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interferonα2a on peripheral blood mononuclear cells (PBMC) from chronic hepatitis C patients and healthy donors
104
Posters
I P/CO5/o43 I
EFFECTSOF AMANTADINE AND AMANTADINE/INTERFERONa2a PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) CHRONIC HEPATITIS C PATIENTS AND HEALTHY DONORS
ON FROM
J. Carrerio. Dept. of Hepatology, Fundaci6n Jimenez Dfaz, Madrid, Spain. Amantadine is a potent anti-influenza virus drug with therapeutic potential in chronic hepatitis C virus (HCV) infection. We have studied the effects of amantadine alone (2 PM), IFNaPa alone (1000 lU/ml) and the combination of the two drugs (both kindly provided by F. HoffmannLa Roche, Basel, Switzerland) in cultured PBMC isolated from 15 chronic hepatitis C patients and IO healthy blood donors. PBMC were cultured with or without mitogens (PHA plus LPS) and HCV antigens (core, NS3) for 7 days. Amantadine itself did not affect cell viability and had minor effects on the responses to mitogens by PBMC derived from HCV patients and healthy donors. Four HCV patients (27%) but none of the donors, had HCV antigen-specific proliferative responses. Amantadine suppressed these responses in all cases; this antiproliferative effect was similar to that produced by IFNa. All PBMC cultures from patients, but none from donors, were HCV RNA positive with or without mitogens. Amantadine alone or combined with IFNa reduced the HCV RNA concentration in PBMC by ,70%, as determined by a modification of AMPLICORTM HCV MONITOR assay. One (7%) two (13%) and three (20%) PBMC cultures became HCV RNA negative after treatment with amantadine alone, IFNa alone and the amantadineIFNa combination, respectively. The expression of the inducibleantiviral mediator 2’,5’-oligoadenylate (2-5A) synthetase was studied; in contrast to IFNa alone which increased significantly 2-5A activity (p
ANALYSIS OF THE HBV PRECORE AND ORF-X SEQUENCES IN anti-HBe POSITIVE PATIENTS WITH AND WITHOUT NORMAL ALT LEVELS
M. Cotonat. V. Carretio. Department of Hepatology, Fundaci6n Jimsnez Diaz and Fundacion para el Estudio de las Hepatitis Virales, Madrid, Spain. Serum samples from 20 antiHBe positive patients with and without normal alanine aminotransferase (ALT) levels were included in this study. All patients had serum hepatitis B virus (HBV) DNA only detectable by PCR. To investigate whether mutations in the nucleotide sequences of X and precore gene regions of HBV-DNA might be responsible for the difference in the activity disease and in the levels of viral replication, viral DNA was amplified by PCR using primers that encompassed precore and ORF-X regions and directly sequenced. Serum HBV-DNA was quantitated using the Amplicor HBV monitor test. The HBV-DNA concentration in patients with abnormal ALT levels was higher than in those with normal ALT ones. Furthermore, when all patients were considerated together, HBVDNA concentration correlate with the ALT levels (~~0.05). 72% of the patients had HBV-DNA harboring the 1896 precore stop mutation, and there was a negative correlation between the percentage of precore mutant genotype and the HBV-DNA concentration (p
HBV DNA INHIBITS PRODUCTION OF TGFP 1 WHICH IN TURN INHIBITS HBsAg PRODUCTION. J. M Alexander. University of Cambridge School of ‘Clinical Medicine, Cambridge, UK, CB2 2QQ. *Institue of Liver Studies, Kings College Hospital, London.
3 B.
TGF-beta 1 is a powerful immunomodulatory agent which can also promote fibrogenesis. We have studied the effect of active TGFB 1 on HBsAg production and in addition that of HBV DNA on TGFPl production. Experiments were performed using an Hep G2 cell line transfected with plasmid vector or plasmid vector containing a single copy of HBV DNA cut in the Pal gene, which permits structural protein synthesis but not viral replication. TGFB 1 induced a dose related reduction in HBsAg production (by ELISA) to a maximum 68% of baseline at Sng/ml of active TGFBl for 48 hours (p=O.Ol). There was no effect on HBeAg production. TGFB 1 production by Hep G2 cells (by ELISA) was reduced to 44% baseline in those cells transfected with HBV DNA compared to plasmid vector alone after 6 days culture (p-&001). Since the presence of Cyclosporine at 3000 @ml accentuated this difference and we have shown previously mat this compound increases HBsAg production markedly, we conclude that the effect on TGFB 1 may be mediated through HBsAg. Thus HBV DNA and TGFP 1 are mutually inhibitory. These data suggest that HBV may control, its own microenvironment to maximise HBsAg production.
COMPARISON BETWEEN HCVRNA AND El/E2 ANTIBODIES IN PATIENTS ON IFN TREATMENT la. G.-ens*. LCascavilla. T.SwtonioG, P.Conoscitore and A. Andriulli Gastr Div “CSS” IRCCS f&Giovanni R, $Infect.Dis. Inst. Univ of Bari ITALY, *INNOGENETICS, Ghent, BELGIUM In chronic HCV carriers a positive correlation between El and E2 Abs levels and viremia has been suggested. We analyzed HCVRNA levels and El and E2 Abs titers i n 89 HCVRNA +ve pts who underwent IFN therapy (3-6 MU TIW for 6 mo). Pts were subdivided in 38 long term responders (LTR), 33 non responders (NR) and 27 relapsers (REL).Serum samples were tested, at 0 , 2 and 6 mo on treatment, and at 6 mo during the f.u. Abs were quantified with a research ELISA using recombinant El and E2 antigens derived from HCV genotype Ib (INNOTEST HCVElAb and E2 Ab prototype version). HCVRNA was quantitatively determined by bDNA (Quantiplex HCVRNA 2.0). Results according with the different response to IFN are shown in the table: basal 2 El/RNA LTR 1.56~10~ 3.97 1.16 2.5 ,NR
3.31x105
5.09
6.14
1.5
2.87~10~ 4.50 5.21 1.5 PEL A positive correlation between HCVRNA and El and E2 basal levels was found. El more than E2 reflects circulating HCVRNA levels. El/HCVRNA ratio h i g her than 2 can be predictive of long term response.
Posters
I P/CO5/o43 I
EFFECTSOF AMANTADINE AND AMANTADINE/INTERFERONa2a PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) CHRONIC HEPATITIS C PATIENTS AND HEALTHY DONORS
ON FROM
J. Carrerio. Dept. of Hepatology, Fundaci6n Jimenez Dfaz, Madrid, Spain. Amantadine is a potent anti-influenza virus drug with therapeutic potential in chronic hepatitis C virus (HCV) infection. We have studied the effects of amantadine alone (2 PM), IFNaPa alone (1000 lU/ml) and the combination of the two drugs (both kindly provided by F. HoffmannLa Roche, Basel, Switzerland) in cultured PBMC isolated from 15 chronic hepatitis C patients and IO healthy blood donors. PBMC were cultured with or without mitogens (PHA plus LPS) and HCV antigens (core, NS3) for 7 days. Amantadine itself did not affect cell viability and had minor effects on the responses to mitogens by PBMC derived from HCV patients and healthy donors. Four HCV patients (27%) but none of the donors, had HCV antigen-specific proliferative responses. Amantadine suppressed these responses in all cases; this antiproliferative effect was similar to that produced by IFNa. All PBMC cultures from patients, but none from donors, were HCV RNA positive with or without mitogens. Amantadine alone or combined with IFNa reduced the HCV RNA concentration in PBMC by ,70%, as determined by a modification of AMPLICORTM HCV MONITOR assay. One (7%) two (13%) and three (20%) PBMC cultures became HCV RNA negative after treatment with amantadine alone, IFNa alone and the amantadineIFNa combination, respectively. The expression of the inducibleantiviral mediator 2’,5’-oligoadenylate (2-5A) synthetase was studied; in contrast to IFNa alone which increased significantly 2-5A activity (p
ANALYSIS OF THE HBV PRECORE AND ORF-X SEQUENCES IN anti-HBe POSITIVE PATIENTS WITH AND WITHOUT NORMAL ALT LEVELS
M. Cotonat. V. Carretio. Department of Hepatology, Fundaci6n Jimsnez Diaz and Fundacion para el Estudio de las Hepatitis Virales, Madrid, Spain. Serum samples from 20 antiHBe positive patients with and without normal alanine aminotransferase (ALT) levels were included in this study. All patients had serum hepatitis B virus (HBV) DNA only detectable by PCR. To investigate whether mutations in the nucleotide sequences of X and precore gene regions of HBV-DNA might be responsible for the difference in the activity disease and in the levels of viral replication, viral DNA was amplified by PCR using primers that encompassed precore and ORF-X regions and directly sequenced. Serum HBV-DNA was quantitated using the Amplicor HBV monitor test. The HBV-DNA concentration in patients with abnormal ALT levels was higher than in those with normal ALT ones. Furthermore, when all patients were considerated together, HBVDNA concentration correlate with the ALT levels (~~0.05). 72% of the patients had HBV-DNA harboring the 1896 precore stop mutation, and there was a negative correlation between the percentage of precore mutant genotype and the HBV-DNA concentration (p
HBV DNA INHIBITS PRODUCTION OF TGFP 1 WHICH IN TURN INHIBITS HBsAg PRODUCTION. J. M Alexander. University of Cambridge School of ‘Clinical Medicine, Cambridge, UK, CB2 2QQ. *Institue of Liver Studies, Kings College Hospital, London.
3 B.
TGF-beta 1 is a powerful immunomodulatory agent which can also promote fibrogenesis. We have studied the effect of active TGFB 1 on HBsAg production and in addition that of HBV DNA on TGFPl production. Experiments were performed using an Hep G2 cell line transfected with plasmid vector or plasmid vector containing a single copy of HBV DNA cut in the Pal gene, which permits structural protein synthesis but not viral replication. TGFB 1 induced a dose related reduction in HBsAg production (by ELISA) to a maximum 68% of baseline at Sng/ml of active TGFBl for 48 hours (p=O.Ol). There was no effect on HBeAg production. TGFB 1 production by Hep G2 cells (by ELISA) was reduced to 44% baseline in those cells transfected with HBV DNA compared to plasmid vector alone after 6 days culture (p-&001). Since the presence of Cyclosporine at 3000 @ml accentuated this difference and we have shown previously mat this compound increases HBsAg production markedly, we conclude that the effect on TGFB 1 may be mediated through HBsAg. Thus HBV DNA and TGFP 1 are mutually inhibitory. These data suggest that HBV may control, its own microenvironment to maximise HBsAg production.
COMPARISON BETWEEN HCVRNA AND El/E2 ANTIBODIES IN PATIENTS ON IFN TREATMENT la. G.-ens*. LCascavilla. T.SwtonioG, P.Conoscitore and A. Andriulli Gastr Div “CSS” IRCCS f&Giovanni R, $Infect.Dis. Inst. Univ of Bari ITALY, *INNOGENETICS, Ghent, BELGIUM In chronic HCV carriers a positive correlation between El and E2 Abs levels and viremia has been suggested. We analyzed HCVRNA levels and El and E2 Abs titers i n 89 HCVRNA +ve pts who underwent IFN therapy (3-6 MU TIW for 6 mo). Pts were subdivided in 38 long term responders (LTR), 33 non responders (NR) and 27 relapsers (REL).Serum samples were tested, at 0 , 2 and 6 mo on treatment, and at 6 mo during the f.u. Abs were quantified with a research ELISA using recombinant El and E2 antigens derived from HCV genotype Ib (INNOTEST HCVElAb and E2 Ab prototype version). HCVRNA was quantitatively determined by bDNA (Quantiplex HCVRNA 2.0). Results according with the different response to IFN are shown in the table: basal 2 El/RNA LTR 1.56~10~ 3.97 1.16 2.5 ,NR
3.31x105
5.09
6.14
1.5
2.87~10~ 4.50 5.21 1.5 PEL A positive correlation between HCVRNA and El and E2 basal levels was found. El more than E2 reflects circulating HCVRNA levels. El/HCVRNA ratio h i g her than 2 can be predictive of long term response.