GASTROENTEROLOGY 1991;100:756-761

Interferon Alfa Therapy in Patients With Chronic Hepatitis B Virus Infection Effects on Hepatitis B Virus DNA in the Liver A N N A S. F. LOK, OLIVER C. K. MA, a n d JOHNSON Y. N. LAU Department of Medicine, Universityof Hong Kong, Queen Mary Hospital, Hong Kong

Pretrial and posttrial liver biopsy samples from 124 adult patients who participated in two randomized, controlled trials of interferon affa therapy for chronic hepatitis B virus (HBV) infection were analyzed to determine the effects of interferon on the replication of HBV in the liver. Replicative forms of HBV DNA were detected in the pretrial biopsy samples from all and posttrial biopsy samples from 74% treated patients and 86% controls. Replicative forms of HBV DNA were detected in the posttrial biopsy samples from all patients w h o remained positive for hepatitis B e antigen and HBV DNA in the serum, in 77% treated patients and 80% controls w h o cleared HBV DNA in the serum but w h o remained positive for hepatitis B e antigen, but in only 19% treated patients and 40% controls who cleared HBV DNA as well as hepatitis B e antigen in the serum. Serum alanine aminotransferase levels were significantly lower in patients whose posttrial biopsies did not contain replicative forms of HBV DNA. In summary, we demonstrated that in most patients with chronic HBV infection treated with interferon alfa, serological response was associated with the disappearance of replicative forms ofHBV DNA in the liver.

'nterferon alfa has been shown to be the most lhepatitis .promising antiviral agent in the treatment of chronic B virus (HBV) infection (1-6). In most studies, a successful antiviral response was defined as the sustained clearance of hepatitis B e antigen (HBeAg) and I-rBV DNA with or without clearance of hepatitis B surface antigen (HBsAg) in the serum 1 year after entry into the trial. None of the controlled trials on interferon correlated the serological response with clearance of replicative forms of I-IBV DNA in the liver tissues, although Hoofnagle et al. reported the disappearance of hepatitis B core antigen (HBcAg), a viral protein frequently associated with the presence of replicative forms of HBV DNA (7), in the posttreat-

ment liver biopsy samples in 9 of 10 responders (4) and Saracco et al. reported the clearance of intrahepatic HBcAg in all 23 responders (6). DiBisceglie et al. also noted that replicative forms of HBV DNA could not be detected in the posttreatment liver biopsy samples from 12 patients who became HBeAgnegative but noted no significant change in the amount of replicative forms of HBV DNA in 16 patients who remained HBeAg-positive (8). In this study, there was a wide variation in the interval (11-45 months) between the pretrial and posttreatment biopsies. In addition, the patients received one or more courses of antiviral and or immunomodulatory therapy before the second biopsies. We analyzed pretrial and posttrial liver biopsy samples from adult patients who participated in two randomized, controlled trials of interferon alfa therapy (a) to evaluate the effects of interferon alfa on HBV DNA in the liver tissues and (b) to correlate the changes in HBV DNA in the liver tissues with the serological response to interferon treatment.

Patients and Methods One hundred fifty-eight Chinese patients with chronic HBV infection who participated in two randomized, controlled trials of recombinant interferon alfa therapy have been followed up for 1-5 years (9,10). Liver biopsy specimens were obtained within 6 months of entry and again 12-18 months after entry into the trial using disposable Menghini needles (Hepafix, 1.6 x 90 mm; B Braun, Germany). The protocol was approved by the Ethical Committee of the University of Hong Kong. The terminal 2 cm of the plastic styler inside the biopsy needle was cut. A single pass biopsy was performed on all patients unless there was inadequate liver tissue for histological diagnosis. In 80% of patients, the length of the biopsied material exceeded 3 cm.

© 1991 by the American GastroenterologicalAssociation 0016-5085/91/$3.00 .

March 1991

Biopsy samples from these patients were divided into two portions; a 1.5-2-cm portion was sent for routine histological examination, and the remaining 1-2 c m was frozen and stored at -70°C for H B V D N A analysis. In patients whose

biopsy samples were less than 3 cm in length, the entire specimen was sent for histology. In 124 patients (89 men and 35 women), aged 18-46 years (median, 29 years), sufficient pretrial and/or posttrial liver tissues were available for HBV DNA analysis. They included 86 treated patients and 38 controls. Sara were tested for HBsAg, HBeAg, and antibody to HBeAg (anti-HBe) by enzyme immunoassays (Abbott Laboratories, Chicago, IL}. Hepatitis B virus DNA was detected by a modification of the method of Matsuyama et al. (11). Briefly, 20 ~L serum was directly spotted onto nitrocellulose {Schleicher and Schull, Dassel, Germany) or nylon (Hybond; Amersham, Aylesbury, Buckinghamshire, England) membranes, denatured, renatured, prehybridized, and then hybridized to 32p-labeled HBV DNA probes. The probes were prepared by nick translation of the complete genome after EcoRI digestion and gel purification from plasmid vector pBR322. The specific activities were 2-4 x 108 cpm/p,g DNA. Autoradiography was allowed to proceed for 3-5 days. The sensitivituy limit of the assay was 0.1 pg HBV DNA per 20 "p,L serum. Serum HBV DNA levels were visually scored as 1-4 (1, 0.2-1 pg; 2, 1-5 pg; 3, 5-25 pg; and 4, > 25 pg HBV DNA/20 p.L serum). Liver biopsy samples were minced in lysis buffer (10 mmol/L Tris hydrochloride, 10 mmolfL Na 2 ethylenediaminetetraacetic acid (EDTA}, and 150 mmol/L NaC1, pH 8) with 1% sodium dodecyl sulphate and 0.5 mg/mL proteinase K (Beckman, Fullerton, CA} and were digested overnight at 37°C. The samples were extracted twice with phenol and chloroform and then precipitated with ethanol. The nucleic acid pellets were resuspended in 10 mmol/L Tris hydrochloride (pH 7.4) and 1 mmol/L Na 2 EDTA. The amount of cellular DNA obtained from each biopsy sample varied frcim 15 to 50 p,g. Five micrograms of cellular DNA was directly spotted onto nylon membranes together with serial standards containing 0.2-20 pg cloned HBV DNA for semiquantitative assessment of HBV DNA in the liver tissues. The amount of HBV DNA in the liver biopsy sample visually scored as 1-3 (1, 4 pgHBV DNA per microgram of cellular DNA). Whenever available, cellular DNA from paired pretrial and posttrial biopsy samples was spotted onto the same membrane to minimize interassay variability. For analysis of the molecular state of HBV DNA in the liver tissues, 15-20 p,g cellular DNA was electrophoresed in 1% agarose gels after digestion with EcoRI (New England Biolab, Beverly, MA) and was then transferred onto nitrocellulose or nylon membranes. The membranes were prehybridized and then hybridized to 22p-labeled HBV DNA probes in 50% formamide at 42°C. Autoradiography was allowed to proceed for 7-10 days. The sensitivity limit of this technique was 1 pg HBV DNA. Statistical analysis was performed by Xz test and Fisher's Exact Test with Yates correction. Comparisons of serum ALT levels were made by Student's t test or analysis of variance after logarithmic transformation of the ALT values.

EFFECTS OF INTERFERON O N H B V D N A IN T H E LIVER

757

Results

Hepatitis B Virus D N A in Pretrial Liver Biopsies In 99 (66 treated a n d 33 control) patients, pretrial biopsies yielded sufficient liver tissues for HBV DNA analysis. Replicative forms of HBV DNA (3.2-kb b a n d w i t h s m e a r of replicative intermediates) c o u l d be detected in the liver tissues f r o m all 99 patients. Three patients had, in 'addition, smears in the h i g h - m o l e c u l a r - w e i g h t regions. T h e s e smears m a y be c a u s e d b y trapping of replicative forms of HBV DNA or diffuse integration of HBV DNA into the host genome. Thus, in m o s t patients, the a m o u n t of HBV DNA in the liver tissues assessed by direct spotting of cellular DNA reflected the a b u n d a n c e of replicative forms of HBV DNA. The a m o u n t s of HBV DNA in the liver tissues were scored as I in 11 (11%) patients, 2 in 21 (21%) patients, a n d 3 in 67 (68%) patients. There was no correlation b e t w e e n the a m o u n t of HBV DNA in the liver tissues and the histological diagnosis (Table 1}. Patients w i t h larger a m o u n t s of HBV DNA in the liver tissues tended to h a v e higher s e r u m alanine a m i n o t r a n s f e r a s e (ALT) levels, but the difference was not significant a n d the range of ALT levels w a s wide. All the patients w h o h a d liver tissue HBV DNA scores of I h a d low levels of HBV DNA in the s e r u m in contrast to half of those w i t h scores of 2 a n d 3 (P = 0.003). T h e antiviral r e s p o n s e to interferon alfa therapy, defined as the sustained clearance of HBeAg a n d HBV DNA w i t h or w i t h o u t clearance of HBsAg in the s e r u m

Table 1. Corm~oh'on Between the Amount of Hepatitis B Virus DNA in Pretrial Liver Biopsies and Histological Diagnosis, Serum Alonine Aminotransferase, and Hepatitis B Virus DNA Levels Liver tissue HBV DNA score

n

Histology Minimal change CPH CAH CAH + cirrhosis Cirrhosis Serum ALT {IU/L}

Mean +- SD Range Log mean Serum HBV D N A score

1(%)

2{%}

3(%}

11 (11)

21 (21)

67 (68)

1 (9) 2 (18} 5 (46) 3 (27)

2 {10) 7 (33} 7 (33) 5 {24)

9 (13) 22 (33} 32 (48) 3 (4)

0 {0)

0 {0)

1 (2}

+- 54 3-188 35

115 -+ 57 12-518 61

56

113 +- 58 7-529 56

1

10 (91)

7 (33)

19 (28}

2 3

1 (9) 0

5 (24) 7 (33}

15 {22.5} 18 (2~

4

0

2 (10)

15 (22.S}

CPH, chronic persistent hepatitis; CAH, chronic active hepatitis; ALT, alanine aminotransferase.

758 LOK ET AL.

GASTROENTEROLOGYVol. 100, No. 3

I year after entry into the trial, was higher in patients w h o had smaller amounts (scores of 1) o f H B V DNA in the pretrial liver biopsies (Table 2).

Hepatitis B Virus DNA in Posttrial Liver Biopsies

Posttrial liver biopsies

In 89 (60 treated and 29 control) patients, posttrial biopsies yielded sufficient liver tissues for HBV DNA analysis. Hepatitis B virus DNA could be detected in the liver tissues from 52 (87%) treated patients and 27 (93%) controls by direct spotting (Table 3). In 44 (74%) treated patients and 25 (86%) controls, replicative forms of HBV DNA were present (P = 0.27). In 2 treated patients, HBV DNA was detected by direct spotting but was absent by Southern blot hybridization. In 5 treated patients and 1 control, Southern blot hybridization s h o w e d a single band at the 3.2-kb region, but no rep!icative intermediates could be detected. In 1 treated patient and 1 control, smears were present in the high-molecularweight regions, but replicative forms of HBV DNA were not found• Hepatitis B virus DNA was not detected by either direct spotting or S o u t h e r n blot hybridization in 8 (13%) treated patients and 2 (7%) controls (P = 0.61). Replicative forms of HBV DNA were detected in the posttrial liver biopsies from all (treated and control) patient~ w h o remained positive for HBeAg and HBV DNA in the serum including five patients w h o had transient clearance of HBeAg during treatment (Table 4). Replicative forms of HBV DNA were still f o u n d in m~st (77% treated and 80% control) patients who cleared HBV DNA in the serum but who remained positive for HBeAg. However, replicative forms of HBV DNA could only be detected in 19% treated patients and 40% controls w h o cleared HBeAg and HBV DNA in the serum. • Among the 21 patients w h o cleared HBeAg, replicative forms of HBV DNA could not be detected in 10 (91%) of 11 treated patients and 2 (67%) of 3 controls w h o u n d e r w e n t posttrial biopsies w i t h i n 1 2 - 1 4

Table 2. CorrelationBetween the Amount of Hepatitis B Virus DNA in PretrialLiver Biopsies and the Serological Response to Interferon Alfa Therapy Liver tissue HBV DNA score Treated patients (n = 66) No clearance of HBeAg Sustained clearance of HBeAg Sustained clearance of HBsAg Controls (n = 3 3 ) No clearance of HBeAg Sustained clearance of I-IBeAg Sustained clearance of HBsAg

Table 3. Hepatitis B Virus DNA in Posttrial Liver Biopsy Specimens From Interferon-Treated and Control Patients

1 (%)

2 {%)

3 (%)

4 C57) 2 (29) 1 {14)

18 (86) 3 (14) 0 {0)

32 (84) 6 C16) 0 CO}

4 C100) 0 Co} 0 CO)

0 {0) 0 (0) 0 {0)

24 C83) 5 (17) 0 (0)

Treated Liver tissue patients Controls HBV DNA score n(%) n(%)

HBV DNA + Replicative intermediates +

44 (74) 25 (86} 7 (12) 4 (14} 12 {20} 4 {14} 25 {42} 17 {58) 8 [13] 2 (7)

Replicative intermediates HBV DNA Total

8 {13)

60

2 (7)

29

m o n t h s of entry and in 3 (60%) of 5 treated patients and 1 (50%) of 2 controls w h o u n d e r w e n t posttrial biopsies within 14-18 m o n t h s of entry. In both treated patients and controls, there was no correlation between the presence or absence of replicative forms of HBV DNA in the liver tissues and histological diagnosis, but serum ALT levels were significantly lower in patients w h o s e posttrial biopsies did not s h o w replicative forms of HBV DNA (log m e a n ALT, 26 vs. 50 IU/L; P = 0.005). On long-term follow-up (3-54 m o n t h s after the posttrial biopsies; median, 30 months), sustained clearance of HBeAg occurred in 7 (17%) of 41 treated patients and in 3 (13%) of 23 controls who did and 2 (67%) of 3 treated patients and 1 control w h o did not have replicative forms of HBV DNA in the posttrial liver biopsy samples (Table 5).

Comparison of lJ'ver Tissue Hepatitis B Virus DNA in Paired Biopsies In 66 (41 treated and 25 control) patients, sufficient liver tissues from both pretrial and posttrial

Table 4. Correlation Between the Presence of Replicative Forms of Hepatitis B Virus DNA in the Posttrial Liver Biopsy Samples and Serological Response to Interferon Alfa Therapy Liver tissue HBV DNA replicative intermediates

Serum HBeAg and HBV DNA

Present

Absent

status

n (%)

n {%)

31 (100) 10 C77] 3 (19)

0 3 [23] 13" {81)

19 [100) 4 (80} 2 (40)

0 1 {20) 3 {60)

Treated patients {n = 60) HBeAg + HBV DNA + HBeAg + HBV DNA HBeAg - HBV DNA Controls {n = 29) I-IBeAg+ HBV DNA + HBeAg + HBV D N A -

HBeAg - HBV DNA -

"One patient cleared both I--IBeAgand HBsAg.

Mar ch 1991

EFFECTS OF INTERFERON ON HBV DNA IN THE LIVER

759

Table 5. Comparison of L i v e r Tissue Hepatitis B Virus DNA in Paired Biopsies and Correlation With Serological Response to In terferon Alfa Therapy Liver t i s s ue HBV DNA replicative intermediates Present S e r u m HBeAg a n d HBV DNA s t a t u s Tr eated p a t i e n t s {n -- 41} HBeAg + HBV DNA + HBeAg + HBV DNA HBeAg - HBV DNA Controls (n = 25) HBeAg + HBV DNA + HBeAg + HBV DNA HBeAg - HBV DNA -

Total

Increased amount

Similar amount

Decreased amount

Absent

24 9 8

1 0 0

20 7 1

3 1 1

0 1 6

15 5 5

0 0 0

15 3 0

O 1 2

0 1 3

biopsies were available for HBV DNA analysis (Figure 1). In most patients, the amounts of HBV DNA in the pretrial and posttrial liver biopsy samples were similar. In 5 treated patients and 3 controls, there was a significant decrease in the amount of replicative forms of HBV DNA (from a score of 3 to 1) in the posttrial biopsy samples. Of these, 1 treated patient and 2 controls had cleared HBeAg. In 7 treated patients and 4 controls, replicative forms of HBV DNA were present in the pretrial but not in the posttrial biopsy samples. Among these 11 patients, 6 treated patients and 3 controls had cleared HBeAg. Discussion

This is the first report on a systematic analysis of the effects of interferon alfa therapy on the replication of HBV in the liver tissues and the correlation between clearance of replicative forms of HBV DNA in the liver and the clearance of HBeAg and HBV DNA in the serum in patients with chronic HBV infection. One of the main aims of antiviral therapy in chronic HBV infection is to eliminate HBV replication. Nevertheless, clearance of HBeAg and HBV DNA in the serum does not necessarily reflect the eradication of HBV replication. Several investigators have demonstrated the presence of replicative forms of HBV DNA in the liver tissues from anti-HBe---positive patients with chronic liver disease (12-14). Reactivation of HBV replication with or without clinical hepatitis has also been reported in anti-HBe-positive patients either spontaneously or during immunosuppressive therapy (15-19}. We found that replicative forms of HBV DNA disappeared from the liver in most but rmt all patients who became HBeAg-negative and HBV DNA-negative in the serum. Nevertheless, in 38% of these patients, HBV DNA could still be detected in the liver tissues in the form of a smear in the high-molecular-weight region or as a single band in the 3.2-kb region. It is not

clear whether these forms of HBV DNA represent latent virus that can subsequently replicate or intermediates in the process of virus elimination or integration. Persistence of the supercoiled form of HBV DNA after disappearance of replicative intermediates has been observed in livers from humans as well as chimpanzees (20,211. None of our patients had serological evidence of reactivation of HBV relJlication after 3-42 months of follow-up. In the majority of patients who remained HBeAgpositive, there was no significant change in the amount of replicative forms of HBV DNA in the liver, although HBV DNA was no longer detectable in the serum in some patients. In the few patients who remained HBeAg-positive but who no longer have replicative forms of HBV DNA in the liver, the chance of clearing HBeAg during follow-up was higher. The close correlation between the elimination of replicative forms of HBV DNA in the liver and the clearance of HBeAg and HBV DNA in the serum was true whether the clearance of HBeAg was spontaneous or interferon induced. Similar findings have been reported by Yokosuka et al. on liver biopsies obtained 2 weeks after interferon treatment (22}. Replicative forms of HBV DNA were more likely to be absent in the posttrial liver biopsies in the interferon-treated patients than in the controls. Nevertheless, elimination of HBV replication in the liver was achieved in only 27% of our interferon-treated patients. The results may be different ff the biopsies were performed during or at the end of the course of therapy. Our finding accords with the low serological response that we have observed in Chinese patients {9,10}. The poor response is not related to the integration of HBV DNA into the host genome secondary to the long duration of infection, because definite integration in the form of high-molecular-weight band~ could not be detected in the pretrial or posttrial biopsies of any patient. A smear in the high-molecularweight region was found in the pretrial biopsies of

760

LOK ET AL.

®

GASTROENTEROLOGY Vol. 100, No. 3

A 1

B

D

C 2

2

e

Q

e

F

G

1

2

H 2

0

A Kb

1

1

H

C

D 2

1

2

2

1

2

23.1 Kb

23-1 -

9.49.46 -7 -

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three and posttrial biopsies of five patients. In six biopsies, the smears were found in the presence of replicative forms of HBV DNA and may represent trapping of replicative intermediates, although it is possible that in these six and the other two biopsies the smears represent diffuse integration of the viral genome.

1

•

2

Figure 1. Comparative analysis of HBV DNA in paired pretrial and posttrial liver biopsy samples by (A) direct spot hybridization of 5 p.g cellular DNA and (B) Southern blot h y b r i d i z a t i o n of 15-20 p.g cellular DNA after digestion with EcoRl. Patients A-D received interferon alfa therapy, and patients E-H were controls. A, B, E, F remained HBeAg-positive, whereas C, D, G, H were HBeAg-negative w h e n the posttrial biopsies were performed. 1, pretrial biopsy; 2, posttrial biopsy.

Patients with a smaller amount of replicative forms of HBV DNA in the pretrial biopsies tended to have lower levels of HBV DNA in the serum and were more likely to respond to interferon therapy. Some investigators have also noted that patients with low pretreatment serum HBV DNA levels respond more favorably to interferon treatment (5,9,22). There was very little correlation between the amount ofHBV DNA in the liver tissues and the liver histology or serum ALT levels, thus confirming previous findings that liver injury in chronic HBV infection is predominantly immune-mediated and not directly cytopathic (23-26). Nevertheless, the elimination of replicative forms of HBV DNA in the liver was associated with significantly lower serum ALT levels, indicating that viral replication is a necessary although not a direct factor in liver injury. This is consistent with the clinical observations of decreased serum ALT levels and histological activity index in patients who have a serological response to interferon therapy (1,3,4,9,21,27-29). In summary, we demonstrated that interferon alfa therapy can eliminate replicative forms of HBV DNA in the liver in patients with chronic HBV infection. Moreover, there was good correlation between serological response and elimination of virus replication in the liver.

References 1. Lok ASF, Novick DM, Karayiannis P, Dunk A, Sherlock S, Thomas HC. A randomized study of the effects of adenine arabinoside 5'-monophosphate (short or long courses) and lymphoblastoid interferon in hepatitis B virus infection. Hepatology 1985;5:1132-1138. 2. Alexander GJM, Brahm J, Pagan EA, Smith HM, Daniels HM,

March 1991

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16.

17.

Eddleston ALWF, Williams R. Loss of HBsAg with interferon therapy in chronic hepatitis B virus infection. Lancet 1987;2:6669. Scully LJ, Shein R, Karayiannis P, McDonald JA, Thomas HC.. Lymphoblastoid interferon therapy of chronic HBV infection: a comparison of 12 vs 24 weeks of thrice weekly treatment. J Hepato11987;5:51-58. Hoofnagle JH, Peters M, Mullen KD, Jones DB, Rustgi V, DiBisceglie AM, Hallahan C, Park Y, Meschievitz C, Jones EA. Randomized, controlled trial of recombinant human alphainterferon in patients with chronic hepatitis B. Gastroenterology 1988;95:1318-1325. Brook MG, Chan G, Yap I, Karayiannis P, Lever AML, Jacyna M, Main J, Thomas HC. Randomized controlled trial of lymphoblastold interferon alfa in Europid men with chronic hepatitis B virus infection. Br Med J 1989;299:652-656. Saracco G, Mazzella G, Rosina F, Cancellieri C, Lattore V, Raise E, Rocca G, Giorda L, Verme G, Gasbarnni G, Barbara L, Bonino F, Rizzetto M, Roda E. A controlled trial of human lymphoblastold interferon in chronic hepatitis B in Italy. Hepatology 1989;10:336-341. Omata M, Yokosuka O, lmazeki F, Ito Y, Mort J, Uchiumi K, Okuda K. Correlation of hepatitis B virus DNA and antigens in the liver: a study in chronic liver disease. Gastroenterology 1987;92:192-196. DiBisceglie AM, Waggoner JG, Hoofnagle JH. Hepatitis B virus deoxyribonucleic acid in liver of chronic carriers: correlation with serum markers and changes associated with loss of hepatitis B e antigen after antiviral therapy. Gastroenterology 1987;93:1236-1241. Lok ASF, Lai CL, Wu PC, Leung EKY. Long-term follow-up in a randomized controlled trial of recombinant alpha-interferon in Chinese patients with chronic hepatitis B infection. Lancet 1988;2:298-302. Lok ASF, Lai CL, Wu PC, Lau JYN, Leung EKY, Wong LSK. Treatment of chronic hepatitis B with interferon: experience in Asian patients. Semin Liver Dis 1989;4:249-253. Matsuyama Y, Omata M, Yokosuka O, Imazeki F, Ito Y, Okuda K. Discordance of hepatitis B e antigen/antibody and hepatitis B virus deoxyribonucleic acid in serum. Gastroenterology 1985 ;89:1104-1108. Harrison TJ, Anderson MG, Murray-Lyon IM, Zuckerman AJ. Hepatiti~ B virus DNA in the hepatocyte: a series of 160 biopsies. J Hepatol 1986;2:1-10. Brechot C, Degos F, Lugassy C, Thiers V, Zafrani S, Franco D, Bismuth H, Trepo C, Benhamou JP, Wands J, Isselbacher K, Tiollais P, Berthelot P. Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. N Engl J Med 1985;312:270-276. Fowler MJF, Monjardino J, Weller IVD, Lok ASF, Thomas HC. Analysis of the molecular state of HBV-DNA in the liver and serum of patients with chronic hepatitis or primary liver cell carcinoma and the effect of therapy with adenine arabinoside. Gut 1984;25:611-618. Lok ASF, Lai CL, Wu PC, Leung EKY, Lam TS. Spontaneous hepatitis B e antigen to antibody seroconversion and reversion in Chinese patients with chronic hepatitis B virus infection. Gastroenterology 1987;92:1839-1843. Lau JYN, Lai CL, Lin HJ, Lok ASF, Liang RHS, Wu PC, Chan TK, Todd D. Fatal reactivation of chronic hepatitis B virus infection following chemotherapy withdrawal in lymphoma patients. QJ Med 1989;73:911-917. Hoofnagle JH, Dusheiko GM, Schafer DF, Jones EA, Micetich

EFFECTS OF INTERFERON ON HBV DNA IN THE LIVER 761

KC, Young RC, Costa J. Reactivation of chronic hepatitis B virus infection by cancer chemotherapy. Ann Intern Med 1982:96:447-449. Davis GL, Hoofnagle JH, Waggoner JG. Spontaneous reactivation of chronic hepatitis B virus infection. Gastroenterology 1984;86:230-235. Perrillo RP, Campbell CR, Sanders GE, Reganstein FG, Bodicky CJ. Spontaneous clearance and reactivation of hepatitis B virus infection among male homosexuals with chronic type B hepatitis. Ann Intern Med 1964;100:43-46. Yokosuka O, Omata M, Imazeki F, O'kuda K. Active and inactive replication of hepatitis B virus deoxyribonucleic acid in chronic liver disease. Gastroenterology 1985;89:610-616. Ruiz-Opazo N, Chakraborty PR, Shafritz DA. Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum. Dane particles: possible implications in persistent HBV infection. Cell 1982;29:129-138. Yokosuka O, Omata M, Imazeki F, Okuda K, Summers J. Changes of hepatitis B virus DNA in liver and serum caused by recombinant leukocyte interferon treatment: analysis of intrahepatic replicative hepatitis B virus DNA. Hepatology 1985:5: 726-734. Perrillo RP, Regenstein FG, Peters MG, DeSchsyver-Kecskemeti K, Bodicky CJ, Campbell CR, Kuhns MC. Prednisone withdrawal followed by recombinant alpha interferon in the treatment of chronic type B hepatitis: a randomized, controlled trial. Ann Intern Med 1988;109:95-100. Mondelli M, Mieli-Vergani G, Alberti A, Vergani D, Portmann B, Eddleston A, Williams R. Specificity o f ' T lymphocyte cytotoxicity to autologous hepatocytes in chronic hepatitis B virus infection: evidence that T cells are directed against HBV core antigen expressed on hepatocytes. J Immunol 1982;129: o

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25. Chisari IV, Milich DR, Tiollais P. Hepatitis B virus infection: a model for immunologically mediated hepatocellular injury. In: Keppler D, Reutter W, Bianchi L, eds. Mechanism of hepatocyte injury and death. Lancaster, England: MTP, 1964;293297. 26. Ferrari C, Penna P, Degli Antoni A, Fiaccadori F. Cellular immune response to hepatitis B virus antigens. An overview. J Hepato11988;7:21-33. 27. Dienstag JL. Immunologic mechanisms in chronic viral hepatitis. In: Vyas GN, Dienstag JL, Hoofnagle JH, eds. Viral hepatitis and liver disease. Philadelphia: Grune & Stratton, 1984:135166. 28. Brook MG, Petrovic L, McDonald JA, Scheuer PJ, Thomas HC. Histological improvement after anti-viral treatment for chronic hepatitis B virus infection. J Hepatol 1989:8:218-225. 29. Scullard GH, Andres LL, Greenberg HB, Smith JL, Sawhney VK, Neal EA, Mahal AS, Popper H, Merigan TC, Robinson WS, Gregory PB. Antiviral treatment of chronic hepatitis B virus infection: improvement in liver disease with interferon and adanine arabinoside. Hepatology 1961 ;1:226-232.

Received February 28, 1990. Accepted August 31, 1990. Address requests for reprints to: Anna S. F, Lok, M.D., Department of Medicine, Queen Mary Hospital, Hong Kong. This study was supported by Strategic Research Grant 336.041.0017 from the University of Hong Kong. The authors are grateful to nurses E. K. Y. Leung, L. S. K. Wong, and Y. L. Fung for their help with running the trials and to A. Peon for her assistance in preparing the manuscript.