- Email: [email protected]
The influences of immunosuppressive agents on HBV replication in vitro
102
Posters
) P/CO5/033 ] TRANSMISSION SHREWS
OF HEPATITIS
C VIRUS INFECTION TO TREE
Z.C. Xie’. J.I. Riezu-Bo?. J.J. Lasarte’. J. Guillen’. J.H. Su’. M.P. Civeira2, and J. Prieto* (1) Department of Preventive Medicine, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; (2) Department of Internal Medicine and (3) Animal services, Faculty of Medicine, University of Navarra, Pamplona, Spain. Although hepatitis C virus (HCV) infection can be reproduced in chimpanzees, these animals are rare and expensive. Tree shrews (tupaias) are small animals, closely related to primates, which adapt easily to laboratory environment. In this work we have investigated the susceptibility of Tupaia belangeri chinensis to HCV infection. Methods: Tupaias caught in the wild in Yunnan (China) were inoculated in China with HCV genotype lb (Study A) and in Spain with a mixture of genotypes lb, la and 3 (Study B). In study B tupaias were divided into three groups: group I was inoculated without previous manipulation, group II received 750 cGy of X-ray whole body irradiation before inoculation and group III was used as control. Results: Transient or intermittent viremia occurred in 34.8% (8/23) and anti-HCV in 30.4% (7123) of tupaias in study A. In study B a transient viremia was detected in 20% (2/10) in group I and in 50% (2/4) in group II. Anti-HCV was found in 1 tupaia from group I and in 3 from group II. Viremia lasted for longer and anti-HCV tended to reach higher titers in animals which received total body irradiation. ALT elevations and unspecific pathological changes occurred in inoculated tupaias but also in some controls. The wild nature of tupaias in this study precludes the interpretation of biochemical and pathological changes as solely due to HCV infection. Conclusion: In summary our results show that Tupaia belangeri chinensis are susceptible to HCV and that whole body irradiation may increase the efficiency of the infection. These animals may serve as an in vivo system to culture HCV and to address pathophysiological and therapeutic issues of HCV infection.
) P/CO5/034
GENETIC POLYMORPHISMS AND SERIC EXPRESSION OF ICAM- IN HCV INFECTED PATIENTS. A.Gallego, C.P&ez, X.Torras, JM.Dedeu, C.G6mez, E.Corral, J.B.A.Crusius*, J.Enriquez. Department of Gastroenterology. Hospital de la Sta. Creu i St. Pau. Barcelona. Spain. *Department of Gastroenterology. Free University Hospital. Amsterdam. Netherlands. Recent works had shown increased levels of soluble ICAM- in a variety of inflammatory conditions of the liver. The aim of this study was to assess genetic polymorphisms of ICAM- and circulating sICAM-I in relation to liver histology in the setting of HCV infection. PATIENTS AND METHODS: We have studied 32 HCV-RNA (+) patients: 14 blood donors with normal ALT and normal or minimal changes in liver histology, and 18 patients with elevated ALT and chronic hepatitis (5 CPH and 13 CAH). Viral load was determined with the kit Amplicor HCV Monito?. Circulating sICAM-1 was determined with an ELISA test (Boehringer Ingelheim). Two ICAM- polymorphisms were studied by SSCP: the first was located in exon 4, codon 241 (Arg or Gly: R/G 241) and the second in exon 6, codon 469 (Lys or Glu: WE 469). RESULTS: Levels of circulating sICAM-1 and HCV-RNA were significantly higher among patients with chronic hepatitis than in those with normal histology or minimal changes: 492,5 + 178,81 vs 288,28 + 66,77 ng/ml and 6,30 + 0,84 vs 4,2S *1,47 logldml respectively @
]
NATURALzKlLLER(NK)cEuLsINPATlENTS~CHRONIC HEPATlTIS C (CHC). F. Lirussi, B. Sanchez*, L. Pellizzari, A. Gup;Lietta*. Inst. of Internal
THE INFLUENCES OF IMMUNOSUPPRESSIVE ON HBV REPLICATION IN VITRO.
Medicine, Universit of Padova, Padova,. Ital and *Dept. of l&rke-Davis Pharmaceutical i&search Division, Immune E athology, Warnerambert Company, Ann Arbor, MI, U.S.A.
Z.J. GonaS. De Mever. C-G. Wu. C. Clarvsse. T. Crabbe, J. Nevts*, E. De Clera*, and S.H. Yap. Dept of Hepatology, U.Z. Gasthuisberg and *Rega Institute, Katholieke Universiteit Leuven, Belgium
Cellular immune response may affect the course and the response to treatment in viral infections. However, the role of NK cells in CHC is not well understood. We have compared the cytotoxic activity of NK cells of healthly volunteers and patients with biopsy proven HCV-
RNA+ve, active CHC using a new method based on a commercially available ELISA kit. Twenty-three healthy controls (M=16; F=7; age=426 yrs k1.6) and 15 CHC patients (M=lO, F=5; age==45.7yrs i3.4) were included in the study. Increasing concentrations (0; 30,000; 125,000; 250,000 cells/ml) of pm-isolated NK cells were incubated for 4 hrs at 37 “C with a fixed concentration (5000 cells/ml) of NKsensitive human cell line (K562). Apoptosis was detected by a peroxidasedependent calorimetric reaction using ABTS (2,2’-azinodi-[3-ethylbenzthiazoline sulfonate]) as a substrate, after K562-DNA fragments were trapped between an anti-h&one antibody and an antiDNA-peroxidase antibody. The baseline activity of NK cells-induced apoptosis was defined as a value of 1.0. Results: NK cells caused apoptotic death of target cells which was directly proportional to the concentration of NK cells in both patients and controls (table). NK celI concentration (cells/ml) ActivityofNKds (jmsxbasal~~*SEM) Patients (n=l5) Controls (n=23) P Conclusions: A sitificantlv
30,000
125,000
250,000
4.22+0.72
17.67i3.25
26.45i4.32
8.25&l .22 0.017
29.83i2.88 0.0091
48.07*5.03 0.0038
reduced activitv of NK cells in uatients. with CHC seems-compatible with an im&ired immune rksponse favoring chronicity of the desease. This new ELISA method for the detection of NK cell cytotoxicity has proven to be quick, easy to handle and not hazardous.
AGENTS
Orthotopic liver transplantation has become an important therapeutic modality even for patients with acute liver failure and end-stage chronic liver disease due to HBV infection. However, in transplanted patients with HBV, the high frequency of disease recurrence is a major problem. Most clinical protocols for posttransplant immunosuppression include a combination of corticosteroids and cyclosporine. Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA) is currently also used as an immunosuppressive agent in transplant recipients. After oral admiiistration, MMF is hydrolysed to MPA, the active compound, which is a potent inhibitor of inosine monophosphate dehydrogenase (IMP-DH). In order to investigate the influences of MPA, corticosteroids and cyclosporin on the HBV replicati?, experiments were performed using in vitro HBV infection model m cultures of primary human hepatocytes. After infection, HBV ccc DNA, precore/core and X mRNAs were detectable in cells in control experiments at least for 10 days so far we have investigated. I-LBV DNA and HBsAg were also secreted into the culture medium. In the presence of 10 &ml of MPA in culture medium, HBV cccDNA, precore/core and X mRNAs were undetectable from day 5 after infection. ‘The secretion of HBV DNA and HBsAg were also markedly inhibited by MPA (lo&ml). In contrast, the presence of dexamethsone (lpM/L) in culture medium increased the HBV DNA production while cyclosporin (25Ong/ml) did not influence the HBV replication in vitro. Cytotoxic effect of these drugs were not noted during the experiments in this study. These observed effects of immunosuppressive agents on HBV replication are therefore have clinically important.
Posters
) P/CO5/033 ] TRANSMISSION SHREWS
OF HEPATITIS
C VIRUS INFECTION TO TREE
Z.C. Xie’. J.I. Riezu-Bo?. J.J. Lasarte’. J. Guillen’. J.H. Su’. M.P. Civeira2, and J. Prieto* (1) Department of Preventive Medicine, Guangxi Medical University, Nanning, Guangxi 530021, People’s Republic of China; (2) Department of Internal Medicine and (3) Animal services, Faculty of Medicine, University of Navarra, Pamplona, Spain. Although hepatitis C virus (HCV) infection can be reproduced in chimpanzees, these animals are rare and expensive. Tree shrews (tupaias) are small animals, closely related to primates, which adapt easily to laboratory environment. In this work we have investigated the susceptibility of Tupaia belangeri chinensis to HCV infection. Methods: Tupaias caught in the wild in Yunnan (China) were inoculated in China with HCV genotype lb (Study A) and in Spain with a mixture of genotypes lb, la and 3 (Study B). In study B tupaias were divided into three groups: group I was inoculated without previous manipulation, group II received 750 cGy of X-ray whole body irradiation before inoculation and group III was used as control. Results: Transient or intermittent viremia occurred in 34.8% (8/23) and anti-HCV in 30.4% (7123) of tupaias in study A. In study B a transient viremia was detected in 20% (2/10) in group I and in 50% (2/4) in group II. Anti-HCV was found in 1 tupaia from group I and in 3 from group II. Viremia lasted for longer and anti-HCV tended to reach higher titers in animals which received total body irradiation. ALT elevations and unspecific pathological changes occurred in inoculated tupaias but also in some controls. The wild nature of tupaias in this study precludes the interpretation of biochemical and pathological changes as solely due to HCV infection. Conclusion: In summary our results show that Tupaia belangeri chinensis are susceptible to HCV and that whole body irradiation may increase the efficiency of the infection. These animals may serve as an in vivo system to culture HCV and to address pathophysiological and therapeutic issues of HCV infection.
) P/CO5/034
GENETIC POLYMORPHISMS AND SERIC EXPRESSION OF ICAM- IN HCV INFECTED PATIENTS. A.Gallego, C.P&ez, X.Torras, JM.Dedeu, C.G6mez, E.Corral, J.B.A.Crusius*, J.Enriquez. Department of Gastroenterology. Hospital de la Sta. Creu i St. Pau. Barcelona. Spain. *Department of Gastroenterology. Free University Hospital. Amsterdam. Netherlands. Recent works had shown increased levels of soluble ICAM- in a variety of inflammatory conditions of the liver. The aim of this study was to assess genetic polymorphisms of ICAM- and circulating sICAM-I in relation to liver histology in the setting of HCV infection. PATIENTS AND METHODS: We have studied 32 HCV-RNA (+) patients: 14 blood donors with normal ALT and normal or minimal changes in liver histology, and 18 patients with elevated ALT and chronic hepatitis (5 CPH and 13 CAH). Viral load was determined with the kit Amplicor HCV Monito?. Circulating sICAM-1 was determined with an ELISA test (Boehringer Ingelheim). Two ICAM- polymorphisms were studied by SSCP: the first was located in exon 4, codon 241 (Arg or Gly: R/G 241) and the second in exon 6, codon 469 (Lys or Glu: WE 469). RESULTS: Levels of circulating sICAM-1 and HCV-RNA were significantly higher among patients with chronic hepatitis than in those with normal histology or minimal changes: 492,5 + 178,81 vs 288,28 + 66,77 ng/ml and 6,30 + 0,84 vs 4,2S *1,47 logldml respectively @
]
NATURALzKlLLER(NK)cEuLsINPATlENTS~CHRONIC HEPATlTIS C (CHC). F. Lirussi, B. Sanchez*, L. Pellizzari, A. Gup;Lietta*. Inst. of Internal
THE INFLUENCES OF IMMUNOSUPPRESSIVE ON HBV REPLICATION IN VITRO.
Medicine, Universit of Padova, Padova,. Ital and *Dept. of l&rke-Davis Pharmaceutical i&search Division, Immune E athology, Warnerambert Company, Ann Arbor, MI, U.S.A.
Z.J. GonaS. De Mever. C-G. Wu. C. Clarvsse. T. Crabbe, J. Nevts*, E. De Clera*, and S.H. Yap. Dept of Hepatology, U.Z. Gasthuisberg and *Rega Institute, Katholieke Universiteit Leuven, Belgium
Cellular immune response may affect the course and the response to treatment in viral infections. However, the role of NK cells in CHC is not well understood. We have compared the cytotoxic activity of NK cells of healthly volunteers and patients with biopsy proven HCV-
RNA+ve, active CHC using a new method based on a commercially available ELISA kit. Twenty-three healthy controls (M=16; F=7; age=426 yrs k1.6) and 15 CHC patients (M=lO, F=5; age==45.7yrs i3.4) were included in the study. Increasing concentrations (0; 30,000; 125,000; 250,000 cells/ml) of pm-isolated NK cells were incubated for 4 hrs at 37 “C with a fixed concentration (5000 cells/ml) of NKsensitive human cell line (K562). Apoptosis was detected by a peroxidasedependent calorimetric reaction using ABTS (2,2’-azinodi-[3-ethylbenzthiazoline sulfonate]) as a substrate, after K562-DNA fragments were trapped between an anti-h&one antibody and an antiDNA-peroxidase antibody. The baseline activity of NK cells-induced apoptosis was defined as a value of 1.0. Results: NK cells caused apoptotic death of target cells which was directly proportional to the concentration of NK cells in both patients and controls (table). NK celI concentration (cells/ml) ActivityofNKds (jmsxbasal~~*SEM) Patients (n=l5) Controls (n=23) P Conclusions: A sitificantlv
30,000
125,000
250,000
4.22+0.72
17.67i3.25
26.45i4.32
8.25&l .22 0.017
29.83i2.88 0.0091
48.07*5.03 0.0038
reduced activitv of NK cells in uatients. with CHC seems-compatible with an im&ired immune rksponse favoring chronicity of the desease. This new ELISA method for the detection of NK cell cytotoxicity has proven to be quick, easy to handle and not hazardous.
AGENTS
Orthotopic liver transplantation has become an important therapeutic modality even for patients with acute liver failure and end-stage chronic liver disease due to HBV infection. However, in transplanted patients with HBV, the high frequency of disease recurrence is a major problem. Most clinical protocols for posttransplant immunosuppression include a combination of corticosteroids and cyclosporine. Mycophenolate mofetil (MMF), the morpholinoethyl ester of mycophenolic acid (MPA) is currently also used as an immunosuppressive agent in transplant recipients. After oral admiiistration, MMF is hydrolysed to MPA, the active compound, which is a potent inhibitor of inosine monophosphate dehydrogenase (IMP-DH). In order to investigate the influences of MPA, corticosteroids and cyclosporin on the HBV replicati?, experiments were performed using in vitro HBV infection model m cultures of primary human hepatocytes. After infection, HBV ccc DNA, precore/core and X mRNAs were detectable in cells in control experiments at least for 10 days so far we have investigated. I-LBV DNA and HBsAg were also secreted into the culture medium. In the presence of 10 &ml of MPA in culture medium, HBV cccDNA, precore/core and X mRNAs were undetectable from day 5 after infection. ‘The secretion of HBV DNA and HBsAg were also markedly inhibited by MPA (lo&ml). In contrast, the presence of dexamethsone (lpM/L) in culture medium increased the HBV DNA production while cyclosporin (25Ong/ml) did not influence the HBV replication in vitro. Cytotoxic effect of these drugs were not noted during the experiments in this study. These observed effects of immunosuppressive agents on HBV replication are therefore have clinically important.