- Email: [email protected]
Lack of relation between viral hepatitis and autoimmune liver diseases
$37
Viral hepatitis; it!fections, diagnostic procedures, therapy I P1 C1/57 I
I P1 Cl/59 I
AUTOANTIBODY (AA) PROFILE IN CHH.DREN WITH CHRONIC
IDENTIFICATION OF THE 35-kDa PRES1-BINDING PROTEIN AS GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE: INVOLVEMENT IN THE INFECTION PROCESS OF HUMAN HEPATOCYTES WITH HEPATITIS B VIRUS
HEPATITIS B VIRUS (HBV) INFECTION TREATED WITH ALPHA INTERFERON (a-IFN) GV GregorioI, J Jones~, A Vegnente3, F Bortolotti4, G Mieli-Vergani 2, D Vergani I. IDept of Immunology & R2hild Health, King's College School of Medicine and Dentistry, London SF_.5 9PJ; 3Dept of Paadiatrics, Universita' di Napoli; 4Inst. Medicino Clinica, Universita di Padova, Italy. A potentially serious side effect of o-IFN is the induction of autoimmunity. To determine whether a-IFN induces autoantibody (AA) formation, we have investigated the presence of nuclear (ANA), smooth muscle (ASM), liver-kidney microsomal (I..KM-1), mitochondrial (A.MA), liver eytosol antigen (LCA), gastric parietal cell (GPC), thyroid microsomai (TMA), thyroglobulin (TGA), extractable nuclear antigen (ENA) AA, using standard techniques, in the sera of 50 children (median age:7 (2-17) yrs) with chronic HBV infection before, during and up to 2 yrs after treatment with either 4 months lymphoblastoid (n=44) or 6 months recombinant a-IFN (n=6). 20 were given prednlsolone for 4 wks before ~-IFN (grp A) and 30 were not (grp B). cr-IFN dose was 5 MU/m2 3x/week. All were HBeAg +ve for at least I yr and HBV-DNA + r e before oe-IFN; 34 had chronic active hepatitis (CAH), 9 ehrunie persistent hepatitis (CPH) and 7 CAH/CPH. 7 (35%) in grp A and 13 (43%) in grp B were + r e for at least 1 AA before o-IFN: 8 (4 in grp A) had ANA (titre range: 1/10-1/I60), 7 (2 in grp A) SMA (1/10-1/40) and 8 (2 in grp A) GPC (1/10-1/40). Of these, 15 (5 in grp A) remained AA+ve during and after c~-IFN with an increase in a one-dilution ANA titre in only 1 from each grp (grp A:l/40 to 1/80, grp B:l/40 to 1/80) whilst 5 (2 in grp A) lost the AA during or after a-IFN, including I who had a temporary increase in ANA (1/40 to 1/80). 6 (3 in grp A), AA-ve before a-IFN, became transiently +ve at 1/10 during ct-IFN (4 SMA, 1 ANA, l GPC). 1 in grp B became ?LNA and GPC (1/10) + r e during and remained +ve 24 months after ctIFN. 9 (2 in grp A) became +ve at 1/10 (5 GPC, 3 SMA, 1 ANA) from 6-24 months after ex-IFN. Presence of AA was not associated with biochemical nor clinical deterioration of liver disease. There was no correlation between AA positivity, steroid pretreatment and response to a-IFN, a-IFN can induce low titre AA in children with chronic HBV infection but is not associated with clinically significant autoimmunity.
H. Mab=t , F. Caoel*• C. Vons , S. Dubanche~;*= D. Franco**. M.-A. Petit*. INSERM Unit 131" and Surgical Liver Un=t , Hbpital A.-B~cl~re, Clamart, France. Previously, we have demonstrated the presence of a 3S-kDa preSl-specific binding protein (preS1-BP3S) at the membrane of human hepatoma cells (HepG2). In this study, we identified this protein by partial amino acid sequence analysis of a preparatively isolated 35-kDa protein, obtained from extracts of HepG2 cells or normal human hepatocytes (NH-Hep) in primary cultures, by continuous elution electrophoresis. This preS1-BP3S was found to be glyceraldehyde-3-phosphate dehydrogenase (GAPD), a key enzyme in glycolysis, in both cases. We showed significant GAPD-binding activity (up to 1-0.1 rtg/ml) of native HBV particles (three different isolates) which led to productive infection of NH-Hep, but not for one non infectious HBV isolate (having low HBV DNA content). Surprisingly, GAPD did not inhibit binding of the preS1 region of HBV to intact NH-Hep. GAPD was then analyzed for its capability to interfere with HBV infection of NH-Hep. Intriguingly, presence of GAPD during infection: (i) greatly increased production of viral DNA into cells, (ii) reduced (~,S0%) secretion of HBV surface antigens and (iii) improved HBeAg's into the medium 5 days after infection. Our findings indicate that GAPD might be involved in the HBV infection process, independently of its glycolytic activity, perhaps as a protein kinase associated with the membranes.
I P1 Cl/58 I
I P1 C1/60 I
CHARACTERIZATION OF THE PRIMING OF REVERSE TRANSCRIPTION IN DUCK HEPATITIS B VIRUS (DHBV): A N E W T A R G E T FOR A N T I V I R A L T H E R A P Y ~ 1 , 2 . C. T r t o o 2. O. H a n t z 2. C. See~er 1. Fox ChaseC a n c e r Center, Philadelphia 1 and I N S E R M U271, Lyon 2. H e p a t i t i s B v i r u s replicates its D N A g e n o m e via r e v e r s e transcription of a pregenomie RNA. W e have used an in vitro translation reaction for the expression of an enzymatically active D H B V p o l y m e r a s e to c h a r a c t e r i z e the p r i m e r of r e v e r s e transcription. Our results demonstrated that, unlike all the other k n o w n reverse transcriptases, the D H B V p o l y m e r a s e uses a conserved tyrosine residue as a primer for D N A synthesis. Viral D N A synthesis is initiated by the formation of a covalent bond between the polymerase and dGMP, followed by the addition of T - A - A in a template d e p e n d e n t m a n n e r (Zoulim and Seeger, J.Virol. 68 : 6-13). W e have tested different polymerase inhibitors for their efficacy to inhibit the p r i m i n g o f D H B V replication in an in vitro asay. W e f o u n d that the p y r o p h o s p h a t e analog, PFA, as well as nucleotide triphosphate analogs, ddGTP, (+) carbovir-TP, (-) carbovir-TP inhibit D N A synthesis at different extent. H o w e v e r these compounds have no efect on the priming.reaction in our in vitro conditions. Carbocyclic 2'-deoxyguanosme (2'-CDG)-TP, w h i c h c o n t a i n s an intact 3'OH, was the only analog w h i c h exhibits a very potent inhibitory effect on the priming reaction. T h i s in vitro effect m a y explain the unusual antiviral activity of 2 ' - C D G observed in the duck model. Furthermore G T P had no inhibitory effect on the incorporation of 3 2 p - d G T P and C D G T P gave a similar inhibition to that by cold dGTP. Our data obtained with the inhibition of the enzymatic activity o f the D H B V p o l y m e r a s e c o n f i r m that the p r i m i n g of D N A synthesis is b i o c h e m i c a l l y distinct from D N A elongation and m a y therefore represent a new target for antiviral therapy.
LACK OF RELATION BETWEEN VIRAL HEPATITIS AND AUTOIMMUNE LIVER DISEASES. A.W. Loh~, G. Gerken, H. Mohr, A. Grolle, H. LObr, U. Treichel, H.P. Dienes*, K.H. Meyer zum Btischenfelde. I. Dept. of Met. and *Pathol., Joh.Gutenberg-Univ.; Malnz, FRG Infections with hepatitis viruses A, B or C have been considered a possible trigger for autoimmune liver diseases. In particular the hepatitis C virus has recently been considered to be associated with autoimmune liver diseases, mostly with autoimmune hepatitis. We have studied the relationship of hepatitis A, B or C virus infection and autoimmune liver diseases in 352 patients with autoimmune liver disease (154 with autoimmune hepatitis, 25 with primary sclerosing cholangitis and 173 with primary biliary cirrhosis), 307 of them under the care of our department. 27 of these 352 patients tested positive in the second generation anti-HCV ELISA, but only 4 patients (1 with autoimmune hepatitis, 1 with primary sclerosing cholangitis and 2 with primary biliary cirrhosis) were positive in confirmatory anti-HCV assays, and only in these could HCV-RNA be isolated. We also studied 201 consecutive patients with hepatitis C, 38 of whom (19 %) had significant titles of autoantibodies, and 306 with hepatitis B, 42 of whom (14%) had autoantibodies. LKM autoantibody was found in only 1 of these 201 HCV patients, but of 22 LKM-positive patients 9 were suffering from hepatitis C virus infection and 13 from autoimmune hepatitis. Distinction between autoimmune hepatitis and viral hepatitis C both in LK_M-positive patients and in patients with other autoantibodies could be made reliably on clinical and laboratory grounds, and was conf'umed by hepatitis C virus serology. Evidence of past or present hepatitis B virus and past hepatitis A virus infection was most common in the hepatitis C virus patients and least common in autoimmune hepatitis• Conclusion: These data show that a link between hepatitis A, B or C virus infection and autoimmune liver diseases is very unlikely. Although false positive anti-HCV ELISA results are found in about 5% of patients with autoimmune liver diseases, true HCV-infection is a rare event. Autoantibodies can be frequently found in hepatitis C as well as in hepatitis B and are therefore only of tittle help in the differential diagnosis. Viral and autoimmune liver disease are nonetheless very distinct entities.
Viral hepatitis; it!fections, diagnostic procedures, therapy I P1 C1/57 I
I P1 Cl/59 I
AUTOANTIBODY (AA) PROFILE IN CHH.DREN WITH CHRONIC
IDENTIFICATION OF THE 35-kDa PRES1-BINDING PROTEIN AS GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE: INVOLVEMENT IN THE INFECTION PROCESS OF HUMAN HEPATOCYTES WITH HEPATITIS B VIRUS
HEPATITIS B VIRUS (HBV) INFECTION TREATED WITH ALPHA INTERFERON (a-IFN) GV GregorioI, J Jones~, A Vegnente3, F Bortolotti4, G Mieli-Vergani 2, D Vergani I. IDept of Immunology & R2hild Health, King's College School of Medicine and Dentistry, London SF_.5 9PJ; 3Dept of Paadiatrics, Universita' di Napoli; 4Inst. Medicino Clinica, Universita di Padova, Italy. A potentially serious side effect of o-IFN is the induction of autoimmunity. To determine whether a-IFN induces autoantibody (AA) formation, we have investigated the presence of nuclear (ANA), smooth muscle (ASM), liver-kidney microsomal (I..KM-1), mitochondrial (A.MA), liver eytosol antigen (LCA), gastric parietal cell (GPC), thyroid microsomai (TMA), thyroglobulin (TGA), extractable nuclear antigen (ENA) AA, using standard techniques, in the sera of 50 children (median age:7 (2-17) yrs) with chronic HBV infection before, during and up to 2 yrs after treatment with either 4 months lymphoblastoid (n=44) or 6 months recombinant a-IFN (n=6). 20 were given prednlsolone for 4 wks before ~-IFN (grp A) and 30 were not (grp B). cr-IFN dose was 5 MU/m2 3x/week. All were HBeAg +ve for at least I yr and HBV-DNA + r e before oe-IFN; 34 had chronic active hepatitis (CAH), 9 ehrunie persistent hepatitis (CPH) and 7 CAH/CPH. 7 (35%) in grp A and 13 (43%) in grp B were + r e for at least 1 AA before o-IFN: 8 (4 in grp A) had ANA (titre range: 1/10-1/I60), 7 (2 in grp A) SMA (1/10-1/40) and 8 (2 in grp A) GPC (1/10-1/40). Of these, 15 (5 in grp A) remained AA+ve during and after c~-IFN with an increase in a one-dilution ANA titre in only 1 from each grp (grp A:l/40 to 1/80, grp B:l/40 to 1/80) whilst 5 (2 in grp A) lost the AA during or after a-IFN, including I who had a temporary increase in ANA (1/40 to 1/80). 6 (3 in grp A), AA-ve before a-IFN, became transiently +ve at 1/10 during ct-IFN (4 SMA, 1 ANA, l GPC). 1 in grp B became ?LNA and GPC (1/10) + r e during and remained +ve 24 months after ctIFN. 9 (2 in grp A) became +ve at 1/10 (5 GPC, 3 SMA, 1 ANA) from 6-24 months after ex-IFN. Presence of AA was not associated with biochemical nor clinical deterioration of liver disease. There was no correlation between AA positivity, steroid pretreatment and response to a-IFN, a-IFN can induce low titre AA in children with chronic HBV infection but is not associated with clinically significant autoimmunity.
H. Mab=t , F. Caoel*• C. Vons , S. Dubanche~;*= D. Franco**. M.-A. Petit*. INSERM Unit 131" and Surgical Liver Un=t , Hbpital A.-B~cl~re, Clamart, France. Previously, we have demonstrated the presence of a 3S-kDa preSl-specific binding protein (preS1-BP3S) at the membrane of human hepatoma cells (HepG2). In this study, we identified this protein by partial amino acid sequence analysis of a preparatively isolated 35-kDa protein, obtained from extracts of HepG2 cells or normal human hepatocytes (NH-Hep) in primary cultures, by continuous elution electrophoresis. This preS1-BP3S was found to be glyceraldehyde-3-phosphate dehydrogenase (GAPD), a key enzyme in glycolysis, in both cases. We showed significant GAPD-binding activity (up to 1-0.1 rtg/ml) of native HBV particles (three different isolates) which led to productive infection of NH-Hep, but not for one non infectious HBV isolate (having low HBV DNA content). Surprisingly, GAPD did not inhibit binding of the preS1 region of HBV to intact NH-Hep. GAPD was then analyzed for its capability to interfere with HBV infection of NH-Hep. Intriguingly, presence of GAPD during infection: (i) greatly increased production of viral DNA into cells, (ii) reduced (~,S0%) secretion of HBV surface antigens and (iii) improved HBeAg's into the medium 5 days after infection. Our findings indicate that GAPD might be involved in the HBV infection process, independently of its glycolytic activity, perhaps as a protein kinase associated with the membranes.
I P1 Cl/58 I
I P1 C1/60 I
CHARACTERIZATION OF THE PRIMING OF REVERSE TRANSCRIPTION IN DUCK HEPATITIS B VIRUS (DHBV): A N E W T A R G E T FOR A N T I V I R A L T H E R A P Y ~ 1 , 2 . C. T r t o o 2. O. H a n t z 2. C. See~er 1. Fox ChaseC a n c e r Center, Philadelphia 1 and I N S E R M U271, Lyon 2. H e p a t i t i s B v i r u s replicates its D N A g e n o m e via r e v e r s e transcription of a pregenomie RNA. W e have used an in vitro translation reaction for the expression of an enzymatically active D H B V p o l y m e r a s e to c h a r a c t e r i z e the p r i m e r of r e v e r s e transcription. Our results demonstrated that, unlike all the other k n o w n reverse transcriptases, the D H B V p o l y m e r a s e uses a conserved tyrosine residue as a primer for D N A synthesis. Viral D N A synthesis is initiated by the formation of a covalent bond between the polymerase and dGMP, followed by the addition of T - A - A in a template d e p e n d e n t m a n n e r (Zoulim and Seeger, J.Virol. 68 : 6-13). W e have tested different polymerase inhibitors for their efficacy to inhibit the p r i m i n g o f D H B V replication in an in vitro asay. W e f o u n d that the p y r o p h o s p h a t e analog, PFA, as well as nucleotide triphosphate analogs, ddGTP, (+) carbovir-TP, (-) carbovir-TP inhibit D N A synthesis at different extent. H o w e v e r these compounds have no efect on the priming.reaction in our in vitro conditions. Carbocyclic 2'-deoxyguanosme (2'-CDG)-TP, w h i c h c o n t a i n s an intact 3'OH, was the only analog w h i c h exhibits a very potent inhibitory effect on the priming reaction. T h i s in vitro effect m a y explain the unusual antiviral activity of 2 ' - C D G observed in the duck model. Furthermore G T P had no inhibitory effect on the incorporation of 3 2 p - d G T P and C D G T P gave a similar inhibition to that by cold dGTP. Our data obtained with the inhibition of the enzymatic activity o f the D H B V p o l y m e r a s e c o n f i r m that the p r i m i n g of D N A synthesis is b i o c h e m i c a l l y distinct from D N A elongation and m a y therefore represent a new target for antiviral therapy.
LACK OF RELATION BETWEEN VIRAL HEPATITIS AND AUTOIMMUNE LIVER DISEASES. A.W. Loh~, G. Gerken, H. Mohr, A. Grolle, H. LObr, U. Treichel, H.P. Dienes*, K.H. Meyer zum Btischenfelde. I. Dept. of Met. and *Pathol., Joh.Gutenberg-Univ.; Malnz, FRG Infections with hepatitis viruses A, B or C have been considered a possible trigger for autoimmune liver diseases. In particular the hepatitis C virus has recently been considered to be associated with autoimmune liver diseases, mostly with autoimmune hepatitis. We have studied the relationship of hepatitis A, B or C virus infection and autoimmune liver diseases in 352 patients with autoimmune liver disease (154 with autoimmune hepatitis, 25 with primary sclerosing cholangitis and 173 with primary biliary cirrhosis), 307 of them under the care of our department. 27 of these 352 patients tested positive in the second generation anti-HCV ELISA, but only 4 patients (1 with autoimmune hepatitis, 1 with primary sclerosing cholangitis and 2 with primary biliary cirrhosis) were positive in confirmatory anti-HCV assays, and only in these could HCV-RNA be isolated. We also studied 201 consecutive patients with hepatitis C, 38 of whom (19 %) had significant titles of autoantibodies, and 306 with hepatitis B, 42 of whom (14%) had autoantibodies. LKM autoantibody was found in only 1 of these 201 HCV patients, but of 22 LKM-positive patients 9 were suffering from hepatitis C virus infection and 13 from autoimmune hepatitis. Distinction between autoimmune hepatitis and viral hepatitis C both in LK_M-positive patients and in patients with other autoantibodies could be made reliably on clinical and laboratory grounds, and was conf'umed by hepatitis C virus serology. Evidence of past or present hepatitis B virus and past hepatitis A virus infection was most common in the hepatitis C virus patients and least common in autoimmune hepatitis• Conclusion: These data show that a link between hepatitis A, B or C virus infection and autoimmune liver diseases is very unlikely. Although false positive anti-HCV ELISA results are found in about 5% of patients with autoimmune liver diseases, true HCV-infection is a rare event. Autoantibodies can be frequently found in hepatitis C as well as in hepatitis B and are therefore only of tittle help in the differential diagnosis. Viral and autoimmune liver disease are nonetheless very distinct entities.