- Email: [email protected]
Virus hepatitis C typing: Comparison between genotypes and serotypes in patients with chronic infection
396A
AASLD ABSTRACTS
1157
ALPHA-IFN TREATMENT IN NCV CAH: RELATIONSHIP WITH THE PRESENCE OF AUTOIMMUNE MARKERS. A Biglino, G C a r i t i , D P i n i , M Depaoli, P G i o a n n i n i . Institute o f I n f e c t i o u s Diseases, U n i v e r s i t y of Turin, I t a l y .
1 158
'VIRUS HEPATITIS C T~(PING : COMPARISON G E N O T Y P E S AND S E R O T Y P E S IN PATIENTS WITH
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(pos/tot) .
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ANA Anti:DNA SMA AMA Anti-HCV .
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(pos/tot) .
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55/92 0/81 36/67 2/31 IgM 67/110 .
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1159 ALTERED HEPATOCYTE TRANSPORT IN TWO RODENT MODELS OF THE ACUTE PHASE RESPONSE. U. Bolder, S. Mnsunuri, C. Schteingart, LR. Hagey, E. Frick, HT. Ton-Nu, M. Ookhtens 1, AF. Hoflnatm. University California, Dept. Medicine, San Diego, CA. tUSC School of Medicine, Los Angeles, CA. Conditions known to induce an acute phase response of the hepatoeyte such as inflammation or postsurgical sepsis are often associated with cholestasis. We have performed experiments aimed at quantifying hepatoeyte transport function for conjugated bile acids in two rodent models of acute phase response. Methods: In a sterile abscess medel (SA), turpentine (2.5 mi/100g) was injected intramuacularly. In a sepsis medea, E. coli lipepolysaccarids (LPS) (0.1 mg/100g) was injected intraperitoneally. Control animals received sham injections with saline. Transport experiments were performed at 18, 36 and 72 h in SA bilinry fistula rats, and at 12 h in isolated perfused rat livers (IPRL) after acute phase induction by LpS. T ~ values for two omjugated bile acids (BA), cholyltanrine (C-tau) a non-toxic BA and chenodenxycholyltaurine (CDC-tau), a cholestatic BA were determined by measuring biliaty sacretien alter stepwise increments in the rate of bile acid infusion. Bile flow, bilimy bile acids, electrolytes, HCO 3" and glutathione were also measured. Results: TE values for conjugated bile acids were decreased with both models. This effect was more pronounced if expressed in relation to the greater liver weight in SAanimals. In the SA model, bile acid independent flow (BAIF) was significantly higher compared to rxmtrols, partly mediated by an increased glutathione output. In the LPS model T ~ values for the two bile acids were markedly reduced. In addition BAIF was also diminished in LPS treated animals, Conh'ol 18h(SA) 36h(SA) 72h(SA) !Con~'ol 12h (J.,PS) C-Tan ~mol/kg rain 18.16 16.35 15.27" 14.42" 7.1 2.8:[: CDC-Tan praol/kg rain 4.73 4.28 4.1" 4.73 1.8 0.7:[: BAIF ~d/kg rain 79.3 124.9"-[: Ill.6~: 86.2 33.5 19.7:[: GSHtot nmol/kg rain 107:[:: 203:~ 158::[: 272~ ND ND Liver weight %BW 3.59 4.31:[: 4.41~ 4.02.1: 3.41 3.94 *P<0.05,:t.P<0.0lto control,~ K L Condnsian: Decreased hepatocyte transport function of bile acids in the rat occurs with an acute phase response. With sterile inflammation an increase of biliary glutathiooe output countered cholestasis by augmentation of BAlE With a sepsis model, bile acid transport and BAIF were markedly reduced. Further studies are required to elucidate whether defective transport is either at the canalicular level, which should cause hepatotoxicity, or on the basolateral level which should cause hepatoprotection.
Recent datasuggast that HCV genotypes may differ in their biologic effect, such as response to interferon therapy or severity of disease. A new typing assay (Murex) based ~m the use of type specific synthetic peptides from the NS4 region of HCV has been recently proposed. Aim of this study was to compare genotyping and serotyping. Methods : Sere olyuained from 15 patients with hepatitis C chronic ipfection were analyzed. Their positive anti HCV status was proven by EIA (Abbott or Ortho) RIBATM (Oftho) 3.0 assays. Genotypes were determined by analysis of PeR products generoted by qualitative AmplicorTM HCV RNA assay (Roche) on a reverse hybridization format (INNO LiPA TM HCV, Innogenetics). The serotyping kit, an EIA-based assay developped by Murex, detects antibodies raised against serotype-specific epitopes of HCV NS4 protein. At the moment, this method does not allow distinction of subtypes nor determination of types other than 1, 2 or 3. Besides the 15 chronic HCV infected patients, as a high prevalence of anti-HCV antibodies have been reported in hemodialyzed patients, we tried to perform detection of type-specific antibodies in some patients undergoing hemodialysts. Results : Gcnotype was determined in the 15 patients tested. Suhtyping was unsaceessfull for one patient having HCV genotype 1. Serotype-specific antibodies were detectedin 12 patients (80%), including the one having an indeterminate HCV subtype. Serotypes : 7 were deficedas type 1, om'respouding to four genotypas la, two lb and one indeterminatetype 1, respectively. The remaining determined serotypes were two type 2 (genotype 2a) and one type 3 (genotype 3a). The three indeterminate serotypes were one I;enotype 4 or 5, one lb, and one 3a, respectively. Preliminary results obtained in ,'~mtyping hemodialyzed patients were less successfall. DisFuanion : The serotypes were in concordance with genotypes in all patients typed. Them was no correlation between the presence or absence of specific antibodies raised against the NS4 epitopes included in the RIBATM 3.0 assay and in those detected in the semtyping assay. Difficulties encountered in serotyping hemodialyzed patients were prolmhly causedby their immunological status i.e. a low antibodies response. Moreover the average signal to noise ratio measured in EIA 3.0 assays performed in these hemodialyzed patients appeared to be lower than for the 15 other patients. Conclusion : Even if the results are in good concctdence with genotyping, the use of semtyping does not yet allow discrimination hetwcen subtypes. If subtyping were possible, this could raise the question of using or not a more informative semtyping test rather than the RIBATM assay, although the serotyping assay is not yet as much sensible as the RIBATM assay. Regarding hemodialymdpatients, studies are in process to improve the sensitivity of the semtyping test in this specific population.
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Anti-SSA/Ro 0/16 ~nti-LKM1 1/76 ~nti-GOR 23/55 C r y o g l o b u l i n e m i a 12/41 Anti-IFN 5/27 .
BETWEEN CHRONIC
]INFECTION. M B o ~ i , M Loevct. Centre Hospitalier, Meanx. France.
Auteimmune markers (AM) are f r e q u e n t l y associated w i t h HCV CAH, The aim of t h i s study was te i n v e s t i g a t e the r e l a t i o n s h i p between AM and sustained response to t r e a t m e n t w i t h alpha-IFN in p a t i e n t s w i t h HCV CAH. 110 p a t i e n t s (74 males; 36 females) with h i s t o l o g i c a l l y documented HCV CAH w e r e s t u d i e d . A l l w e r e HCV-RNA o o s i t i v e . The f o l l o w i n g AM were considered f o r a n a l y s i s : ~NA, anti-DNA a n t i bodies, 8MA, AMA, anxi-SSA/Ro a n t i b o d i e s , a n t i LKM1 a n t i b o d i e s , anti-GOR a n t i b o d i e s , c r y o g l o b u l i nemia, a n t i core HCV-IgM and a n t i - I F N a n t i b o d i e s . AIDha-IFN was a d m i n i s t e r e d as an i.m. i n j e c t i o n at a oose of 3 MU three times/week fo - 24 weeks. The absence of anti-GOR and of a n t i core HCV-IgM were found ~o me s i g n i f i c a n t l y associated with s u s t a i ned response to aloha-IFN ( r e s p e c t i v e l y , P=0.014 ano P=O.O00 by Chi=so. t e s t ) . No other r e l a t i o n s h i p s between AM and t r e a t m e n t w i t h alpha-IFN w e r e oDserveo° F i n a l l y , c i r c u l a t i n g a n t i b o d i e s Eo IFN did not seem to i n f l u e n c e the response te IFN t r e a t m e n t . Results
HEPATOLOGY October 1995
1160
ASSESSMENT OF ISOLATED RAT HEPATOCYTE SUSPENSIONS AS A MODEL SYSTEM TO EXAMINE THE EFFECT OF P-GLYCOPROTEIN INI-HBITORS ON DAUNORUBICIN AND ETOPOSIDE HEPATOBILIARY DISPOSITION. CL Booth, K R Brouwer*, and K L R Brouwer. Division of Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, and *Department of Drug Metabolism, Glaxo Wellcome, Research Triangle Park, NC. P-Glyooprotcin (P-G), the multidrug resistance gene product, is localized on the canaliealar domain in rat hepatocytes. Previous studies suggest that P-G function and expression may be limited in primary rut hepatoeyte cultures during the first 48 hr. The purpose of the present study was to test the hypothesis that freshly isolated hepatocytes in suspension could aoeumulate P-G substrates, and that P-G inhibitors could modulate substrute egress. Hepatoeytes were isolated by collagonase perfusion, viability was assessed by trypan blue exclusion, and data were corrected for the adherent id volume as determined by hepatoey~c incubation with [carboxyl]dextran. The effect of hepatocyte pre-incubation with the P-G inhibitors rupamil (10 [aM) or GF120918 (~0 nM) on the uptake of P-G substrates -daunorub~cin (1000 ~M) or ['H]-etoposide (50 ~M) was determined. In addition, [°H]-daunorubicin (1000 p.M) egress in the presenc~ of verapamil (10 - 20 ~tM) or GF120918 (200 - 400 nM) was evaluated. [ H]Daunorubicin was taken up readily by hepatocytes (0.205 + 0.048 pmol/mg/sec; 30-120 sec) and to a significant extent (48.7 _+ 3.3% after a 30min incubation). Pre-incubation with verapamil or GF120918 did not alter the initial rate of uptake (0.221 + 0.070 pmol/mg/soe or 0.188 + 0.021 pmol/mg/se¢, respectively) or the intraoellular accumulation at 30 min (50.5 _+ 1.8 % or 50.0 + 4.4 %, respectively). The initial rute of etoposide uptake (3.95 + 0.73 pmol/mg/sec; 15-90 sec) was not altered after pre-incubation with GF120918 (4.58 -+ 1.02 pmol/mg/sec). After a 30-min incubation, intraoellular etoposide was only 3.2%, and was not altered by GF120918 (3.5%). The initial rate of daanorubicin egress (0.247 + 0.080 pmol/mg/sec; 15-90 sec) was not affected by the presence of either verupamil (0.232 _+ 0.071 pmol/mg/sec) or GF120918 (0.286 _+0.072 pmol]mg/sac). Isolated rut hepatoeyte suspensions can accumulate model P43 substrates, but egress of these substrates does not appear to be altered by P-G inhibitors. This study demonstrates that the freshly isolated hapatoeyte suspension is not an appropriate model system to examioe the effects o f P-G modulation on bepatobiliary drug disposition. Funded by N1H grant GM41935 and Glaxo Wellcome.
AASLD ABSTRACTS
1157
ALPHA-IFN TREATMENT IN NCV CAH: RELATIONSHIP WITH THE PRESENCE OF AUTOIMMUNE MARKERS. A Biglino, G C a r i t i , D P i n i , M Depaoli, P G i o a n n i n i . Institute o f I n f e c t i o u s Diseases, U n i v e r s i t y of Turin, I t a l y .
1 158
'VIRUS HEPATITIS C T~(PING : COMPARISON G E N O T Y P E S AND S E R O T Y P E S IN PATIENTS WITH
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(pos/tot) .
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ANA Anti:DNA SMA AMA Anti-HCV .
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55/92 0/81 36/67 2/31 IgM 67/110 .
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1159 ALTERED HEPATOCYTE TRANSPORT IN TWO RODENT MODELS OF THE ACUTE PHASE RESPONSE. U. Bolder, S. Mnsunuri, C. Schteingart, LR. Hagey, E. Frick, HT. Ton-Nu, M. Ookhtens 1, AF. Hoflnatm. University California, Dept. Medicine, San Diego, CA. tUSC School of Medicine, Los Angeles, CA. Conditions known to induce an acute phase response of the hepatoeyte such as inflammation or postsurgical sepsis are often associated with cholestasis. We have performed experiments aimed at quantifying hepatoeyte transport function for conjugated bile acids in two rodent models of acute phase response. Methods: In a sterile abscess medel (SA), turpentine (2.5 mi/100g) was injected intramuacularly. In a sepsis medea, E. coli lipepolysaccarids (LPS) (0.1 mg/100g) was injected intraperitoneally. Control animals received sham injections with saline. Transport experiments were performed at 18, 36 and 72 h in SA bilinry fistula rats, and at 12 h in isolated perfused rat livers (IPRL) after acute phase induction by LpS. T ~ values for two omjugated bile acids (BA), cholyltanrine (C-tau) a non-toxic BA and chenodenxycholyltaurine (CDC-tau), a cholestatic BA were determined by measuring biliaty sacretien alter stepwise increments in the rate of bile acid infusion. Bile flow, bilimy bile acids, electrolytes, HCO 3" and glutathione were also measured. Results: TE values for conjugated bile acids were decreased with both models. This effect was more pronounced if expressed in relation to the greater liver weight in SAanimals. In the SA model, bile acid independent flow (BAIF) was significantly higher compared to rxmtrols, partly mediated by an increased glutathione output. In the LPS model T ~ values for the two bile acids were markedly reduced. In addition BAIF was also diminished in LPS treated animals, Conh'ol 18h(SA) 36h(SA) 72h(SA) !Con~'ol 12h (J.,PS) C-Tan ~mol/kg rain 18.16 16.35 15.27" 14.42" 7.1 2.8:[: CDC-Tan praol/kg rain 4.73 4.28 4.1" 4.73 1.8 0.7:[: BAIF ~d/kg rain 79.3 124.9"-[: Ill.6~: 86.2 33.5 19.7:[: GSHtot nmol/kg rain 107:[:: 203:~ 158::[: 272~ ND ND Liver weight %BW 3.59 4.31:[: 4.41~ 4.02.1: 3.41 3.94 *P<0.05,:t.P<0.0lto control,~ K L Condnsian: Decreased hepatocyte transport function of bile acids in the rat occurs with an acute phase response. With sterile inflammation an increase of biliary glutathiooe output countered cholestasis by augmentation of BAlE With a sepsis model, bile acid transport and BAIF were markedly reduced. Further studies are required to elucidate whether defective transport is either at the canalicular level, which should cause hepatotoxicity, or on the basolateral level which should cause hepatoprotection.
Recent datasuggast that HCV genotypes may differ in their biologic effect, such as response to interferon therapy or severity of disease. A new typing assay (Murex) based ~m the use of type specific synthetic peptides from the NS4 region of HCV has been recently proposed. Aim of this study was to compare genotyping and serotyping. Methods : Sere olyuained from 15 patients with hepatitis C chronic ipfection were analyzed. Their positive anti HCV status was proven by EIA (Abbott or Ortho) RIBATM (Oftho) 3.0 assays. Genotypes were determined by analysis of PeR products generoted by qualitative AmplicorTM HCV RNA assay (Roche) on a reverse hybridization format (INNO LiPA TM HCV, Innogenetics). The serotyping kit, an EIA-based assay developped by Murex, detects antibodies raised against serotype-specific epitopes of HCV NS4 protein. At the moment, this method does not allow distinction of subtypes nor determination of types other than 1, 2 or 3. Besides the 15 chronic HCV infected patients, as a high prevalence of anti-HCV antibodies have been reported in hemodialyzed patients, we tried to perform detection of type-specific antibodies in some patients undergoing hemodialysts. Results : Gcnotype was determined in the 15 patients tested. Suhtyping was unsaceessfull for one patient having HCV genotype 1. Serotype-specific antibodies were detectedin 12 patients (80%), including the one having an indeterminate HCV subtype. Serotypes : 7 were deficedas type 1, om'respouding to four genotypas la, two lb and one indeterminatetype 1, respectively. The remaining determined serotypes were two type 2 (genotype 2a) and one type 3 (genotype 3a). The three indeterminate serotypes were one I;enotype 4 or 5, one lb, and one 3a, respectively. Preliminary results obtained in ,'~mtyping hemodialyzed patients were less successfall. DisFuanion : The serotypes were in concordance with genotypes in all patients typed. Them was no correlation between the presence or absence of specific antibodies raised against the NS4 epitopes included in the RIBATM 3.0 assay and in those detected in the semtyping assay. Difficulties encountered in serotyping hemodialyzed patients were prolmhly causedby their immunological status i.e. a low antibodies response. Moreover the average signal to noise ratio measured in EIA 3.0 assays performed in these hemodialyzed patients appeared to be lower than for the 15 other patients. Conclusion : Even if the results are in good concctdence with genotyping, the use of semtyping does not yet allow discrimination hetwcen subtypes. If subtyping were possible, this could raise the question of using or not a more informative semtyping test rather than the RIBATM assay, although the serotyping assay is not yet as much sensible as the RIBATM assay. Regarding hemodialymdpatients, studies are in process to improve the sensitivity of the semtyping test in this specific population.
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Anti-SSA/Ro 0/16 ~nti-LKM1 1/76 ~nti-GOR 23/55 C r y o g l o b u l i n e m i a 12/41 Anti-IFN 5/27 .
BETWEEN CHRONIC
]INFECTION. M B o ~ i , M Loevct. Centre Hospitalier, Meanx. France.
Auteimmune markers (AM) are f r e q u e n t l y associated w i t h HCV CAH, The aim of t h i s study was te i n v e s t i g a t e the r e l a t i o n s h i p between AM and sustained response to t r e a t m e n t w i t h alpha-IFN in p a t i e n t s w i t h HCV CAH. 110 p a t i e n t s (74 males; 36 females) with h i s t o l o g i c a l l y documented HCV CAH w e r e s t u d i e d . A l l w e r e HCV-RNA o o s i t i v e . The f o l l o w i n g AM were considered f o r a n a l y s i s : ~NA, anti-DNA a n t i bodies, 8MA, AMA, anxi-SSA/Ro a n t i b o d i e s , a n t i LKM1 a n t i b o d i e s , anti-GOR a n t i b o d i e s , c r y o g l o b u l i nemia, a n t i core HCV-IgM and a n t i - I F N a n t i b o d i e s . AIDha-IFN was a d m i n i s t e r e d as an i.m. i n j e c t i o n at a oose of 3 MU three times/week fo - 24 weeks. The absence of anti-GOR and of a n t i core HCV-IgM were found ~o me s i g n i f i c a n t l y associated with s u s t a i ned response to aloha-IFN ( r e s p e c t i v e l y , P=0.014 ano P=O.O00 by Chi=so. t e s t ) . No other r e l a t i o n s h i p s between AM and t r e a t m e n t w i t h alpha-IFN w e r e oDserveo° F i n a l l y , c i r c u l a t i n g a n t i b o d i e s Eo IFN did not seem to i n f l u e n c e the response te IFN t r e a t m e n t . Results
HEPATOLOGY October 1995
1160
ASSESSMENT OF ISOLATED RAT HEPATOCYTE SUSPENSIONS AS A MODEL SYSTEM TO EXAMINE THE EFFECT OF P-GLYCOPROTEIN INI-HBITORS ON DAUNORUBICIN AND ETOPOSIDE HEPATOBILIARY DISPOSITION. CL Booth, K R Brouwer*, and K L R Brouwer. Division of Pharmaceutics, School of Pharmacy, University of North Carolina, Chapel Hill, and *Department of Drug Metabolism, Glaxo Wellcome, Research Triangle Park, NC. P-Glyooprotcin (P-G), the multidrug resistance gene product, is localized on the canaliealar domain in rat hepatocytes. Previous studies suggest that P-G function and expression may be limited in primary rut hepatoeyte cultures during the first 48 hr. The purpose of the present study was to test the hypothesis that freshly isolated hepatocytes in suspension could aoeumulate P-G substrates, and that P-G inhibitors could modulate substrute egress. Hepatoeytes were isolated by collagonase perfusion, viability was assessed by trypan blue exclusion, and data were corrected for the adherent id volume as determined by hepatoey~c incubation with [carboxyl]dextran. The effect of hepatocyte pre-incubation with the P-G inhibitors rupamil (10 [aM) or GF120918 (~0 nM) on the uptake of P-G substrates -daunorub~cin (1000 ~M) or ['H]-etoposide (50 ~M) was determined. In addition, [°H]-daunorubicin (1000 p.M) egress in the presenc~ of verapamil (10 - 20 ~tM) or GF120918 (200 - 400 nM) was evaluated. [ H]Daunorubicin was taken up readily by hepatocytes (0.205 + 0.048 pmol/mg/sec; 30-120 sec) and to a significant extent (48.7 _+ 3.3% after a 30min incubation). Pre-incubation with verapamil or GF120918 did not alter the initial rate of uptake (0.221 + 0.070 pmol/mg/soe or 0.188 + 0.021 pmol/mg/se¢, respectively) or the intraoellular accumulation at 30 min (50.5 _+ 1.8 % or 50.0 + 4.4 %, respectively). The initial rute of etoposide uptake (3.95 + 0.73 pmol/mg/sec; 15-90 sec) was not altered after pre-incubation with GF120918 (4.58 -+ 1.02 pmol/mg/sec). After a 30-min incubation, intraoellular etoposide was only 3.2%, and was not altered by GF120918 (3.5%). The initial rate of daanorubicin egress (0.247 + 0.080 pmol/mg/sec; 15-90 sec) was not affected by the presence of either verupamil (0.232 _+ 0.071 pmol/mg/sec) or GF120918 (0.286 _+0.072 pmol]mg/sac). Isolated rut hepatoeyte suspensions can accumulate model P43 substrates, but egress of these substrates does not appear to be altered by P-G inhibitors. This study demonstrates that the freshly isolated hapatoeyte suspension is not an appropriate model system to examioe the effects o f P-G modulation on bepatobiliary drug disposition. Funded by N1H grant GM41935 and Glaxo Wellcome.