LETTERSTO THE EDITOR overt thyroid dysfunction during ~-IFN therapy. They also suggest a possible link between HCV and autoimmune thyroiditis. These facts need, however, to be confirmed in a larger number of patients.
References I Burman P, Totterman TH, Oberg K, Karlsson FA. Thyroid autoimmunity in patients on long term therapy with leukocytederived interferon. J Clin Endocrinol Metab 1986; 63: 1086-90. 2 Conlon KC, Urba W J, Smith II JW, Steis RG, Longo DL, Clark JW. Exacerbation of symptoms of autoimmune disease in patients receiving alpha-interferon therapy. Cancer 1990; 65: 2237-42. 3 Rosa F, Fellous M. The effect of gamma-interferon on MHC antigens. Immunol Today 1984; 5: 261-2. 4 Burman P, Karlsson FA, Oberg K, Aim G. Autoimmune thyroid disease in interferon-treated patients. Lancet 1985; 2: 100-1. 5 Mayet W J, Hess G, Gerken G, et al. Treatment of chronic type B hepatitis with recombinant a-interferon induces autoantibodies not specific for chronic hepatitis. Hepatology 1989; 10: 24-8.
245 D. Pateron, D.J. Hartmann a, J.C. Duclos-Vallee, H. Jouanolle b and M. Beaugrand Service d'H~pato-gastroent~rologie, Hrpital Jean Verdier, 93143 Bondy, *Centre de Radioanalyse, CNRS URA-1459 Institut Pasteur, 69007 Lyon and bClinique des maladies du foie, Hrpital Pontchaillou. 35033 Rennes, France
6 Renault PF, Hoofnagle JH. Side effects of alpha interferon. Semin Liver Dis 1989; 9: 273-7. 7 Berris B, Feinmann SV. Thyroid dysfunction and liver injury following alpha-interferon treatment of chronic viral hepatitis. Dig Dis Sci 36: 1657-60. 8 Magrin S, Craxi A, Fiorentino G. Is autoimmune chronic active hepatitis a HCV related disease? J Hepatol 1991; 13: 128-31. 9 Haddad J, Deny P, Munz C, et al. Chronic lymphocytic sialadenitis characteristic of Sjfgren's syndrome in patients with HCV chronic hepatitis or cirrhosis. Lancet 1992; 339: 321-3. I0 Prentice LM, Phillips DIW, Sarsero D, Beever K, McLachlan S, Smith BR. Geographical distribution of subclinical autoimmune thyroid disease in Britain: A study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinol 1990; 123: 493-8.
HEPAT 01168
Monitoring of interferon-a treatment in chronic active hepatitis B We present a case showing that the use of more sensitive serological tests for the diagnosis of the hepatitis B virus (HBV) might permit better monitoring of the antiviral activity of interferon-~ treatment. A 69-yearold male patient with severe chronic active hepatitis B and with signs of beginning fibrosis on liver biopsy was treated with interferon-~ (IntronA, 1.5 MU s.c.) thrice a week for 4 months. Serological markers of HBV, hepatitis C and hepatitis 6 were determined by commercially available enzyme immunoassays: anti-HBs (test A), anti-6 and antihepatitis C antibodies respectively by AUSAB, AntiDelta EIA and Anti-HCV EIA (c-i00-3) from Abbott Laboratories, U.S.A.; HSsAg, anti-HBc (results not shown), anti-HBs (test B (1)), HBe and anti-HBe by the corresponding enzyme immunoassays from Hoffman-La Roche, Switzerland. Semi-quantitative analysis of HBsAg was performed by counter-immunodiffusion. HBV-DNA was measured by a hybridization technique using a digoxigenin-labelled probe (2). HBV genome amplification was carried out by polymerase chain reaction (PCR) as previously described (3). Two oligonucleotide primers were used for hybridization to HBs sequences (5'-CTCTCAATTTTCTAGGGGGA-3' posi-
tion 267-286 and 5'-AGCAGCAGGATGAAGAGGAA-3' position 401-420 according to Tiollais (4)) together with a commercial amplification kit (Cetus). Amplified DNA was characterized by gel electrophoresis followed by transfer to a nylon membrane and hybridization with a HBV-specific probe labelled with digoxigenin (5). Test B and PCR were performed retrospectively on frozen serum samples. Results are shown in Table 1. The patient was negative for hepatitis C and ~ (results not shown). HBe/anti-HBe seroconversion was observed after the complete 4-month course of interferon-c~ treatment. HBs antigen remained positive though a slight decrease in titer was observed. HBV-DNA when measured by the conventional hybridization became negative after the third month. The retrospective use of PCR showed that HBV elimination was only partial and HBV-DNA remained detectable for 36 months. This is in contrast with the findings of Carman (6) who has shown by PCR the disappearance of viremia 13-18 months after the end of interferon treatment. Anti-HBs remained persistently negative with test A, but low titers were observed after 2 months with test B. These antibodies decreased slowly until complete disappearance at the
LETTERS TO THE EDITOR
246 TABLE I Serological markers of hepatitis B during interferon treatment Interferon treatment
H Bs-titer immuno-diffusion
H Be-Ag
Anti-HBe
Before During (month 1) During (month 2) During (month 3) During (month 4) After (month 3) After (month 6) After (month 12) After (month 16) After (month 18) After (month 24) After (month 36)
I/32 1/16 1/16 1/16 1/8 1/8 I/8 1/16 1/8 1/16 1/8 1/4
pos. pos. pos. pos. pos. pos. neg. neg. neg. neg. neg. neg.
neg. neg. neg. neg. neg. pos. pos. pos. pos. pos. pos. pos.
(IU/ml)
HBV-DNA (pg/ml)
HBV PCR a
325 225 192 188 147 normal normal normal normal normal normal normal
1000 400 80 40 0 0 0 0 3 0 0 0
ND ND ND ++++ ++++ ++++ ND +++ ND + + + + + +
ALAT
Anti-HBs Test A
Test B
0 0 ND 0 0 0 0 ND ND ND ND ND
ND 21 17 ND ND 7 5 6 ND I neg. neg.
" Results by visual evaluation of the spot colour intensity: + + + + = very strongly positive, + + + =strongly positivie, + + =positive, - = negative. N D = n o t done.
end of the treatment, indicating that they were only temporarily induced. These findings could be interpreted as the beginning of an immune response potentially capable of completely eliminating HBV and that this response was abrogated after the (too early?) end of the treatment. The prospective use of these tests should have lead us to continue interferon for a longer period and/or with a higher dosage until the possible complete HBV elimination. Thus interferon-or treatment induced a substantial inhibition of HBV replication and seroconversion from HBe to anti-HBe. This was to the benefit of the patient whose liver enzyme values returned to normal and who
experienced a considerable improvement in quality of life. Liver histology was not repeated. We conclude that a battery of sensitive serological tests would permit a better and more individual adaptation of the dose and the duration of interferon-or treatment in chronic active hepatitis B. The case presented took part in the Swiss multicenter study for the treatment of chronic hepatitis B run by the Swiss Association for the Study of the Liver.
References
for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J Clin Microbiol 1989; 27(9): 1930-3. 4 Tiollais P, Charnay P, Vyas GN. Biology of hepatitis B virus. Science 1981; 213: 406-I I. 5 Guo K J, Bowden DS. Digoxigenin-labeled probes for the detection of hepatitis B virus DNA in serum. J Clin Microbiol 1991; 29: 506-9. 6 Carman WF, Dourakis S, Karayiannis P, Crossey M, Drobner R, Thomas HC. Incidence of hepatitis B viraemia, detected using the polymerase chain reaction, after successful therapy of hepatitis B virus carriers with interferon-alpha. J Med Virol 1991; 34:114-8.
1 Lavanchy D, Peitrequin R, Vu DH, Frei PC. Measurement of antiHBs production after vaccination: comparison of results obtained by two different immunoassays. In: Coursaget P, Tong M J, eds. Progress in Hepatitis B Immunization. John Libbey/INSERM, 1990; 194: 73-7. 2 Seibl R, Holtke H J, Ruger R, et al. Non-radioactive labeling and detection of nucleic acids. III. Applications of the digoxigenin system. Biol Chem Hoppe Seyler 1990; 371: 939-51. 3 Kaneko S, Feinstone SM, Miller RH. Rapid and sensitive method
D..Lavanchya, C. Mayerat a, P.C. FreP and A.L. Blum b °Division of Immunology and Allergy and bDivision of Gastroenterology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
HEPAT 01169
Function of partial liver allografts after heterotopic transplantation In the field of liver transplantation both heterotopic grafting and the regular use of clearance studies in the follow-up of patients are far from commonplace. We routinely performed indocyanine green (ICG) and anti-
pyrine (AP) elimination tests in 10 patients who received heterotopic partial liver allografts because of end-stage chronic liver disease (1). Four of these patients had chronic hepatitis-B virus infections, two with f-virus
References I Burman P, Totterman TH, Oberg K, Karlsson FA. Thyroid autoimmunity in patients on long term therapy with leukocytederived interferon. J Clin Endocrinol Metab 1986; 63: 1086-90. 2 Conlon KC, Urba W J, Smith II JW, Steis RG, Longo DL, Clark JW. Exacerbation of symptoms of autoimmune disease in patients receiving alpha-interferon therapy. Cancer 1990; 65: 2237-42. 3 Rosa F, Fellous M. The effect of gamma-interferon on MHC antigens. Immunol Today 1984; 5: 261-2. 4 Burman P, Karlsson FA, Oberg K, Aim G. Autoimmune thyroid disease in interferon-treated patients. Lancet 1985; 2: 100-1. 5 Mayet W J, Hess G, Gerken G, et al. Treatment of chronic type B hepatitis with recombinant a-interferon induces autoantibodies not specific for chronic hepatitis. Hepatology 1989; 10: 24-8.
245 D. Pateron, D.J. Hartmann a, J.C. Duclos-Vallee, H. Jouanolle b and M. Beaugrand Service d'H~pato-gastroent~rologie, Hrpital Jean Verdier, 93143 Bondy, *Centre de Radioanalyse, CNRS URA-1459 Institut Pasteur, 69007 Lyon and bClinique des maladies du foie, Hrpital Pontchaillou. 35033 Rennes, France
6 Renault PF, Hoofnagle JH. Side effects of alpha interferon. Semin Liver Dis 1989; 9: 273-7. 7 Berris B, Feinmann SV. Thyroid dysfunction and liver injury following alpha-interferon treatment of chronic viral hepatitis. Dig Dis Sci 36: 1657-60. 8 Magrin S, Craxi A, Fiorentino G. Is autoimmune chronic active hepatitis a HCV related disease? J Hepatol 1991; 13: 128-31. 9 Haddad J, Deny P, Munz C, et al. Chronic lymphocytic sialadenitis characteristic of Sjfgren's syndrome in patients with HCV chronic hepatitis or cirrhosis. Lancet 1992; 339: 321-3. I0 Prentice LM, Phillips DIW, Sarsero D, Beever K, McLachlan S, Smith BR. Geographical distribution of subclinical autoimmune thyroid disease in Britain: A study using highly sensitive direct assays for autoantibodies to thyroglobulin and thyroid peroxidase. Acta Endocrinol 1990; 123: 493-8.
HEPAT 01168
Monitoring of interferon-a treatment in chronic active hepatitis B We present a case showing that the use of more sensitive serological tests for the diagnosis of the hepatitis B virus (HBV) might permit better monitoring of the antiviral activity of interferon-~ treatment. A 69-yearold male patient with severe chronic active hepatitis B and with signs of beginning fibrosis on liver biopsy was treated with interferon-~ (IntronA, 1.5 MU s.c.) thrice a week for 4 months. Serological markers of HBV, hepatitis C and hepatitis 6 were determined by commercially available enzyme immunoassays: anti-HBs (test A), anti-6 and antihepatitis C antibodies respectively by AUSAB, AntiDelta EIA and Anti-HCV EIA (c-i00-3) from Abbott Laboratories, U.S.A.; HSsAg, anti-HBc (results not shown), anti-HBs (test B (1)), HBe and anti-HBe by the corresponding enzyme immunoassays from Hoffman-La Roche, Switzerland. Semi-quantitative analysis of HBsAg was performed by counter-immunodiffusion. HBV-DNA was measured by a hybridization technique using a digoxigenin-labelled probe (2). HBV genome amplification was carried out by polymerase chain reaction (PCR) as previously described (3). Two oligonucleotide primers were used for hybridization to HBs sequences (5'-CTCTCAATTTTCTAGGGGGA-3' posi-
tion 267-286 and 5'-AGCAGCAGGATGAAGAGGAA-3' position 401-420 according to Tiollais (4)) together with a commercial amplification kit (Cetus). Amplified DNA was characterized by gel electrophoresis followed by transfer to a nylon membrane and hybridization with a HBV-specific probe labelled with digoxigenin (5). Test B and PCR were performed retrospectively on frozen serum samples. Results are shown in Table 1. The patient was negative for hepatitis C and ~ (results not shown). HBe/anti-HBe seroconversion was observed after the complete 4-month course of interferon-c~ treatment. HBs antigen remained positive though a slight decrease in titer was observed. HBV-DNA when measured by the conventional hybridization became negative after the third month. The retrospective use of PCR showed that HBV elimination was only partial and HBV-DNA remained detectable for 36 months. This is in contrast with the findings of Carman (6) who has shown by PCR the disappearance of viremia 13-18 months after the end of interferon treatment. Anti-HBs remained persistently negative with test A, but low titers were observed after 2 months with test B. These antibodies decreased slowly until complete disappearance at the
LETTERS TO THE EDITOR
246 TABLE I Serological markers of hepatitis B during interferon treatment Interferon treatment
H Bs-titer immuno-diffusion
H Be-Ag
Anti-HBe
Before During (month 1) During (month 2) During (month 3) During (month 4) After (month 3) After (month 6) After (month 12) After (month 16) After (month 18) After (month 24) After (month 36)
I/32 1/16 1/16 1/16 1/8 1/8 I/8 1/16 1/8 1/16 1/8 1/4
pos. pos. pos. pos. pos. pos. neg. neg. neg. neg. neg. neg.
neg. neg. neg. neg. neg. pos. pos. pos. pos. pos. pos. pos.
(IU/ml)
HBV-DNA (pg/ml)
HBV PCR a
325 225 192 188 147 normal normal normal normal normal normal normal
1000 400 80 40 0 0 0 0 3 0 0 0
ND ND ND ++++ ++++ ++++ ND +++ ND + + + + + +
ALAT
Anti-HBs Test A
Test B
0 0 ND 0 0 0 0 ND ND ND ND ND
ND 21 17 ND ND 7 5 6 ND I neg. neg.
" Results by visual evaluation of the spot colour intensity: + + + + = very strongly positive, + + + =strongly positivie, + + =positive, - = negative. N D = n o t done.
end of the treatment, indicating that they were only temporarily induced. These findings could be interpreted as the beginning of an immune response potentially capable of completely eliminating HBV and that this response was abrogated after the (too early?) end of the treatment. The prospective use of these tests should have lead us to continue interferon for a longer period and/or with a higher dosage until the possible complete HBV elimination. Thus interferon-or treatment induced a substantial inhibition of HBV replication and seroconversion from HBe to anti-HBe. This was to the benefit of the patient whose liver enzyme values returned to normal and who
experienced a considerable improvement in quality of life. Liver histology was not repeated. We conclude that a battery of sensitive serological tests would permit a better and more individual adaptation of the dose and the duration of interferon-or treatment in chronic active hepatitis B. The case presented took part in the Swiss multicenter study for the treatment of chronic hepatitis B run by the Swiss Association for the Study of the Liver.
References
for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J Clin Microbiol 1989; 27(9): 1930-3. 4 Tiollais P, Charnay P, Vyas GN. Biology of hepatitis B virus. Science 1981; 213: 406-I I. 5 Guo K J, Bowden DS. Digoxigenin-labeled probes for the detection of hepatitis B virus DNA in serum. J Clin Microbiol 1991; 29: 506-9. 6 Carman WF, Dourakis S, Karayiannis P, Crossey M, Drobner R, Thomas HC. Incidence of hepatitis B viraemia, detected using the polymerase chain reaction, after successful therapy of hepatitis B virus carriers with interferon-alpha. J Med Virol 1991; 34:114-8.
1 Lavanchy D, Peitrequin R, Vu DH, Frei PC. Measurement of antiHBs production after vaccination: comparison of results obtained by two different immunoassays. In: Coursaget P, Tong M J, eds. Progress in Hepatitis B Immunization. John Libbey/INSERM, 1990; 194: 73-7. 2 Seibl R, Holtke H J, Ruger R, et al. Non-radioactive labeling and detection of nucleic acids. III. Applications of the digoxigenin system. Biol Chem Hoppe Seyler 1990; 371: 939-51. 3 Kaneko S, Feinstone SM, Miller RH. Rapid and sensitive method
D..Lavanchya, C. Mayerat a, P.C. FreP and A.L. Blum b °Division of Immunology and Allergy and bDivision of Gastroenterology, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland
HEPAT 01169
Function of partial liver allografts after heterotopic transplantation In the field of liver transplantation both heterotopic grafting and the regular use of clearance studies in the follow-up of patients are far from commonplace. We routinely performed indocyanine green (ICG) and anti-
pyrine (AP) elimination tests in 10 patients who received heterotopic partial liver allografts because of end-stage chronic liver disease (1). Four of these patients had chronic hepatitis-B virus infections, two with f-virus
