Autoantibody prevalence in children with liver ... - Wiley Online Library

Clin Exp Immunol 1998; 112:471–476

Autoantibody prevalence in children with liver disease due to chronic hepatitis C virus (HCV) infection G. V. GREGORIO*†, P. PENSATI‡, R. IORIO‡, A. VEGNENTE‡, G. MIELI-VERGANI† & D. VERGANI* *Institute of Hepatology, University College London Medical School and †Department of Child Health, King’s College School of Medicine & Dentistry, London, UK, and ‡Dipartimento di Pediatria, Universita` degli Studi di Napoli Federico II, Napoli, Italy

(Accepted for publication 19 January 1998)

SUMMARY HCV infection and interferon-alpha (IFN-a) therapy have been associated with autoimmunity. To assess whether chronic liver disease (CLD) due to HCV infection or its treatment with IFN-a cause autoimmune manifestations, the prevalence of tissue autoantibodies in 51 children with chronic HCV infection and 84 with other CLD was analysed by standard techniques. Sixty-five percent of patients with chronic HCV infection, 66% with chronic hepatitis B infection and 60% with Wilson’s disease were positive for at least one autoantibody. In the 51 subjects with chronic HCV infection (29 treated with IFN-a, 22 untreated), tested on 165 occasions over a median of 9 months (range 5–42 months), autoantibodies to nuclei (ANA), smooth muscle (SMA), gastric parietal cell (GPC) and/or liver kidney microsomal type 1 (LKM-1) were similarly prevalent in treated and untreated patients (90% versus 68%, P ¼ 0·12). Positivity for SMA was present in 67%, GPC in 32%, ANA in 10%, LKM-1 in 8% of cases. Treatment with IFN-a had to be suspended due to transaminase elevation in one SMA-positive, one ANA-positive but in three of four LKM-1-positive patients. Our results show that: (i) autoantibodies are common in viral-induced hepatitis and Wilson’s disease; (ii) positivity for SMA, GPC, ANA is part of the natural course of chronic HCV infection, their prevalence being unaffected by IFN-a; and (iii) IFN-a should be used cautiously in the treatment of LKM-1/HCV-positive patients. Keywords

autoimmunity

interferon

hepatitis C

INTRODUCTION Since its discovery in 1989 [1], HCV has been increasingly associated with autoimmunity [2,3]. The presence of serological markers of autoimmunity in patients with chronic hepatitis due to HCV infection has been investigated in few adult series [4–8] and in only one paediatric cohort [9], yielding discrepant results. Thus, the reported prevalence of smooth muscle autoantibodies (SMA) ranges from 12% to 66%, that of anti-nuclear autoantibodies (ANA) from 6% to 21% and that of liver kidney microsomal type 1 autoantibodies (LKM-1) from 0% to 10%. The difference in autoantibody prevalence between the various studies may derive from technical reasons and/or differences in the populations investigated. Moreover, some studies [10–12] have suggested that interferon-alpha (IFN-a) treatment induces the appearance of serological markers of autoimmunity or even triggers the

Correspondence: Professor Diego Vergani, Institute of Hepatology, University College London Medical School, 69–75 Chenies Mews, London WC1E 6HX, UK. q 1998 Blackwell Science

autoantibodies

clinical manifestations of autoimmune hepatitis in patients with chronic HCV infection. Others [7,8] have suggested that IFN-a treatment does not induce clinically overt autoimmune disease even in the presence of autoimmune serum markers. Neither type of study has, however, analysed the behaviour of autoimmune serological markers over time comparing patients with chronic HCV infection treated with IFN-a with patients remaining untreated over a similar period of time. To assess whether chronic liver disease due to HCV infection is associated with autoimmune manifestations, we studied the prevalence of tissue autoantibodies in 51 children with HCVþ chronic liver disease and compared it with that of patients with other chronic liver disorders. In addition, to investigate whether IFN-a induces serological signs of autoimmunity, we analysed retrospectively the prevalence of tissue autoantibodies in 165 serial serum samples collected over a median period of 9 months from the 51 HCVþ children, 29 of whom had been treated with IFN-a while 22 had remained untreated. The presence of thyroglobulin and thyromicrosomal autoantibodies was also tested in the cohort of patients with chronic liver disease due to HCV infection, as

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Table 1. Clinical and immunological features of 51 children with chronic liver disease due to HCV, 84 children with other chronic liver disorders and 24 healthy controls at entry to the study

Autoantibody tire $1/10 Diagnosis

n

Chronic HCV infection Chronic HBV infection Wilson’s disease Alagille’s syndrome a1-antitrypsin deficiency Healthy controls

51 24 20 20 20 24

Median age (range), years

Sex (F/M)

Median AST levels (U/l)* ANA

8 (2–16) 6 (2–17) 13 (6–16) 5 (1–16) 3 (0·1–13) 9 (2–14)

24/27 7/17 3/17 10/10 11/9 8/16

47 (16–168) 48 (21–138) 49 (22–301) 170 (48–312) 67 (18–32) 27 (8–39)

3 2 5 0 0 0

SMA

ANA/SMA

LKM-1

GPC

Patients positive for one or more autoantibodies, n (%)

25 8 5 0 0 0

1 2 0 0 0 0

4 0 0 0 0 0

15 3 2 0 0 0

33 (65) 16 (66) 12 (60) 0 0 0

*Normal value #50 U/l; n, number of patients; ANA, antinuclear antibody; GPC, gastric parietal cell; HBV, hepatitis B virus; LKM-1, liver kidney microsomal type 1 autoantibody; SMA, anti-smooth muscle antibody.

these autoantibodies have been previously reported in this group of patients [4] and to be possibly induced by IFN-a [13]. PATIENTS AND METHODS Patients A total of 135 children with chronic liver disease was investigated (Table 1). Fifty-one had chronic liver disease due to HCV infection. These were consecutive patients referred to the Department of Paediatrics, Naples, Italy (n ¼ 42), and to the Paediatric Liver Service, King’s College Hospital, London, UK (n ¼ 9). All patients were anti-HCVþ (second generation ELISA; United Biomedical Inc., Hauppage, NY and Sanofi Pasteur, Marnes-la-Coquette, France) and HCV-RNAþ (Amplicor; Hoffmann La Roche, Basel, Switzerland). All had sufficient amount of stored serum, collected over a median period of 9 months (range 5–42 months), for serial

analysis. None of the patients had serological evidence of coinfection with hepatitis B (microparticle enzyme immunoassay; Abbott, Chicago, IL), delta (EIA; Abbott) and HIV-1 (Access Immunoassay; Sanofi Diagnostic Pasteur). None had clinically overt autoimmune disease. Of the 51 patients, 42 underwent percutaneous liver biopsy (Table 2). Twenty-four (57%) patients had histological evidence of chronic hepatitis with mild to moderate activity, nine (21%) had chronic hepatitis with minimal activity and nine (21%) had non-specific reactive hepatitis. None of these patients had histological evidence of cirrhosis. Liver biopsy was not performed in nine patients because of absence of parental consent in three, presence of associated medical disorders in two cases (tetralogy of Fallot and seizure disorder) and persistently normal aminotransferase levels in four. In the latter, positivity for HCV RNA had been demonstrated on at least two occasions over a follow-up period of 7, 13, 24 and 96 months.

Table 2. Clinical, biochemical and histological features of children with chronic liver disease due to HCV infection at entry into the study divided according to the presence or absence of autoantibodies

Autoantibody positive (n ¼ 33) Median age (years) Males, n (%) Median AST (normal value # 50 U/l) Possible mode of transmission, n (%) Parenteral Vertical Unknown Liver histology Chronic hepatitis, mild to moderate activity Chronic hepatitis, minimal activity* Non-specific reactive hepatitis

Autoantibody negative (n ¼ 18)

8 (2–16) 18 (54) 40 (17–168)

7 (3–16) 11 (61) 50 (26–64)

22 (67) 8 (34) 3 (9) n ¼ 29 15 (52)

13 (72) 4 (22) 1 (6) n ¼ 13 9 (69)

7 (24)

2 (15)

7 (24)

2 (15)

No significant differences between the two groups. *Formerly chronic persistent hepatitis. q 1998 Blackwell Science Ltd, Clinical and Experimental Immunology, 112:471–476

Autoantibodies in childhood chronic HCV infection Clinical history suggested that HCV infection was acquired by parenteral route in 35 (69%) patients, vertical transmission in 12 (24%), while in four (7%) the mode of infection was unknown. Twenty-nine patients received IFN-a treatment three times weekly for 12 months; 19 with recombinant IFN-a2b (Viraferon; Schering Plough Ltd, Kenilworth, NJ) at 5 million units/m2 and 10 with lymphoblastoid IFN-a (Wellferon; Wellcome Ltd, Beckenham, UK) at 3 million units/m2. Criteria for IFN-a treatment were as follows: positivity for HCV-RNA, aminotransferase levels exceeding or the upper limit of normal for at least 1 year and liver histology compatible with chronic hepatitis. Patients were evaluated monthly during the treatment period and every 3 months thereafter. Response to treatment was defined as disappearance of serum HCV-RNA (virological response) or aminotransferase normalization occurring during and persisting until cessation of IFN-a treatment (biochemical response). Of the 84 patients with anti-HCV¹ (second generation ELISA; Sanofi Pasteur) chronic liver disorders, 24 had chronic hepatitis B virus infection. All were HBV-DNAþ (dot blot assay; Abbott) and HBeAgþ (microparticle enzyme immunoassay; Abbott) for at least 6 months with features of chronic hepatitis on liver biopsy. Twenty children had Wilson’s disease (abnormal levels of 24-h urinary copper excretion before and after penicillamine challenge [14]), 20 Alagille’s syndrome and 20 a1-antitrypsin deficiency (AATD) (PIZZ phenotype by isoelectric focusing). These pathological controls were selected to represent a wide range of liver pathology from cholestasis (Alagille’s syndrome) to chronic hepatitis (Wilson’s disease) and progressive fibrosis (AATD). Sera from 24 age-matched healthy children (median age 9 (2– 14) years; 16 boys) were tested as controls. Autoantibody detection Serum samples from HCVþ children and pathological and healthy controls were tested for the presence of autoantibodies by a single investigator blinded to clinical details. One hundred and sixty-five sequential serum samples (median of three serum samples per patient) were available from children with chronic liver disease due to HCV infection. Amongst the 29 patients treated with IFN-a, serum samples were taken before initiation of treatment and two additional samples were obtained during and/or after IFN-a (median time 9 (5–41) months). The samples from untreated patients were collected over a period of 6– 24 months (median 10 months). Blood samples were taken for management purposes and aliquots of serum were stored at ¹708C for future analysis. Patient sera were screened at a dilution of 1:10 in PBS by indirect immunofluorescence on frozen rat liver, kidney, stomach as substrate, allowing the detection of nuclear, smooth muscle, LKM-1, mitochondrial, liver cytosolic antigen and gastric parietal cell (GPC) autoantibodies using a standard technique [15]. Tissuebound autoantibodies were visualized using fluorescent polyvalent anti-human immunoglobulin (IgG, A, M; The Binding Site, Birmingham, UK) as a second reagent, diluted 1:20 in PBS, using an Aristoplan Leica microscope (Milton Keynes, UK) equipped with the filter system 12/3 with excitation at 450–490 nm and emission at 515 nm. All positive sera were titred to extinction. The immunoglobulin isotype of the autoantibody-positive sera on initial sample was defined using fluorescent anti-human IgG (The Binding Site) and IgM (The Binding Site), diluted 1:20 in PBS. Furthermore, for ANA-positive cases, the ANA pattern was characterized using HEp-2 cells (Biodiagnostics, Upton-upon-Severn,

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Follow up positive: 19

positive: 23 negative: 4 IFN-α treated

29 positive: 3 negative: 6 negative: 3 positive: 7 positive: 10 negative: 3

No treatment

22 positive: 5 negative: 12 negative: 7

Fig. 1. Autoantibody positivity in 51 children with chronic HCV infection, at entry into the study and at follow up.

UK) as substrate under the same conditions used for antibody detection on rodent substrate. In the cohort of patients with chronic liver disease due to HCV infection, additional tests included thyroglobulin and thyromicrosomal antibodies using a passive particle agglutination kit according to the manufacturer’s instructions (Fujirebio, Tokyo, Japan), and the results were reported as positive or negative. Statistical analysis Fisher’s exact or Student’s t-tests were used as appropriate for statistical comparison. Correlation study between autoantibody titres and transaminase levels was done using Spearman’s correlation coefficient. P < 0·05 was considered significant. RESULTS Autoantibody prevalence in HCVþ patients before IFN-a treatment versus HCV¹ patients Sixty-five percent, 66% and 60% of the patients with chronic HCV, chronic HBV and Wilson’s disease, respectively, were positive for one or more of the following autoantibodies: ANA, SMA, LKM-1 and GPC (Table 1). The autoantibody isotype was IgG in all cases. In contrast, all sera from patients with Alagille’s syndrome, a1antitrypsin deficiency and healthy controls were negative for any of the autoantibodies tested. Autoantibody positivity at entry into the study and at follow up is summarized in Fig. 1 for the entire patient cohort. SMA was the most frequent autoantibody detected in both HCVþ and HBVþ chronic liver disease, being present in 51% and 41% of patients, respectively. There was no difference in the median SMA titres of the two groups (l/10 (range 1/10–1/40) for both). In contrast, in patients with Wilson’s disease, ANA (titres 1/10–1/640 (median 1/40)) and SMA (titres 1/10–1/40 (median 1/10)) were similarly prevalent. Positivity for GPC was observed in 29% of patients with HCV infection, and to a lesser extent in patients with chronic HBV infection (13%) and Wilson’s disease (10%). The presence of LKM-1 autoantibody was observed in four (8%) patients, all with chronic liver disease due to HCV infection. LKM-1 titre was 1/10 in two cases, 1/40 in one and 1/80 in one. Reactivity to cytochrome P4502D6 (CYP2D6) was observed in all four LKM-1þ cases using

q 1998 Blackwell Science Ltd, Clinical and Experimental Immunology, 112:471–476

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a eukaryotically expressed CYP2D6 in a radioligand immunoassay, which allows detection of antibodies targeting both linear and conformational epitopes [16]. A homogeneous immunofluorescence pattern was observed in all ANAþ patients with chronic HCV and chronic HBV infection. In the five patients with Wilson’s disease, the nuclear pattern was homogeneous in three, nucleolar in one and both homogeneous and nucleolar in one. Effect of IFN-a on the autoimmune serology of patients with chronic liver disease due to HCV infection At entry into the study, no difference was observed in the clinical, biochemical and histological features of autoantibody-positive and autoantibody-negative patients. Thirty-three children (65%) were positive for one or more autoantibodies, with titres ranging from 1/ 10 to 1/80 (median of 1/10). Of these, 23 were subsequently treated with IFN-a and 10 remained untreated. Between 6 and 12 months after initial testing (corresponding to months 6–12 of IFN-a therapy in treated patients), no difference was observed in the autoantibody prevalence between treated and untreated patients (treated 22/29 (76%) versus untreated 13/22 (59%), P ¼ 0·33). Of the 23 IFN-a-treated patients who were autoantibody-positive initially, four (17%) became autoantibodynegative, while three of six patients who were initially autoantibody-negative and were treated with IFN-a (two with recombinant IFN-a2b and one with lymphoblastoid IFN-a) became autoantibody-positive after 1, 3 and 6 months of treatment. The autoantibodies were SMA in the first, homogeneous ANA and SMA in the second and SMA and GPC in the third, all at a titre of 1/10. Similarly, of the 10 (30%) untreated patients who were initially autoantibody-positive, three became autoantibody-negative while five of 12 (42%) untreated patients who were initially autoantibody-negative became transiently positive for SMA (titre of 1/10 in three and 1/20 in two) between 6 and 12 months after the initial testing but became autoantibody-negative again 12–24 months later. All four LKM-1þ patients on initial testing were subsequently treated with IFN-a (initial AST levels 34, 40, 57 and 69 U/l). All remained LKM-1þ at the same titre during and after IFN-a therapy. In one of the two patients with an LKM-1 titre of 1/10, the AST level became normal from the first week of treatment with persistence of normal values up to month 12 of IFN-a therapy. In the three other patients, IFN-a treatment was discontinued because of at least a three-fold increase in aminotransferase levels not accompanied by any abnormal clinical signs and symptoms. The AST levels returned to normal 3, 6 and 12 months after cessation of IFN-a treatment. None of these patients required immunosuppressive therapy. Over the entire 42-month period of observation, autoantibody positivity for ANA, SMA, LKM-1 and/or GPC was observed on at least one occasion at a dilution $ 1:10 in 41 (80%) patients with no significant difference in autoantibody prevalence between treated (26/29 (88%)) and untreated (15/22 (68%), P ¼ 0·12) patients. Positivity for SMA was noted in 34 (67%) patients, GPC in 16 (32%), ANA in five (10%) and LKM-1 in four (8%). Using a cutoff titre of 1/20 akin to Bortolotti et al. [22], 14 (27%) patients were autoantibody-positive: 11 (21%) for SMA, one for LKM-l and SMA, one for LKM-1 and one for ANA. Of these 14 patients, eight were autoantibody positive $ 1/20 on initial testing, while six became autoantibody positive $ 1/20 during follow up: three on month 3 of IFN-a treatment and three untreated patients 4, 4 and

12 months after initial testing. No patient was found to be positive for antimitochondrial, liver cytosolic antigen type 1, thyroglobulin or thyroid microsomal antibodies in any of the 165 sera tested. None of the autoantibody-positive patients showed clinical deterioration in association with the appearance or rise in titre of ANA, SMA, LKM-1 and/or GPC. There was no correlation between the autoantibody titres and serum transaminase levels (r ¼ 0·2, NS). Of the six patients with biochemical response to IFN-a treatment, three also had a virological response. All six patients were autoantibody-positive on at least one occasion (five for SMA and one for LKM-1). A total of 11 patients discontinued IFN-a treatment; in six because of non-reduction in serum aminotransferase levels after 6 months of therapy, and in five because of at least a three-fold increase in aminotransferase levels during therapy. All five patients who developed hypertransaminasaemia were autoantibody-positive before IFN-a treatment. These included three of the four LKM-1þ (LKM-1 titre of 1/10, 1/40 and 1/80) cases; one of the 17 (4%) SMAþ (SMA titre of 1/10) and one of the four ANAþ (titre of 1/20) patients. The latter two patients had AST levels of 105 and 80 U/l after 4 months of discontinuing IFN-a treatment. DISCUSSION There are three main findings in the present study. First, autoantibodies are frequently found in children with viral-induced hepatitis and Wilson’s disease, but are absent in those with other forms of chronic liver disorders such as Alagille’s syndrome and a1-antitrypsin deficiency. Second, positivity for autoantibodies to smooth muscle (SMA), gastric parietal cells (GPC) and to a lesser extent the nuclear antibody (ANA) is part of the natural course of childhood chronic HCV infection, their prevalence and titre being unaffected by IFN-a therapy. Third, IFN-a may be dangerous in those HCVþ patients who are also positive for LKM-1, independent of its titre. The high prevalence of autoantibodies observed in patients with chronic HCV infection and in two control groups, chronic HBV and Wilson’s disease, but not in patients with other forms of liver disorders of similar severity, albeit with less histological inflammatory activity, suggests that the presence of these autoantibodies is not just a consequence of the chronic liver damage. In chronic HBV infection we have found, by searching protein databases, regional similarities between the HBV-DNA polymerase and the antigenic targets of human nuclear and smooth muscle proteins [17]. By constructing the peptides corresponding to the homologous regions, immunological cross-reactivity was observed between the viral peptides and self homologue, strongly implicating molecular mimicry as a mechanism for the presence of ANA and SMA. The question whether molecular mimicry may also account for positive autoimmune serology in HCV infection is presently being investigated. The cause of autoantibody production in patients with Wilson’s disease remains to be established. However, elevated serum immunoglobulin concentrations and positivity for non-organ-specific autoantibodies have been previously reported in this group of patients [18]. The presence of autoantibodies in 80% of our HCVþ patients on at least one occasion is higher than that reported by Abuaf et al. [4] (46%), Bortolotti et al. [9] (35%) and McFarlane et al. [6] (19%). One possible explanation for the difference is that we have determined the presence of autoantibodies on serial samples taken at least 1 month apart, while most published data have only done a


Autoantibodies in childhood chronic HCV infection single point determination. In view of the fluctuating behaviour of autoantibodies, a single point determination may not provide complete information on autoantibody prevalence in these patients. Additional reasons for the difference in autoantibody prevalence between ours and the cited studies [4–9] may derive from technical differences and differences in the populations investigated. Akin to our study, Abuaf et al. [4] and McFarlane et al. [6] have used the conventional screening dilution of 1/10 to examine the presence of autoantibodies, as originally proposed by Holborow & Johnson [19]; however, these studies [4–8] are not immediately comparable to ours, as they were conducted in adult populations. In the only study in children with chronic HCV infection examining the prevalence of autoimmune serology, Bortolotti et al. [9] reported a positivity for SMA in 18%, LKM-1 in 10% and ANA in 7% using a screening dilution of 1/20. Our results would have been similar to Bortolotti’s [9] had we used a screening dilution of 1/20. However, particularly in the paediatric age group, a starting dilution of 1/10 should be used, as autoantibodies at any titre are rare in healthy children. This is in keeping with the recommendation of the International Autoimmune Hepatitis Group [20]. Thus, of 100 healthy children of the same age range as our study population who were tested for tissue autoantibodies, transient positivity for SMA at a titre of 1/10 was noted in only six cases, concomitant with a viral infection which disappeared on retesting (Akinwinka, unpublished data). Similar to previous reports [4,7,9], the most commonly occurring autoantibodies in HCVþ patients in our study were SMA. The 8% positivity for LKM-1 in this study is higher than that reported in adult patients (0–6%) [4–8]. Conversely, positivity for ANA appears to be lower in children (10% in ours, 7% in Bortolotti’s study) than in adults (14%, 18%, 21%) [4,7,8]. Whether age, immunogenetic or ethnic factors or viral genotypes account for the differences in the prevalence of positivity for ANA and LKM-1 between adults and children with HCV infection remains to be determined. In chronic HCV infection, IFN-a is currently the most effective treatment, a potentially severe side-effect of which is the induction of autoimmunity [21]. The question whether autoantibody production is part of the natural course of HCV infection rather than a consequence of IFN-a therapy was addressed in the present study by testing for the presence of autoantibodies in serial samples from patients both treated and untreated with IFN-a. A parallel analysis of these two groups showed that over a similar median period of observation, a similar proportion of treated and untreated patients develop SMA, GPC and to a lesser extent ANA at a similar titre, suggesting that the production of these autoantibodies represents an autoimmune response triggered by the HCV infection per se rather than by IFN-a therapy. Only one SMAþ and one ANAþ patient who were treated with IFN-a showed biochemical deterioration of liver disease which improved on cessation of therapy. Positivity for these autoantibodies therefore does not appear to be a contraindication to IFN-a treatment. In contrast, our preliminary data suggest that positivity for LKM-1 autoantibody appears to have important clinical implications in view of the significant biochemical deterioration observed in three of the four LKM-1þ patients during IFN-a treatment. Whilst IFN-a is currently the treatment of choice for chronic HCV infection [22], the classical treatment of LKM-1 AIH involves immunosuppressive agents which may interfere with viral clearance if the patient is HCVþ [23,24]. It is important that the type of treatment of LKM-lþ/HCVþ patients is decided after thorough

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investigations to exclude classical AIH. If the decision to treat with IFN-a is made, the patient should be followed up at close intervals with frequent determinations of liver function tests. Alternative treatment options should be considered in patients with worsening liver function. If the patient fulfils the criteria for the diagnosis of AIH [20], IFN-a should be stopped and immunosuppressive treatment may be considered in the presence of persistently abnormal liver function tests. In conclusion, the present study shows that low titre of autoantibodies typically associated with autoimmune liver diseases is a common finding in children with chronic HCV infection and is not due to IFN-a treatment. Determination of autoantibody to LKM-1 is important before starting IFN-a treatment. ACKNOWLEDGMENTS G.M.-V. is supported by the Children’s Liver Disease Foundation (CLDF) (Digbeth, Birmingham B5 6DR, UK). The healthy children were recruited through the Gillingham and Medway Branch of CLDF, Kent, UK. We thank Dr Yun Ma for performing the CYP2D6 radioligand assay.

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