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Autoantibody prevalence in chronic hepatitis C patients treated with interferon alpha and isoprinosine
181 VIRAL HEPATITIS: BASIC ASPECTS
1
vco5/001
1 co5/003
QUANTIFICATION OF LIVER FIBROSIS IN NEEDLE BIOPSY SPECIMENS BY FRACTAL ANALYSIS
IDENTIFICATION OF A NOVEL HGBVC/HGV
F. Grizzi’ and N. Dioquardi’. ‘Researcher of lstituto Clinico Humanitas, ‘Scientific Director of lstituto Clinic0 Humanitas. Rouano, Milano,ltaly. Hepatic fibrosis is the most common outcome of all the chronic disorders having a viral, toxic or metabolic origin. Its histological evaluation, is currently carried out by semiquantitative analysis. The latter, howewer, is largely subjective and not fully reproducible. Aim of this study is to propose a reliable and reproducible fractal scoring system, to evaluate the morphological complexity of the fibrosis in needle liver biopsy specimens. Liver biopsy specimens were obtained from 26 patients with HCV related chronic liver disease. Five pm-thick sections were stained with Masson’s trichrome and then digitized using an image analysis system. For each sample fradal dimension (D,) and spectral dimension (DS) were calculated automatically by a simple fractal software. The results show that for all the cases the mean fractal dimension Dr = 1.42SO.1948 exceeded the topological dimension, so that empirically, over the range of scales examined, hepatic fibrosis fulfilled the mathematical definition of fractal structure. The obtained spectral dimension (DS) was 0.9502+0.13. This suggested that a fractal-based scoring system could be sensitive enough to detect subtle changes in collagen accumulation between morphologically similar liver samples. This study demonstrates the feasibility to quantitate liver fibrosis by investigating its morphological irregularity. Furthermore, this approach can also be of help to evaluate the pathogenesis of liver fibrosis and its progression.
DETERMINATION OF MINIMAL HEPATITIS C VIRUSSPECIFIC INHIBITORY ANTISENSE OLIGODEOXYNUCLEOTIDES IN AN IN VITRO AND A CELLULAR SYSTEM T. Lehmann”. M. Alt’. E. Bijraelt’. B. Voosen. T. Sauerbruch. W.H. Caselmann, Dept. of Medicine, University of Bonn; *MPI f. Biochemie, Dept. of Virus Research, Martinsried; Dept. of Organic Chemistry, University of Frankfurt, Germany A 23-mer antisense phosphorothioate oligodeoxynucleotide S-0DN4 directed against HCV nts. 326-348 has been shown to effectively inhibit HCV expression (Hepatology 1995; 22:707). To delineate minimal inhibitory sequences we further truncated S-0DN4. The inhibitory potential of these truncated terminally (t) modified tS-ODNs was tested in a previously described in vitro translation system. Over 95% inhibition of HCV translation was seen with a 2Omer and 17-mer tS-ODN in this system. In parallel, a HCV-recombinant HepG2 cell sytem was generated. Stable transfection with a HCV DNA construct containing the CMV promotor, the S’NCR and 66 nts. of HCV core fused in frame to the luciferase cDNA which lacks the endogenous ATG was performed. HepG2 cells transfected with the same construct missing the HCV sequences were used as controls. G418 resistant colonies were tested by PCR and Southern blot analyses for integration of the transfected DNA and luciferase activities were analysed. Two clones (basal luciferase expression 3.1~10~ and 15.8~10~ RLU for the HCV containing clones and 7.9 x105 and 6.6~10~ RLU for the control cells) were M. Serwe.
used for continous propagation. These data suggest that antisense S-ODNs may become a useful tool to control HCV expression, if effective ODN delivery and specific targeting can be achieved.
SUBTYPE
.M. L&ez-Alcorocho’, I. Castillo’. J.F. Tom&* and V. Carreiio’. ‘Dent. of HeDatoloCW, Fundaci6n Jimbnez Diaz and Fundacidn Estidio Hepatitis Virz&s; ‘Dept of Hematology, Hospital La Princesa. Madrid, Spain. Backaround: The existence of 5 heoatitis GB tvoe C/heoatitis G virus (HGBV-C/HGV) subtypes related tb geograp&al locaiion has been reported. Aim: To determine the subtype of the HGBV-C/HGV isolated from 5 multitransfused patients infected with this virus, prior to bone marrow transplantation (BMT). Methods: Total RNA was extracted from serum samples taken before BMT and the entire HGBV-C/HGV 5’-UTR was amplified by RT-PCR, using degenerated primers deduced from all published sequences. RT-PCR prbducts w&e cloned and several clones from each patient (5 in 2 patients, 4 in 2 cases and 1 in the remaining patient) were automatically sequenced. Nucleotide sequences were aligned and compared with thirty-nine 5’-UTR sequences obtained from GenBank (7 from subtype la, 9 subtype 1b, 12 subtype 2a, 9 subtype 2b and 2 subtype 3). Phylogenetic and bootstrap analysis were performed using the PHYLIP package 3.5~. Results: Comparison of evolutionary distances among all analyzed clones showed a aenomic variabilitv at two levels: one level represented by the heterogeneity obse’tved within the isolates from each infected oatient (distances from 0.0065 to 0.0171) and other one among the isdlates frbm different patients (distances’from 0.0237 to 0.0832). On the other hand, the evolutionary distances among the clones of our patients and the 39 sequences obtained from GenBank ranged from 0.0876 to 0.2062 (0.1633-0.1932 with subtype la, 0.17560.2062 with subtype 1b, 0.0876-0.1091 with subtype 2a, 0.1001-0.1275 with subtype 2b and 0.1036-0.1333 with subtype 3). The phylogenetic tree showed that the isolates from our patients segregated into an unique branch which was different to those corresponding to the 5 described subtypes. This result was supported by the bootstrap analysis of 100 replicates. Condusions: The genomic heterogeneity observed within the isolates from each patient suggests the existence of HGBV-C/HGV quasispecies. A novel HGBV-C/HGV subtype has been found in the patients included in this study.
AUTOANTIBODY PREVALENCE IN CHRONIC HEPATITIS C PATIENTS TREATED WITH INTERFERON ALPHA AND ISOPRINOSINE N.Osna, E.Hapina, G.Silonova, V. Kuse, L.Viksna, ASochnev, Latvian Medical Academy, Riga, Latvia
This fragment is a part of EUROHEP study on combined treatment of chronic hepatitis C (CHC) with rIFNa2b and isoprinosine. The aim of this work is to study autoantibody prevalence in dynamics of CHC patients’ treatment with IFNa alone and in combination with isopronosine. Thirty-two CHC patients were randomized into 2 groups: A.-treated with IFNa (3MUx3W, 24 weeks)+ isoprinosine; B.-treated with IFNa + placebo. Autoantibody (Aab) prevalence was tested in sera samples in IFA. Before treatment 42% of patients were Aab negative, 20% -ANA +, 27% -SMA+, 2%-LKM+. In 2-3 months of treatment Aab have appeared in almost all the patients without any link to clinical, biochemical or virological course of CHC. In A. group 63% of responders had SMA (titers > 1: 160) and 38%- ANA (titers >1:40). In nonresponders of A. group 86% became SMA+(titers > 1: 160), and 57%-ANA + (titers >1:40). Both responders and nonresponders of B. group had elevation of SMA (86%) and ANA (57%). We conclude that rIFNa treatment increase Aab level (titers of SMA and ANA) without any link to treatment in patients treated with rIFNa+isoprinosine efficacy; combination titers of SMA and ANA in seemed to be lower in responders versus non-reponders and patients treated with rIFNa+placebo
1
vco5/001
1 co5/003
QUANTIFICATION OF LIVER FIBROSIS IN NEEDLE BIOPSY SPECIMENS BY FRACTAL ANALYSIS
IDENTIFICATION OF A NOVEL HGBVC/HGV
F. Grizzi’ and N. Dioquardi’. ‘Researcher of lstituto Clinico Humanitas, ‘Scientific Director of lstituto Clinic0 Humanitas. Rouano, Milano,ltaly. Hepatic fibrosis is the most common outcome of all the chronic disorders having a viral, toxic or metabolic origin. Its histological evaluation, is currently carried out by semiquantitative analysis. The latter, howewer, is largely subjective and not fully reproducible. Aim of this study is to propose a reliable and reproducible fractal scoring system, to evaluate the morphological complexity of the fibrosis in needle liver biopsy specimens. Liver biopsy specimens were obtained from 26 patients with HCV related chronic liver disease. Five pm-thick sections were stained with Masson’s trichrome and then digitized using an image analysis system. For each sample fradal dimension (D,) and spectral dimension (DS) were calculated automatically by a simple fractal software. The results show that for all the cases the mean fractal dimension Dr = 1.42SO.1948 exceeded the topological dimension, so that empirically, over the range of scales examined, hepatic fibrosis fulfilled the mathematical definition of fractal structure. The obtained spectral dimension (DS) was 0.9502+0.13. This suggested that a fractal-based scoring system could be sensitive enough to detect subtle changes in collagen accumulation between morphologically similar liver samples. This study demonstrates the feasibility to quantitate liver fibrosis by investigating its morphological irregularity. Furthermore, this approach can also be of help to evaluate the pathogenesis of liver fibrosis and its progression.
DETERMINATION OF MINIMAL HEPATITIS C VIRUSSPECIFIC INHIBITORY ANTISENSE OLIGODEOXYNUCLEOTIDES IN AN IN VITRO AND A CELLULAR SYSTEM T. Lehmann”. M. Alt’. E. Bijraelt’. B. Voosen. T. Sauerbruch. W.H. Caselmann, Dept. of Medicine, University of Bonn; *MPI f. Biochemie, Dept. of Virus Research, Martinsried; Dept. of Organic Chemistry, University of Frankfurt, Germany A 23-mer antisense phosphorothioate oligodeoxynucleotide S-0DN4 directed against HCV nts. 326-348 has been shown to effectively inhibit HCV expression (Hepatology 1995; 22:707). To delineate minimal inhibitory sequences we further truncated S-0DN4. The inhibitory potential of these truncated terminally (t) modified tS-ODNs was tested in a previously described in vitro translation system. Over 95% inhibition of HCV translation was seen with a 2Omer and 17-mer tS-ODN in this system. In parallel, a HCV-recombinant HepG2 cell sytem was generated. Stable transfection with a HCV DNA construct containing the CMV promotor, the S’NCR and 66 nts. of HCV core fused in frame to the luciferase cDNA which lacks the endogenous ATG was performed. HepG2 cells transfected with the same construct missing the HCV sequences were used as controls. G418 resistant colonies were tested by PCR and Southern blot analyses for integration of the transfected DNA and luciferase activities were analysed. Two clones (basal luciferase expression 3.1~10~ and 15.8~10~ RLU for the HCV containing clones and 7.9 x105 and 6.6~10~ RLU for the control cells) were M. Serwe.
used for continous propagation. These data suggest that antisense S-ODNs may become a useful tool to control HCV expression, if effective ODN delivery and specific targeting can be achieved.
SUBTYPE
.M. L&ez-Alcorocho’, I. Castillo’. J.F. Tom&* and V. Carreiio’. ‘Dent. of HeDatoloCW, Fundaci6n Jimbnez Diaz and Fundacidn Estidio Hepatitis Virz&s; ‘Dept of Hematology, Hospital La Princesa. Madrid, Spain. Backaround: The existence of 5 heoatitis GB tvoe C/heoatitis G virus (HGBV-C/HGV) subtypes related tb geograp&al locaiion has been reported. Aim: To determine the subtype of the HGBV-C/HGV isolated from 5 multitransfused patients infected with this virus, prior to bone marrow transplantation (BMT). Methods: Total RNA was extracted from serum samples taken before BMT and the entire HGBV-C/HGV 5’-UTR was amplified by RT-PCR, using degenerated primers deduced from all published sequences. RT-PCR prbducts w&e cloned and several clones from each patient (5 in 2 patients, 4 in 2 cases and 1 in the remaining patient) were automatically sequenced. Nucleotide sequences were aligned and compared with thirty-nine 5’-UTR sequences obtained from GenBank (7 from subtype la, 9 subtype 1b, 12 subtype 2a, 9 subtype 2b and 2 subtype 3). Phylogenetic and bootstrap analysis were performed using the PHYLIP package 3.5~. Results: Comparison of evolutionary distances among all analyzed clones showed a aenomic variabilitv at two levels: one level represented by the heterogeneity obse’tved within the isolates from each infected oatient (distances from 0.0065 to 0.0171) and other one among the isdlates frbm different patients (distances’from 0.0237 to 0.0832). On the other hand, the evolutionary distances among the clones of our patients and the 39 sequences obtained from GenBank ranged from 0.0876 to 0.2062 (0.1633-0.1932 with subtype la, 0.17560.2062 with subtype 1b, 0.0876-0.1091 with subtype 2a, 0.1001-0.1275 with subtype 2b and 0.1036-0.1333 with subtype 3). The phylogenetic tree showed that the isolates from our patients segregated into an unique branch which was different to those corresponding to the 5 described subtypes. This result was supported by the bootstrap analysis of 100 replicates. Condusions: The genomic heterogeneity observed within the isolates from each patient suggests the existence of HGBV-C/HGV quasispecies. A novel HGBV-C/HGV subtype has been found in the patients included in this study.
AUTOANTIBODY PREVALENCE IN CHRONIC HEPATITIS C PATIENTS TREATED WITH INTERFERON ALPHA AND ISOPRINOSINE N.Osna, E.Hapina, G.Silonova, V. Kuse, L.Viksna, ASochnev, Latvian Medical Academy, Riga, Latvia
This fragment is a part of EUROHEP study on combined treatment of chronic hepatitis C (CHC) with rIFNa2b and isoprinosine. The aim of this work is to study autoantibody prevalence in dynamics of CHC patients’ treatment with IFNa alone and in combination with isopronosine. Thirty-two CHC patients were randomized into 2 groups: A.-treated with IFNa (3MUx3W, 24 weeks)+ isoprinosine; B.-treated with IFNa + placebo. Autoantibody (Aab) prevalence was tested in sera samples in IFA. Before treatment 42% of patients were Aab negative, 20% -ANA +, 27% -SMA+, 2%-LKM+. In 2-3 months of treatment Aab have appeared in almost all the patients without any link to clinical, biochemical or virological course of CHC. In A. group 63% of responders had SMA (titers > 1: 160) and 38%- ANA (titers >1:40). In nonresponders of A. group 86% became SMA+(titers > 1: 160), and 57%-ANA + (titers >1:40). Both responders and nonresponders of B. group had elevation of SMA (86%) and ANA (57%). We conclude that rIFNa treatment increase Aab level (titers of SMA and ANA) without any link to treatment in patients treated with rIFNa+isoprinosine efficacy; combination titers of SMA and ANA in seemed to be lower in responders versus non-reponders and patients treated with rIFNa+placebo