|Year : 2002 | Volume
| Issue : 1 | Page : 12-15
Correlation of autoimmune reactivity with hepatitis B and C virus (HBV and HCV) infection in histologically proven chronic liver diseases
S Shantha , SP Thyagarajan , RK Premavathy , RG Sukumar , K VK Mohan , KR Palanisamy , P Rajasambandam
Dept. of Immunology, Stanley Medical College, Chennai - 600 001, India
Dept. of Immunology, Stanley Medical College, Chennai - 600 001, India
PURPOSE: To comprehensively study the possibility of autoimmune reactivity by hepatitis viruses B and C (HBV & HCV) in Indian chronic liver disease (CLD) patients. METHODS: One hundred and sixty histopathologically proven CLD cases and 100 matched controls were analysed for viral serology for HBV and HCV and autoimmune serology for antinuclear antibody (ANA), anti smooth muscle antibody (ASMA) and Liver kidney microsomal antibody (LKM) using standard immunofluorescence technique. RESULTS: 43.7% of cases were chronic hepatitis B while 16.2% were positive for HCV. CLD-B cases showed ANA positivity in 27.1% and ASMA positivity in 25.7%. CLD-C cases revealed 26.9%, 46.1% and 11.1% positivity for ANA, ASMA and LKM antibodies respectively. These rates and titres of autoantibodies were statistically significant (p=<0.02) when compared with that of controls. Conclusions: Based on the pattern of autoantibody positivity, it could be concluded that chronic HBV infection may induce autoimmune hepatitis (AIH) type I and chronic HCV infection might trigger AIH - Type II in Indian CLD cases.
|How to cite this article:|
Shantha S, Thyagarajan S P, Premavathy R K, Sukumar R G, Mohan K V, Palanisamy K R, Rajasambandam P. Correlation of autoimmune reactivity with hepatitis B and C virus (HBV and HCV) infection in histologically proven chronic liver diseases. Indian J Med Microbiol 2002;20:12-5
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Shantha S, Thyagarajan S P, Premavathy R K, Sukumar R G, Mohan K V, Palanisamy K R, Rajasambandam P. Correlation of autoimmune reactivity with hepatitis B and C virus (HBV and HCV) infection in histologically proven chronic liver diseases. Indian J Med Microbiol [serial online] 2002 [cited 2020 Aug 4];20:12-5. Available from: http://www.ijmm.org/text.asp?2002/20/1/12/8331
The concept of virus induced autoreactivity is not unique to liver diseases. Despite many years of research, the questions on precise mechanism and whether such reactions inevitably lead to tissue damage are still the subject of much debate., Several of the hepatotropic viruses seem not to be directly cytopathic and there is increasing evidence to show that liver damage may be due to host immune responses against virus infected cells or due to concomitant virus induced autoreactions against hepatocytes. The role of hepatitis viruses in the induction of autoimmune chronic active hepatitis (AICAH) or for autoreactivity as a mechanism contributing to liver damage largely remains circumstantial.
In the continued search for the triggering agents for autoimmune diseases, microbial or otherwise, hepatitis viruses especially Hepatitis C Virus (HCV) has been recently implicated most often in autoimmune hepatitis.,,
The present study thus has attempted to comprehensively study the possibility of autoimmune reactivity by hepatitis viruses B and C in Indian chronic liver disease patients with appropriate parameters.
| ~ Materials and Methods|| |
One hundred and sixty histopathologically proven chronic liver disease cases attending the gastroenterology units of Govt. Stanley Hospital (GSH) and Govt. General Hospital (GGH), Chennai constituted the study materials. Fifty healthy controls and 50 age matched patients attending medical outpatient departments of GSH for minor illnesses without liver involvement were also included in the study. Under aspetic precautions, 5 mL of blood was collected by venepuncture, serum separated in three aliquots and stored at -70°C. All Chronic liver disease cases were clinically analysed for extrahepatic autoimmune features. None of them had typical history except for Hashimoto's thyroiditis and Juvenile diabetes mellitus. Liver biopsies from all chronic liver disease cases were studied for histopathological staining as described by Boyer and Reuben. The serum samples were tested for viral markers of HBV and HCV (Hepatitis B surface antigen - HBsAg; Hepatitis B e antigen - HBeAg; antibody to HBeAg - anti HBe and antibody to HCV - anti HCV) using standard ELISA test kits from Organon Technika and Ortho-diagnostics. The Anti - HCV ELISA kits of Ortho-diagnostics used in the study was of the third generation assay type incorporating core (structural), NS3 -
Protease / Helicase (non - structural), NS4(non - structural) and NS5 replicase (non - structural) proteins. Eventhough third generation assays are said to be of improved sensitivity and specificity, all the samples were confirmed by RIBA 3.0 kit (Recombinant Immunobinding assay) of Ortho-diagnostics. PCR for HBV-DNA & RT PCR for HCV - RNA were carried out on all the samples and their comparitive correlation has been presented separately (In Press). The present analysis was based on Anti - HCV ELISA with RIBA confirmation.
Composite tissue blocks were prepared using rat liver, kidney and stomach which were snap frozen after harvest. In a clean petridish the stomach tissue was spread out and cut into strips long enough and wide enough to go round kidney and liver. A wedge of kidney and liver were kept on a strip of stomach which was turned inside out and rolled up. The composite block was mounted. 7 µm cryostat sections were cut and mounted onto slides and stored at -20°C. Antihuman FITC IgG conjugate of “Binding site Birminham, U.K.” was used for the immunofluorescence test as described by Johnson et al.
Complete absence of fluorescence in 1:20 dilution of serum samples were considered negative for autoantibodies, sera showing apple green fluorescence of the nuclei were considered positive for antinuclear antibody (ANA); fluorescent staining of the muscularis layers of the stomach together with any arterial muscle layer present in other tissue sections was considered as positive for anti smooth muscle antibody (ASMA). Serum samples showing strong to moderate fluorescence of the cytoplasm of the liver cells and proximal renal tubules were considered positive for liver kidney microsomal (LKM) antibody. All sera tested positive were further quantitated by dilution.
The results obtained were statistically analysed by Chi square test.
| ~ Results|| |
Out of 160 histopathologically proven CLD cases 70 (43.7%) were positive for HBV markers, 26 (16.2%) were positive for anti HCV and 64 (40%) were negative for both HBV and HCV markers [Table - 1].
ANA and ASMA in low titre (around 1:20) seem to be present in the healthy and matched controls of the study (ANA: 10 & 22%) (ASMA: 4.0 & 8.0%). LKM antibodies are not present in the controls analysed. When this pattern is compared with the positivity rate and titre of auto antibodies in CLD, both are statistically significant (p < 0.02). The baseline cut off titre as generated in the study in controls has revealed significant levels of ANA, ASMA and LKM antibodies in chronic hepatitis C and CLD- NBNC cases [Table - 2].
The analysis for auto antibody positivity pattern as per histological staging has shown that none of the CPH cases to be positive for autoantibodies. The presence of auto antibodies were generally seen in the later stages of CLD like CAH, cirrhosis and Hepatocellular carcinoma. However, no significant difference was observed within these types with regard to the level of autoantibody positivity [Table - 3].
| ~ Discussion|| |
Current theories of mechanism of virus induced auto immunity fall into three categories which are not mutually exclusive, (a) Auto antigen modification (b) Disturbance of host immune mechanism involved in control of autoreactivity and (c) Molecular mimicry.
In genetically susceptible individuals autoreactions may continue despite successful elimination of the virus. Plotz and coworkers8 have proposed an alternative form of molecular mimicry involving production of antiidiotypes against virus neutralizing antibodies. As the mirror images of the latter, these anti idiotypes would resemble the viruses and combine with the complementary virus receptors on host cells thereby acting as autoantibodies that could participate in tissue damaging immune reactions.
Low levels of ANA and ASMA in apparently healthy population, observed in the present study, correlates with the reports of Wood et al  and Konikoff et al who have reported frequent occurrence of ANA in normal subjets in low titre. The absence of LKM antibody in healthy and age matched controls is also consistent with the reports of Homberg et al who had reported absence of LKM antibody in 400 healthy individuals studied.
Characteristic laboratory feature of A.I.H, although not universally present, is hypergammaglobulinaemia. This non specific response is associated with presence of circulating autoantibodies against nucleus and/or smooth muscle (Type I A.I.H) or LKM (Type II A.I.H). It is a consensus, that presence of ANA, ASMA or LKM antibodies is particularly helpful in diagnosing A.I.H. However, the initial absence of these should not exclude the diagnosis of A.I.H. as patients may become positive on followup.
The consensus opinion that in Type I- A.I.H, ANA and ASMA are present and in Type II A.I.H. LKM antibodies are predominantly seen, has been proposed by Bellary et al, Mc Farlane et al, and Manns et al. In the present study, auto antibodies were seen in 27.1% of CLD-B patients, 61.5% of CLD-C and 39.06% of CLD-NBNC patients. Thus the autoantibody positivity in CLD-C cases was significantly higher than that of HBV positive group (p < 0.01) and the NBNC group (p < 0.01). Similar observations have been made previously by Manns et al  and Lohse et al. Out of 160 histologically proven CLD cases in the present study 27.5% were positive for ANA, 30.6% for ASMA; 6.9% for LKM. This pattern is similar to the reports of Clifford et al.
Based on the above observations in the present study, wherein higher levels of ANA positivity with absence of LKM antibodies were observed in hepatitis B cases, it may be concluded that in HBV endemic countries like India, HBV may also have a role to trigger autoimmune reactivities in chronic liver diseases. The present study has also established that a significant proportion of CLD-C cases (46.1%) have higher levels of ASMA and LKM antibodies (11.5%). In the background of a comparatively lesser percentage of CLD-C in this part of the country (16.2%), higher levels of autoantibodies in CLD-C poses an important question whether these cases could be considered as HCV induced or associated A.I.H. However, confirmatory studies are required on a multicentric basis to evolve a national picture.
The present study has in addition, indicated the need for analysing chronic liver diseases for both viral and autoimmune serology in the Indian setting. This approach would greatly facilitate institution of either antiviral treatment or cortilcosteroid therapy on the basis of clinical and laboratory parameters.
| ~ Acknowledgements|| |
The financial assistance extended by Indian Council of Medicial Research (ICMR) through an extramural adhoc project grant is gratefully acknowledged.
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