- Email: [email protected]
HCV co-infection
Poster Sessions
108
with tumor progression in some types of human cancer. Short isoforms of Cyclin El mRNA were found in human tissues. In the present studies we investigated if short isoforms of Cyclin El might be linked to murine hepatocarcinogenesis. We first cloned and sequenced the yet unknown distal part of the genomic mouse Cyclin El. Sequence comparison of the mouse and human Cyclin El locus revealed a strong conservation of the in&-on-exon structure, whereas only little homology was found on the DNA level. We next used single and double transgenic mice with hepatocyte-specific expression of c-myc (AATlc-myc) or EGF (ALB/IgEGF) to induce hepatocellular carcinomas. HCC developed 3 to 12 month after birth. HCC nodules were isolated and a RT-PCR approach was performed to study Cyclin El expression. Short isoforms of Cyclin El were only found in the single transgenic, but not in double transgenic animals with fast occurring HCC’s. In HCC from single transgenic mice the full length Cyclin El as well as 3 new isoforms with deletion of either exon 4 or exon 5 or an insertion between exon 2 and 3 were found. In summary, we cloned the murine Cyclin El gene and found alternatively spliced isoforms in murine HCC’s and normal livers. Interestingly, the expression of these short Cyclin El variants was associated with slow tumor development. Further studies are currently underway to characterize functions of Cyclin El isoforms.
Category 5: Viral hepatitis: basic aspects
I364
LIVER CELL APOPTOSIS
IN CHRONIC
HEPATITIS B: ROLE OF
I365
RANTES
(-403
AND HIV/HCV
G+A)
PROMOTER
POLYMORPHISM
IN HCV
CO-INFECTION
G. Ahlenstiel’, A. Iwan2, H. H. Brackmann3, B. Kupfer4, B. Matz’, T. Sauerbruch6, U. Spengler7, R. l? Woitas’. ‘Department Of Internal Medicine I, University Of Bonn, Bonn, Germany; 21nstitute Of Experimental Hematology, University Of Bonn, Bonn, Germany; ‘Institute Of Medical Microbiology And Immunology, University Of Bonn, Bonn, Germany
Background: Chemokine and their receptors are crucial for the immune response in HIV and HCV infection. The natural corn--se of these infections may be altered by a polymorphism within the RANTES promoter (-403 G+A) which leads in vitro to up-regulated RANTES transcription. Methods: We therefore determined the frequency of the RANTES-403 mutation using real time PCR and hybridization probes in patients with HIV infection (n=87), HCV infection (n=129), HIV/HCV co-infection (n=121), and 109 healthy blood donors. Each group was stratified into -403 homozygotes, -403/wildtype heterozygotes and wildtype homozygotes, respectively. Finally, resulting subsets were compared with respect to HIV and HCV loads. Result: The RANTES-403 allele frequency in HIV infected patients (50/124 (28. 7%)) differed significantly from HCV (51/207 (19. 7%), HIV/HCV co-infected patients (37/205 (15. 3%) and the healthy control group (33085 (15. 1%); p < 0. 05). RANTES-403 homozygosity was found in 5 (5. 7%) of the HIV- infected patients compared to 3 in the HCV (2. 3%) and HIV/HCV co-infected groups (2. 5%) and 1 of the healthy blood donors (0. 9%). HIV infected patients carrying the mutant allele had viral loads that were 3 times lower than in the wildtype patients (n.s.), furthermore HCV loads were lower in -403 homozygous patients (ns.). Conclusions: The RANTES-403 mutation is significantly more frequent in HIV infection than in our other cohorts. This mutation is associated with reduced HIV loads. However, our data do not support an association between hepatitis C and the RANTES-403 polymorphism.
FAS-AG AND TGF-j31
D.T. Abdourakhmanov’, E.A. Kogan2, T.N. Nekrasova2, .I. Guardia3. ‘Internal And Occupational Diseases, Moscow Medical Academy, Moscow, Russia; 2Pathology Department, Moscow MedicalAcademy, Moscow, Russia; ‘Hepatology Unit, Hospital General Vail Hebron, Medicine Faculty, Autonomous University, Barcelona, Spain Background: Apoptosis is morphologically distinct form of cell death characterized by chromatin condensation, and nuclear and cellular fragmentation. Its role in viral hepatitis has not been adequately studied. TGFbl has been shown to induce apoptosis in primary hepatocyte cultures and hepatoma cell in vitro. However, the mechanisms leading to TGF-blinduced apoptosis and role of CD95/Fas-Ag expression in regulation of apoptosis are still poorly understood, especially in viva studies. Aims/Methods: To evaluate the degree of apoptosis, and role of Fas-Ag and TGF-bl expression in liver damage in chronic hepatitis B. TUNELtest (ENZO ApopDetec Cell Assay, USA) to detect apoptosis and specific monoclonal antibodies (DAK0 and Biosource, USA) to detect Fas-Ag and TGF-bl in liver biopsy samples of 20 patients with cronic hepatitis B were used. Results: Mean apoptopic index (AI) - number of TUNEL-positive hepatocytes on 3000 cells - was 2, 4&2, 2% (0, 08-8, 1%). AI positively correlate with Histhological Activity Index (by Ishak score) (p=O.O5) and aminotransferase level (p=O.O5). The degree of TGF-bl expression strongly positively correlate with AI (p=O.O25). AI were higher in Fas-Ag-positive cases than in Fas-Ag-negative cases (p
I
366
IN VIVO TRANSCOMPLEMENTATION COMPETENT INDUCED
NUCLEOCAPSIDS
OF HBV REPLICATION
REDUCES
LIVER INJURY IN TRANSGENIC
THE HBSAG MICE
M. Ali’ R. Weth’, C. F. Grimm’, W. 0. Boecher2, H. E. Blum’, U.Sch&z’, M. Geissler’. ‘De@ Of Medicine II, Freiburg; 2Dept Of Medicine I, Mainz, Germany Background and Aims: Transgenic mice expressing HBV envelope proteins in the liver represent an experimental model for some of the histopathological effects of infection in humans, including prolonged hepatocellular injury, necrosis, hyperplasia, and an elevated incidence of liver tumors (Tg(Alb-lHBV)Bri44, Chisari 1990). In order to establish a transgenie mouse model for the evaluation of therapeutic vaccines against HBV and to determine the impact of HBsAg antigenemia on T cell tolerance induction we established a HBV replicating transgenic mouse line lacking the production of small envelope proteins due to a non-stop mutation in the S-ATG codon (Tgl. 4HBV-S-Minus). To transcomplement HBV envelope proteins these mice were cross-bred to Tg(Alb-lHBV)Bri44 resulting in AlbHBs/S-Mut mice. Results and Discussion: Tgl. 4HBV-S-Minus mice were characterized by high level HBV replication and gene expression in the liver and kidney comparable to the well known 1. 3. 46 strain. No cccDNA was detected. Sera of mice were negative for viral particles and HBsAg and strongly positive for HBeAg. In the liver, HBcAg was easily detectable by Western blotting and immunohistochemical staining. PreS 1 and preS2 proteins, but not the small envelope protein were expressed in the liver at low levels. These mice, therefore, are an interesting model to study immune responses to HBV structural and non-structural antigens in absence of HBsAg antigenemia and their effects on intrahepatic HBV replication. Cross-breeding AlbHBs with SMUT mice resulted in Fl mice replicating HBV and export-
108
with tumor progression in some types of human cancer. Short isoforms of Cyclin El mRNA were found in human tissues. In the present studies we investigated if short isoforms of Cyclin El might be linked to murine hepatocarcinogenesis. We first cloned and sequenced the yet unknown distal part of the genomic mouse Cyclin El. Sequence comparison of the mouse and human Cyclin El locus revealed a strong conservation of the in&-on-exon structure, whereas only little homology was found on the DNA level. We next used single and double transgenic mice with hepatocyte-specific expression of c-myc (AATlc-myc) or EGF (ALB/IgEGF) to induce hepatocellular carcinomas. HCC developed 3 to 12 month after birth. HCC nodules were isolated and a RT-PCR approach was performed to study Cyclin El expression. Short isoforms of Cyclin El were only found in the single transgenic, but not in double transgenic animals with fast occurring HCC’s. In HCC from single transgenic mice the full length Cyclin El as well as 3 new isoforms with deletion of either exon 4 or exon 5 or an insertion between exon 2 and 3 were found. In summary, we cloned the murine Cyclin El gene and found alternatively spliced isoforms in murine HCC’s and normal livers. Interestingly, the expression of these short Cyclin El variants was associated with slow tumor development. Further studies are currently underway to characterize functions of Cyclin El isoforms.
Category 5: Viral hepatitis: basic aspects
I364
LIVER CELL APOPTOSIS
IN CHRONIC
HEPATITIS B: ROLE OF
I365
RANTES
(-403
AND HIV/HCV
G+A)
PROMOTER
POLYMORPHISM
IN HCV
CO-INFECTION
G. Ahlenstiel’, A. Iwan2, H. H. Brackmann3, B. Kupfer4, B. Matz’, T. Sauerbruch6, U. Spengler7, R. l? Woitas’. ‘Department Of Internal Medicine I, University Of Bonn, Bonn, Germany; 21nstitute Of Experimental Hematology, University Of Bonn, Bonn, Germany; ‘Institute Of Medical Microbiology And Immunology, University Of Bonn, Bonn, Germany
Background: Chemokine and their receptors are crucial for the immune response in HIV and HCV infection. The natural corn--se of these infections may be altered by a polymorphism within the RANTES promoter (-403 G+A) which leads in vitro to up-regulated RANTES transcription. Methods: We therefore determined the frequency of the RANTES-403 mutation using real time PCR and hybridization probes in patients with HIV infection (n=87), HCV infection (n=129), HIV/HCV co-infection (n=121), and 109 healthy blood donors. Each group was stratified into -403 homozygotes, -403/wildtype heterozygotes and wildtype homozygotes, respectively. Finally, resulting subsets were compared with respect to HIV and HCV loads. Result: The RANTES-403 allele frequency in HIV infected patients (50/124 (28. 7%)) differed significantly from HCV (51/207 (19. 7%), HIV/HCV co-infected patients (37/205 (15. 3%) and the healthy control group (33085 (15. 1%); p < 0. 05). RANTES-403 homozygosity was found in 5 (5. 7%) of the HIV- infected patients compared to 3 in the HCV (2. 3%) and HIV/HCV co-infected groups (2. 5%) and 1 of the healthy blood donors (0. 9%). HIV infected patients carrying the mutant allele had viral loads that were 3 times lower than in the wildtype patients (n.s.), furthermore HCV loads were lower in -403 homozygous patients (ns.). Conclusions: The RANTES-403 mutation is significantly more frequent in HIV infection than in our other cohorts. This mutation is associated with reduced HIV loads. However, our data do not support an association between hepatitis C and the RANTES-403 polymorphism.
FAS-AG AND TGF-j31
D.T. Abdourakhmanov’, E.A. Kogan2, T.N. Nekrasova2, .I. Guardia3. ‘Internal And Occupational Diseases, Moscow Medical Academy, Moscow, Russia; 2Pathology Department, Moscow MedicalAcademy, Moscow, Russia; ‘Hepatology Unit, Hospital General Vail Hebron, Medicine Faculty, Autonomous University, Barcelona, Spain Background: Apoptosis is morphologically distinct form of cell death characterized by chromatin condensation, and nuclear and cellular fragmentation. Its role in viral hepatitis has not been adequately studied. TGFbl has been shown to induce apoptosis in primary hepatocyte cultures and hepatoma cell in vitro. However, the mechanisms leading to TGF-blinduced apoptosis and role of CD95/Fas-Ag expression in regulation of apoptosis are still poorly understood, especially in viva studies. Aims/Methods: To evaluate the degree of apoptosis, and role of Fas-Ag and TGF-bl expression in liver damage in chronic hepatitis B. TUNELtest (ENZO ApopDetec Cell Assay, USA) to detect apoptosis and specific monoclonal antibodies (DAK0 and Biosource, USA) to detect Fas-Ag and TGF-bl in liver biopsy samples of 20 patients with cronic hepatitis B were used. Results: Mean apoptopic index (AI) - number of TUNEL-positive hepatocytes on 3000 cells - was 2, 4&2, 2% (0, 08-8, 1%). AI positively correlate with Histhological Activity Index (by Ishak score) (p=O.O5) and aminotransferase level (p=O.O5). The degree of TGF-bl expression strongly positively correlate with AI (p=O.O25). AI were higher in Fas-Ag-positive cases than in Fas-Ag-negative cases (p
I
366
IN VIVO TRANSCOMPLEMENTATION COMPETENT INDUCED
NUCLEOCAPSIDS
OF HBV REPLICATION
REDUCES
LIVER INJURY IN TRANSGENIC
THE HBSAG MICE
M. Ali’ R. Weth’, C. F. Grimm’, W. 0. Boecher2, H. E. Blum’, U.Sch&z’, M. Geissler’. ‘De@ Of Medicine II, Freiburg; 2Dept Of Medicine I, Mainz, Germany Background and Aims: Transgenic mice expressing HBV envelope proteins in the liver represent an experimental model for some of the histopathological effects of infection in humans, including prolonged hepatocellular injury, necrosis, hyperplasia, and an elevated incidence of liver tumors (Tg(Alb-lHBV)Bri44, Chisari 1990). In order to establish a transgenie mouse model for the evaluation of therapeutic vaccines against HBV and to determine the impact of HBsAg antigenemia on T cell tolerance induction we established a HBV replicating transgenic mouse line lacking the production of small envelope proteins due to a non-stop mutation in the S-ATG codon (Tgl. 4HBV-S-Minus). To transcomplement HBV envelope proteins these mice were cross-bred to Tg(Alb-lHBV)Bri44 resulting in AlbHBs/S-Mut mice. Results and Discussion: Tgl. 4HBV-S-Minus mice were characterized by high level HBV replication and gene expression in the liver and kidney comparable to the well known 1. 3. 46 strain. No cccDNA was detected. Sera of mice were negative for viral particles and HBsAg and strongly positive for HBeAg. In the liver, HBcAg was easily detectable by Western blotting and immunohistochemical staining. PreS 1 and preS2 proteins, but not the small envelope protein were expressed in the liver at low levels. These mice, therefore, are an interesting model to study immune responses to HBV structural and non-structural antigens in absence of HBsAg antigenemia and their effects on intrahepatic HBV replication. Cross-breeding AlbHBs with SMUT mice resulted in Fl mice replicating HBV and export-