Cellular and humoral immune reactions against autoantigens and hepatitis C viral antigens in chronic hepatitis C

Cellular and Humoral Immune Reactions Against Autoantigens and Hepatitis C Viral Antigens in Chronic Hepatitis C JOHN KOSKlNAS,* CHRISTOPHER

BARBARA M. McFARLANE,*

J. TIBBS,*

MASASHI

MIZOKAMI,’

KAYHAN T. NOURI-ARIA,* PETER T. DONALDSON,*

IAN G. McFARLANE,*

and ROGER WILLIAMS* *Institute of Liver Studies, King’s College Hospital, London, England; and ?I Department of Medicine, Nagoya City University, Nagoya, Japan

a different

See editorial on page 1550.

target

antibodies

epitope

associated

also been reported

Bac&round/Aims: Previous reports have suggested that the hepatitis C virus (HCV) may induce autoimmune hepatitis. The aim of this study was to examine this hypothesis by investigating humoral and cellular immune responses to HCV-related antigens and various autoantigens in patients with chronic HCV infections. Methods: Lymphoproliferative responses in vitro and/ or circulating antibodies to an HCV core peptide, the putative autoantigen GOR, the liver-specific hepatic asialoglycoprotein receptor (ASGP-R), and other autoantigens were investigated in 27 adults with chronic hepatitis C. Results: Five patients with HCV (18.5%) showed cellular immune responses to ASGP-R and two others had antibodies to ASGP-R, whereas 6 of 14 patients (42.8%) showed cellular responses to GOR and 7 of 14 patients (50%) showed responses to HCV core. Other autoantibodies were detected in three patients (11%). Nine patients with autoimmune hepatitis studied concurrently for comparison showed cellular and/or humoral responses to ASGP-R but not to GOR. Only 2 of 11 patients with other chronic liver disorders showed immune responses to any antigen tested. Conclusions: Specific immunocompetence against HCV-related antigens can often be shown in patients with chronic hepatitis C but is infrequently accompanied by autoreactions against liver-specific or nonspecific antigens. A reported association between Tcell responses to HCV core and lack of liver damage could not be confirmed.

A

atitis

series of studies, mainly from southern documented

the finding of a high frequency

C virus (HCV)

infection

in patients

areas who present with autoantibodies suggestive

of autoimmune

est frequency patients

hepatitis

these

than

in 44%-80%

the LKM-1

infections

of patients

areas who present with antinuclear

(ANA)

have

from these

and/or smooth

muscle

antibodies

(SMA).‘,3’7*8 This has led to the sug-

gestion

that HCV

may induce AIH and is supported

the observations

that (1) the majority

chronic

C have circulating

hepatitis

with a reportedly designated

naturally

GOR:-”

for antibody

bodies in such patients,” by partial

sequence

including HCV

occurring

homology

genome.10*‘2-‘4

to the core region studies

America

of HCV

seldom

that

prevery

normally

seropositive

(although

mostly at low or moderate

Conversely,

a serial study in our laboratories suggested

This apparent

conflict

or LKMtiters).

of Italian

C progressing

that de novo induction

by HCV

responses

SMA-,

with acute hepatitis

autoreactivity

far obtained

associ-

but a recent report*’ has indicated may be ANA-,

all of the evidence

with

patients

that up to one third

chronicity

to find

viral infections

have the serum autoantibodies

patients presenting

of the

in patients

thought

senting with non-A, non-B hepatitis ated with AIH,“-‘”

of

from elsewhere

have failed

infections

it had been

the targets

of the HCV polyprotein,

However,

and in North

Previously,

be-

to GOR and LKM antibetween

and segments

a high frequency AIH.8,‘5,‘6

pentadecapeptide

and (3) this might be explained

one corresponding

in Europe

with

reacting

(2) there is a close correlation

tween seropositivity

these antibodies

of patients

antibodies

by

to

of humoral

seems to be a very rare event.*l might

be related to the fact that

of HCV-induced

autoreactivity

has come from studies of humoral

to autoantigens.

To determine

so

immune

whether

HCV

and other features (AIH).‘-*

microsomal

recent evidence

of hep-

from

(up to 86%) has been observed

with liver/kidney

ies,’ although

Europe, has

specificity

with AIH.S*6 HCV

indicates

(LKM)

The highin Italian antibod-

that these have

Abbreviationsused in this paper;AIH, autoimmune hepatitis; ANA, antinuclear antibodies; ASGP-R, asialoglycoprotein receptor; LKM, liver-kidney microsomal antibodies; SMA, smooth muscle antibodies. 0 1994 by the American Gastroenterological Association 00165085/94/$3.00

November 1994

AUTOIMMUNITY IN CHRONIC HEPATITIS C

has an inherent important

tion

capability

to know

between

curring other

autoimmune

peptide

study,

have

HCV

hepatic

events

cellular

trigger

of circulating

antibodies

responses

in patients peptides

receptor

as other markers of humoral allotype

and ASGP-R

autoimmunity

as well

and the HLA

with chronic HCV infection

activities <35

(median,

ity for hepatitis Twenty-six cirrhosis

range,

or mildly

dian, 33 g/L; range, 24-44 ity for antibodies

serum aspartate

101 III/L;

IU/L), normal

years)

aminotransferase

40-344

elevated

IU/L.; normal,

serum globulins

g/L; normal,

(me-

<35 g/L), seropositiv-

to HCV and for HCV RNA, and seronegativB and human immunodeficiency

had chronic

active hepatitis

in nine and one with

had a history of previous

virus markers.

containing

microplates

at various

air containing

mean counts

blood transfusions,

Eight

nine were intrave-

10 had no apparent infections).

Indians,

(“sporadic”

two others

were from the Middle

five were Italian,

and the remainder

United

or, in two cases, elsewhere

risk

Two patients

were either

East,

born in the

in northern

Eu-

England)

cultures.

was always

was added to each well,

into DNA

of proliferated

16 hours by liquid

England).

The variation less than

+-lo%.

As a positive

antigen.

For comparisons

between

different

used. A positive

subjects, response

greater than the maximum control

group,

of responses

which

mean

Chemical counts

per

divided

by

cells in the absence

of

of antigen

to the different

the peak stimulation was defined

in separate

indices were calcu-

formula:

of cultured

per

triplicates

(Sigma

cells in the presence

per minute

scintilla-

between

Stimulation

to the following

of cultured

cells

as the mean counts

4 /.tglmL phytohemagglutinin

according

of test

at 37°C in

for the assay, cells were also stimulated

Co., Poole, Dorset, lated

in 96-well

or absence

and incubated

Data were expressed

for any antigen

Pais-

200 /.tg/mL

5% CO*. After 6 days, 0.5 PCi of

incorporated

minute for triplicate

with

in 10%

Then the cells were

in the presence

after an additional

tion counting.

glutamine,

of 1 X 10’ cells/well

(Amersham,

was measured

wells

cen-

cells were washed

streptomycin.

concentrations

by

density-gradient

of 1 X lo6 cells/ml

2 mmol/L

and 100 pg/mL

and radioactivity

control

cells were separated

blood mononuclear

at a concentration

liver damage.

nous drug users, and the remaining

Kingdom

penicillin,

minute

minimal

group.

fetal calf serum in RPM1 1640 (Gibco,

ley, Scotland)

on liver biopsy with

factors for viral hepatitis were West

(14

were based on clinical and histological

elevated

(8 men and 5 years) with nor-

a control

Oslo, Norway)

to a concentration

[3H)thymidine

persistently

workers

blood mononuclear

Peripheral

heat-inactivated

antigens

men and 13 women; median age, 48 years; range, 19-64 criteria,

trifugation. and adjusted

humidified

Diagnoses

laboratory

liver tests comprised

(Nycomed,

flat-bottomed

Patients

were studied.

healthy

age, 35 years; range, 28-40

Peripheral Lymphoprep

cultured

Materials and Methods

patients

in

Proliferation Assay

(ASGP-R),

profiles of the patients.

Twenty-seven

cirrhosis

and

to the presence or absence

to GOR

Thirteen median

ma1 biochemical

which has been shown to be a major target of cellular and humoral autoreactions in AIH.**-*’ The findings in relation

cirrhosis. women;

reported.

showing

all and features of alcoholic hepatitis in four, and two had chronic hepatitis B infection with chronic active hepatitis and

the relation-

to the core and GOR

have been examined

with other chronic liver diseases. Nine of these had alcoholic liver disease with liver biopsy specimens

oc-

immune

not been

we have investigated

asialoglycoprotein

it is

cross-recogni-

reflects

to date,

lymphoproliferative

chronic

to the

the reported

or B-cell levels that might

to the GOR

ships between

autoreactions,

core and GOR responses;

In the present

with

whether

HCV

at the T-cell

reactions

to induce

1437

antigens

indices

as a stimulation

were index

observed with cells from the normal

varied

according

to the antigen

under

test.

rope. Two additional negative history

groups

for anti-HCV

of blood transfusions

for comparison.

median

provisional

or drug addiction,

criteria

were studied

nine patients

for a diagnosis

(two men years)

of AIH28 with

at titers 2 1:80 by immuno-

on rodent tissues and hyperglobulinemia

61 g/L; range, 37 -78

g/L) with selective

elevations

(median, of serum

immunoglobulin G (median, 24.7 g/L; range, 20.0-70.7 g/L; normal, <16 g/L) at presentation. At the time of study, all were receiving and azathioprine dian aspartate

treatment

with prednisolone

(75 mg/day) aminotransferase,

Test Antigens

were sero-

age, 52 years; range, 2 1-75

ANA and/or SMA autoantibodies fluorescence

all of whom

and HCV RNA and had no

One group comprised

and seven women; fulfilling

of patients,

antibodies

(lo-20

mg/day)

but still had active disease (me118 IU/L; range, 60-220

IU/

L; median globulins, 40 g/L; range, 35-56 g/L), and four had cirrhosis. The second group comprised 11 patients (eight men and three women; median age, 55 years; range, 41-68 years)

peptide,

The immunodominant epitope of the HCV core polycorresponding to amino acids 39-73,L9 and GOR

peptide

were synthesized

goya University, proliferation

Nagoya,

at the Department Japan.

of Medicine,

assay at 10 and 30 PglmL,

and GOR

at 10 and 20 /.tg/mL based on preliminary was affinity-purified

from Triton

Na-

HCV core was used in the

X-100

studies. extracts

was used ASGP-R of acetone

powders of normal rabbit and rat liver as described elsewherei” and used at 10 concentrations ranging from 10 ng/mL to 5 PglmL.

Measurement of Antibodies Anti-HCV

and antibody

to GOR

were detected

by

enzyme-linked immunosorbent assays (HCV enzyme immunoassay and GOR enzyme immunoassay; United Biomedical Inc.,

1438

GASTROENTEROLOGY Vol. 107, No. 5

KOSKINAS ET AL.

HLA Typing

Hauppauge, NY) based on synthetic peptides corresponding, respectively, to gene products of the core and NS3/4 regions of the HCV genome

and to the GOR genomic

Both assays were performed instructions

according

with appropriate to ASGP-R

was measured

previously

described.i0

Screening

by indirect

immunofluorescence

and negative

dilution

controls.

by radioimmunoassay

as

for other autoantibodies on frozen sections

of 1:lO were titrated

was

of compos-

ite tissue blocks of rat liver, kidney, and stomach. at the screening

ment-dependent

to the manufacturer’s

positive

Antibody

HLA phenotypes

14.11

sequence.

26 HLA-B

antigens

were determined reaction

Outer

primers

(nucleotide)

Sera positive

GACACTCCACCATAGAT-3’)

by “nested”

of the HCV

(nucleotides and JR19

I-20,

(nucleotides

TCACGCA-3’)

and JR14

35-53,

al.” To avoid false-positive was performed of Kwok negative

significant.

Results Five

5’-GGC-

of the

27 patients

with

HCV

197-

were found to give peak stimulation

and inner prim-

response

to ASGP-R

141-161,

results, polymerase and with

statistically

(18.5%)

as described

with rigid adherence

and Higuchi’4

chain

genome.i2

6.6) compared

S’-GAACTACTGTCT-

(nucleotides

AATTCCGGTGTACTCACC-3’)

after polymerase

with a series of sequence-

poly-

(nucleotides

2 16,5’-CGCCCAAATCTCCAGGCATT-3’), ers were JR13

of DNA

and probing

and

DRB genotypes

Statistics

from the highly conserved

region

used were JR12

reported.i5

specific probes.‘”

was considered

5’ untranslated

comple-

Statistical analysis was performed using Fisher’s Exact Test and the Wilcoxon rank sum test as appropriate. P < 0.05

in serum was detected

merase chain reaction using primers

by analysis

by standard

assay for 14 HLA-A

as previously

amplification

out to negativity.

Detection of HCV RNA HCV RNA

were determined

microcytotoxicity

5’-GGC-

by Romeo

et

chain reaction

to the recommendations appropriate

positive

and

controls.

infection indices

in

3.4 and 10.5 (median,

of between

range, 0.5-3.0).

with the control group (median, 2.0; The peak stimulation index in 4 of these

5 was observed

at a concentration

fifth gave maximum 1). None stimulation trations

response

of the other

of 78 ng/mL,

with

22 patients

156 ng/mL (Figure with

HCV

index > 3.0 at any of the 10 ASGP-R

tested. By comparison,

and the gave a concen-

eight of the nine patients

with AIH (88.9%) gave peak SIs >3.0 (median, 12.9; range, 3.1-46.0) at ASGP-R concentrations between 12 -

150 and 600 pg/mL (Table 1). This difference between patients with AIH and patients with HCV was highly

11 1

significant

both

in terms

of frequency

of positive

re-

sponses (P = 0.0003) and degree of stimulation (P < 0.001). In only 2 of the 11 patients with other chronic liver diseases (both alcoholics) Fourteen

(18%) were responses ob-

(SIs = 4.6 and 5.6)

served to ASGP-R

consecutive

patients

(patients

14-27;

2) of the 27 with HCV were investigated responses to GOR and HCV core peptides.

Table

for cellular Six (42.8%)

showed positive proliferative responses to GOR (i.e., SIs > 2.4; median, 4.2; range, 2.5-8.2) as compared with the normal controls (median, 1.7; range, 0.8-2.4) (Table

10 ASGP-R

100 concentration

loo0

loo00

(&III)

Figure 1. Lymphoproliferative responses to 10 concentrations of ASGP-R. ranging from 10 pg/mL to 5 ng/mL, in the five patients with chronic hepatitis C who showed positive responses to this antigen. Note peak stimulation indices at 78 pg/mL in four patients and at 156 pg/mL in the fifth. Shaded area denotes normal range (stimulation indices < 3.0). 0, patient 3; +, patient 14; A, patient 21; ?? , patient 25; 0, patient 27.

2). Five of the six gave a maximum

response

at

a GOR concentration of 20 PglmL, and the sixth gave a maximum response at 10 pg/mL. Two of these (patients 25 and 27; Figure 2) also responded to ASGP-R. In contrast, none of the patients with AIH or other chronic liver diseases gave positive responses to GOR (median stimulation indices, 1.6 and 1.2; ranges, 1.2-2.3 and 0.3-2.0,

respectively).

Of the same 14 patients with HCV tested against GOR, seven (50%) gave positive responses to HCV core antigen (SIs > 2.4; median, 3.7; range, 3.4-8.0) compared with the normal control group (median, 1.5; range, 0.8-2.4)

(Table

2).

Five

patients

gave

maximum

re-

November 1994

AUTOIMMUNITY

Table1. Details

of Patients

With Chronic

HCV or AIH Who Had Humoral

or Cellular

Immune

AIH

Humoral (titer)

2.1 3.1” 3.1” 7.2” 23.0” 46.0” 20.0” 7.0” 4.4” aPositive responses

= stimulation

Patient no.

I:900

1

I:3700 1:600 I:400 I:350 Negative Negative Negative Negative

10 3 14 21 25 27

Humoral (titer)

2.2 1.8 5.7” 10.5” 7.2” 8.1” 3.4a

1:1600 I:300 Negative Negative Negative Negative Negative

index > 3.0

Antibodies

to GOR

7 of the 27 patients

liferative

to HCV

(median

to HCV

but were not detected

response to GOR and cellular reactivity Thus,

5 of the 7 who responded

to HCV core included

responses patient

the

2) who showed cellular

not GOR.

reactivity

In none of the 11 patients

liver diseases was a response dian stimulation

index,

Table 2. Lymphoproliferative

to ASGP-R

Responses

liver diseases.

(26%)

cutoff, 1.00)

in any of the patients

14; Table

with AIH

Of the 14 patients

but

1:300 further

(me-

and

to HCV Core and GOR Peptides

were

patient

had ANA

found

very

infre-

had SMA (at 1:20),

a

at 1:40, and none had LKM.

with HCV with antibody

and HLA Typing

this latter

to HCV core.

with HCV infections. Only two to ASGP-R (7.4%) (at titers of

1:16OO), two others

The two patients

1.2-2.2).

2) responded

autoantibodies

quently in the patients of the 27 had antibody

with other chronic

to HCV core observed

1.8; range,

other

in sera from

HCV infection

and in one who did not, although (patient

Circulating

two (patients 25 and 27; Figure 2) who responded to both GOR and ASGP-R and a third patient (patient 14; Figure

were found

with HCV tested for cellular reactivity to GOR, antibody to GOR was found in 2 of the 6 who showed proliferative

sponders to HCV core (14.2%) (patient 19; Figure 2) showed cellular reactivity to GOR (P = 0.014). The who responded

peptide

with chronic

OD ratio, 2.38; range, 2.22-3.90;

or the 11 with

core (71.4%) also responded to GOR (patients 17, 18, 22, 25, and 27; Figure 2), whereas only 1 of the 7 nonre-

seven patients

to ASGP-R

Cellular (stimulation index)

sponses with 10 pg/mL and two with 30 PglmL of HCV core peptide. A close correlation was found between procore peptide.

Responses

1439

Chronic hepatitis C

Cellular (stimulation index)

Patient no.

IN CHRONIC HEPATITIS C

Data in 14 Patients

to ASGP-R

With Chronic

HCV Lymphocyte proliferation (stimulation index) Patient no. 14 15 16 17 18 19 20 21 22 23 24 25 26 27

HI-A

Sex (M/F)

Liver histology

HCV core

GOR

Antibody to GOR (OD)

F M F M F M M M F M M M F F

CAH/C CAH/C MLD CAH CAH CAH CAH CAH CAH/C CAH CAH CAH/C CAH CAH/C

3.4” 1.8 3.5” 8.0” 5.9” 1.5 1.8 2.1 3.6” 0.8 1.4 3.7” 0.3 6.5”

1.5 1.6 1.6 4.1” 2.5” 6.3” 1.5 1.9 3.1” 0.9 1.1 8.2” 0.5 4.3”

2.38b 0.25 0.80 0.23 3.90b 2.22b 0.12 0.27 0.17 0.06 0.13 0.39 0.03 0.02

C, cirrhosis; CAH, chronic active hepatitis; MLD, minimal liver damage; ND, not determined. “Positive results = stimulation index > 2.4. bPositive results = OD > 1.00.

A

B

2, 1, 1, 3,

26 29 24 25

5, 17, 8, 18,

49 44 17 50

1, 2, 3, 31, I, I,

3 32 11 30

7, 8, 7. 27, 44, 5, 44, 18, 7, 7,

44 35 61 17 41 44 17

1, 2 2, 30 2, 9 I, 11

DR 13,15 13, 7 3, 8 12.15 1. 15 3, 4 13,15 I, 15 7, 15 8, 11 4, 13 10, ND ND

1440

GASTROENTEROLOGY Vol. 107, No. 5

KOSKINAS ET AL.

also showed cellular agreement

with previous

16 17

18 19 20

21 22 23 24

AIH

sponse to ASGP-R

can stimulate antibodies

of cellular

autoantigen-specific

showing autologous

responses

suppression

HCV had antibody recognition

25

26

27

to ASGP-R

of this antigen

without

apparent

is more difficult

hypergammaglobulinemia,

and

other

GOR were observed tested

with

in 42.8%

concomitant

activation

between

that the immune

quence

homology

4 of the 5 with

1:3700; median,

to ASGP-R

(Table

to ASGP-R

(titers

reactivity

correlations

to ASGP-R,

between

GOR,

cellular

or HCV core

peptide and serum biochemical liver test parameters or the presence or absence of cirrhosis or other histological markers of disease activity (Table 2). There was also no apparent assoclatlon between these cellular and humoral responses in the patients with chronic HCV and either ethnic

background

responses

or any HLA haplotype

or allotype.

Discussion The results of the present study indicate that autoreactions of the type found particularly in AIH are rare in patients with chronic HCV infections. Few had circulating autoantibodies and, whereas nearly 90% of the patients with AIH studied for comparison showed lymphoproliferative responses in vivo to ASGP-R, such responses were found in only 18% of the patients with HCV. Interestingly, these T-cell responses in the patients with HCV were not accompanied by production of antibody to ASGP-R antibodies, which were found in only two patients with HCV. In contrast, all but one of the patients with AIH with circulating antibody to ASGP-R

to the GOR

It seems that HCV

1).

There were no significant or humoral

antibody

1:600) showed cellular responses

with HCV (antibody

responses to GOR and ASGP-R).

by cross-recognition

to

with the AIH group

also

to

GOR response) in one third of these, but this did not correlate with cellular reactivity to ASGP-R (70% dis-

caused

did not have antibodies

autoantibodies

of the patients

B-cell

gesting

1:350-

to explain,

do not seem to be related to immune reactions against the GOR peptide. Lymphoproliferative responses to

erative

in which

Bwith

cellular

These findings

1 and 10; Table 2) did not show cellular reactivity to this antigen, whereas the five who did show prolif-

to ASGP-R

without

of the corresponding

were not found in these two patients.

(patients

2). This contrasts

in vitro.‘*

to ASGP-R

versely, there was a very close correlation

(Table

in re-

B lympho-

but it is unlikely to be caused by a generalized polyclonal B-cell activation because none of the patients had marked

cordance

responses

that cloned T

after proliferation

to ASGP-R

is in

cell response.37m’” The fact that two of the patients

Figure 2. Comparison of lymphoproliferative responses to HCV core, GOR, and ASGP-R in 14 patients with chronic hepatitis C (patients 14-27; Table 2). The response to each antigen is expressed as a stimulation index ratio (peak stimulation index divided by the cutoff for the particular assay). Thus, all ratios > 1 indicate positive responses. W, HCV core; 8, GOR; F3, ASGP-R.

ASGP-R

which

concomitant production of antibodies to ASGP-R in some patients with HCV might be explained by a natural

number

Patient

studies

with

The finding

14 15

to ASGP-R,

cells from patients cytes to produce

___ __ $

reactivity

antigen

and

response

between HCV

HCV

Concellular

core peptides, to GOR

related GOR

between

sug-

is probably

to the partial

se-

and HCV core.‘*

core is the most

for HLA class II-restricted

immunogenic T cells,*“~*’

and preliminary data suggest that the region between amino acids 39-74 (the segment used in the present studies) is the immunodominant epitope.*’ The fact that only 50% of our patients with HCV showed peripheral blood mononuclear cell proliferative responses to this segment of the core protein is in agreement with a recent study showing

that nearly all patients

with chronic hepa-

titis C have serum antibodies to HCV core (c22), but only a proportion show concomitant T-cell responses.“’ In that study,43 It was also found that cellular responses to the whole HCV core protein could be shown in 73% of HCV-infected individuals with no liver disease but in only 11.5% with chronic liver disease, suggesting that T-cell responses to HCV core are associated with a benign course of the infection. However, this was certainly not the case in the present study, in which all but one of our patients with HCV had chronic active hepatitis with or without cirrhosis. A drawback of the present study is the lack of patients with LKM antibodies. None of our patients with HCV had these antibodies and, in common with the experience of investigators in North America, ** LKM is a rare find-

November 1994

AUTOIMMUNITY

ing in adults with AIH in the United

Kingdom.

during the last 15 years, we have had only five such cases regularly attending our Institute. This reinforces the curious geographical

differences

in prevalence

with features of HCV infection bodies

to GOR

chronic

are commonly

HCV infection

of hepatitis

and autoimmunity. present

Anti-

in patients

and are reported

with

between

a segment

of cytochrome

of the HCV polyprotein

gesting

cross-reactivity.‘03”

alence

of

antibodies

HCV

in

in certain

P450IID6

and GOR

However,

patients

although

presenting

the prev-

with

areas is high, the frequency

in patients

from these same areas who present

infections

is very low”;

with HCV infections clear.

sugLKM

of LKM

with HCV

the reason why some patients have LKM antibodies remains un-

non-B viral hepatitis’7-20

lar and

humoral

quently

in patients

is a theoretical in susceptible

autoreactions

and suggest occur

with HCV infections.

possibility individuals,

cannot alone account

that cellu-

relatively

infre-

Although

there

that they might trigger AIH HCV-induced autoreactivity

for the marked associations

HCV and AIH that have been reported

between

1. Esteban JI, Esteban R, Viladomiu L, Lopez-Talavera JC, Gonzalez

3.

4.

5.

11. Michel G, Ritter A, Gerken G. Meyer zum Buschenfelde

KH, Decker R, Manns MP. Anti-GOR and hepatitis C virus in autoimmune liver diseases. Lancet 1992; 339:267-269.

12. Manns MP. Griffin KJ, Sullivan KF, Johnson EF. LKM-1 autoantibodies recognize a short linear sequence in P450llD6, a cytochrome P-450 monooxygenase. J Clin Invest 1991;88:13701378. 13. Okamoto H, Tsuda F, Machida A, Munekata E, Akahane Y, Mashiko K, Mitsui T, Tanaka T, Miyakawa Y, Mayumi bodies against synthetic oligopeptides deduced from the core gene for the diagnosis of hepatitis C virus infection. ogy 1992; 15:180-186.

Y, Sugai M. Antiputative Hepatol-

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Received November 2, 1993. Accepted June 13, 1994. Address requests for reprints to: Ian G. McFarlane, D.Sc., Institute of Liver Studies, King’s College Hospital, Denmark Hill, London SE5 QRS, England. Supported by the South East Thames Regional Health Authority (LORS grant 92/01). The authors thank United Biomedical Inc. for providing the antibody to hepatitis C virus and anti-GOR kits.