual wells on the slides. A positive and negative control was also applied with each test run. The slides were then incubated at room temperature for 30 minutes in a covered moist chamber. After the incubation, the slides were rinsed with PBS using a squirt bottle and then washed for 10 (15 minutes for ASMA) minutes in a Coplin jar containing PBS. The FITC conjugate was then applied (one drop/20-30 mL) to cover each well. This was followed by another incubation period of 30 minutes as well as rinsing as described earlier. For the final wash, a few drops of Evan's blue were added as a counterstain. After the appropriate period of time the slides were drained and mounted using the provided mounting medium. The slides were then viewed with a Zeiss microscope under UV illumination.
For the ASMA test we modified the recommended procedure slightly - we used a serum dilution of 1:40 for the test and the washing was lengthened to 15 minutes. This was done as we found that interpretation was difficult because all the serum samples showed mild non-specific fluorescence when the suggested procedure was followed. Even with the modifications however, there was no change in the appearance of the +ve and -ve controls. ALKM-1 was assayed using the ELISA technique with kits from Elias, Germany.
RESULTS AND DISCUSSION
In all, 18 out of 35 patients tested positive for ANA at a titre of ô 80, which is considered clinically significant. Only 6 of the 18 patients were also afflicted with CRF. One of these six was a patient of systemic lupus erythematosus (SLE), which was the primary cause of renal disease. Only one patient tested positive for ASMA (age 60/F/No CRF, ANA titre 40) at a titre of ô40. None of the patients tested were positive for AMA and ALKM-1. In summary, only 4 of 35 patients had clinically significant positive titres for ANA or ASMA that can be attributed to HCV infection. Our results suggest that ANA positivity, which by itself is uncommon, is more prevalent amongst individuals infected with HCV than ALKM-1 and it is less likely to occur in patients also suffering from CRF.
Several studies have shown that HCV induces the development of autoimmune antibodies and is implicated in the pathogenesis of AIH-2b.10-12 This is probably due molecular mimicry of cytochrome P450 IID6 by certain epitopes in the HCV core antigen. Some reports show that HCV may induce other non-specific markers of autoimmunity such as ASMA and ANA., The production of these non-specific markers may be a result of generalised immune activation. As compared to a study by Clifford et al, we found lower rates of positivity for non-specific markers. That may be due to the fact that their study did not include any immunocompromised patients, such as those with CRF in the present study.
Another study, by Rostaing et al, conducted solely amongst patients with renal transplants found to significant difference in the prevalence of non-specific autoimmune antibodies between HCV RNA positive and HCV RNA negative patients. This and other studies, seem to show little or no relation between HCV and autoimmune disorders.
A review of the literature concerning autoimmunity presents an unclear picture. It however, becomes clear that there probably are several factors that affect HCV - induced autoimmunity, which may include the genotype of the infecting virus. In conclusion, the present study fails to find a significant association between HCV and autoimmune disease, specifically, AIH-2b. Further research is required to throw light upon other factors that may affect autoimmunity in HCV infection.
This study was supported by a grant from the Bombay Hospital Trust, for which we thank them.
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