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Relative quantification and mapping of hepatitis C virus by “in situ” hybridization and digital image analysis
98
Posters
( P/CO5/017 1 RELATIVE QUANTIFICATION AND MAPPING OF HEPATITIS C VlRUS BY ‘in situ’ HYBRIDIZATION AND DlGlfAL IMAGE ANALYSIS E. Rodriauez-lfiiao’. J. GosBlvez2. J Bartolom~‘. JL. Ramiro-Diaz2, JF. Tom&?. and V. Carrefio’. ‘Dept. of Hepatology, Fundacibn imbnez Dlaz and Fundaci6n para el estudio de las Hepatiti virales; P’ GenetIc Unit, Dept of Biology, Universidad Autbnoma, 3Dept of Hematology, Hospital La Princesa. Madrid, Spain. Although several reports concerning the detection of hepatitis C virus (HCV) by “in situ” hybridization have been published, there are no data concerning the relative viral load in infected hepatocytes nor about its relation with serum viremia levels. So, we have developed a simple method based on digital image analysis of “in situ” hybridization signals that allows: 1) the estimation of the relative number of HCV genome copies per infected cell, 2) The analysis of the relationship between the relative amount of HCV in the biopsies and the serum viremia levels. “In situ” hybridization was performed in paraffin embedded liver biopsies from 4 patients with chronic hepatitis C. As probe, a 340 bp cDNA corresponding to the B’non-coding region of the HCV genome was used. The probe was labelled with digoxigenin-ll-ddUTP. The hybridization signals were analyzed by digital image analysis. Hepatitis C virus RNA was detected by “in situ” hybridization in the hepatocytes of the four liver samples. The hybridization signals were found in the cytoplasm although in some hepatocytes nuclear and nucleolar signals were also observed. The relative viral load per infected cell fitted second order polynomial curves in the four cases. The minimum and maximum relative viral load per infected hepatocyte differed in the four cases. However, the mean relative viral load was similar in the four liver samples analyzed (31295, 31090, 31854 and 37996 standard units). The percentage of HCV infected cells ranged from 4.8% to 65.6% in the four cases. The percentage of positive cells for the viral RNA correlates with the serum viremia levels tested by the Amplicor Monitor test (Roche). Our data suggest that HCV viremia in serum does not depend on the relative viral load per infected cell but on the number of infected hepatocytes.
1 PICO5/018
‘. N. A. Pa ’ GO1 -h4asm’ Li& Unit’ and Dept. OfPathology’, St V&at’s
IN HEPATITIS
E. H Hwpital,
C
CO F rrellv’, ERCI, Dublin, Jreland
Hepatitis C virus (HCV) infection is associated with autoimmunity and lymphoprolifemtion. Both CD5 B lymphocytes which produce naturally occurring polyreactive antibodies and @TCR T lymphocytes have been implicated in the pathogenesis of autoimmune diseases. In addition, increased numbers of CD5 B cells in chronic lymphocytic leukaemia, infectious mononucleosis and rheumatoid arthritis are associated with an increase in y8TCR T lymphocytes. Ail: To determine the peripheral blood levels of $TCR T lymphocytes and CD5 B cells in HCV infected individuals and age matched controls. Methods:Peripheral blood mononuclear cells from 13 HCV infected patients and age/sex matched controls were analysed by 3 colour flow cytometry for CD3, CD4, CD8, CD56, y6 and ap TCR. In addition 4 HCV infected individuals, 4 age/sex matched controls and 4 individuals with resolved infection were analysed for CD5 and CD19. Serum from all 12 individuals was analysed by indirect immunofluomsence for anti-nuclear, anti-smooth muscle, anti-parietal cell and antimitochondrial antibodies and by nephelometry for rheumatoid factor. Results: CDS B cells were significantly increased (1~0.02) in HCV infected individuals when compared to HCV RNA negative and normal controls. There was no significant di&rence in T lymphocytes subsets between HCV infected individuals and controls. Of the 4 HCV RNA positive individuals 3 had positive rheumatoid factor, 2 anti-nuclear antibody and 3 anti-smooth muscle antibody while none of the controls had positive auto-antibodies.
~~* .d P-l”.
I
A relatiohshi between iron status and the activity of chronic hepatitis C (H 6 ) has been proposed. Iron overload is frequent in patients with HC, and iron depletion may induce a reduction of transaminases that is not related to a modification of the viral load. A ene in the iron overload of HC
1
CDS B LYMPHOCYTES ARE INCREASED VIRUS INFECTION gM. P.Cu
LIVER IRON DISTRIBUTION, HISTOLOGICAL AClWlTY AND MUTATIONS IN THE HEMOCHROMATOSIS GENE (HFE) IN CHRONIC HEPATITIS C M. Sampietro, R. Boldorini*, N. Corbetta. M. Amato, A. Casatta,
4279
,013
6342
3074
148
4%
3 52
4 76
94w
2.461
1023
64 64
313,
189
291
2 26
419
9563
a02
100
074
0’4
057
006
OL2
OS0
035
Conch~sions: CD5 B cells are increased in chronic HCV infection independently of $iTCR T cells. This increase in CD5 B cells is associated with auto-antibody production and may contribute to the autoimmune phenomenon of HCV infection.
SOLUBLE TUMOR NECROSIS FACTOR RECEPTORS IN CHRONIC HEPATITIS C: A CORRELATION WITH SEVERITY OF LIVER DISEASE. H. Zylberberg’, AC. Rimaniol’, S. Pal’, D. De Groote’, P. Berthelot’, J-F Bach’, C. Br60hot’.3, F. Zavala’. ’ Unlt6 d’HBpatologie, ’ INSERM U-25, 3 INSERM U-370 HBpltal Necker. Tumor necrosis factor-alpha (TNF) IS a mediator of Background: inflammation and cellular Immune response Soluble TNF receptors (sTNFR) sTNF-R55 and sTNF-R75, which compete with cellular receptors for the bindlng of TNF, have been detected at high levels In various mfectlous diseases including HIV and HBV infection Aim:In order to invesbgate the acbvatlon of the TNF system In HCV infecbon, we have analyzed the balance between TNF and sTNF-R I” 60 HCV-Infected subjects according to their clinical. biological, vlrologlcal and histological characteristics. Methods Serum TNF, sTNF-R55 and sTNF-R75 levels were determlned before any therapy and were compared to a control group of 60 healthy subjects. Results: Mean TNF levels were 50 5 + 4.5 pglml In patlents and undetectable (~5 pg/ml) in the control sublects. sTNF-R55 and sTNF-R75 levels were si&A&&tly’higher in HCV-infected patients than in the controls: 2.9? 0.1 nglmlvs 1.3k0.1, (p=O.OOOl), and9.5r0.6 nglmlvs 4.2? 02, (p = 0.0001) respectively. In contrast to other Infectious diseases, there was no correlation between levels of sTNF-R and TNF. sTNF-R75 but not TNF levels were correlated with aminotransferases levels (p = 0.0006 and p = 0.02 for AST and ALT respectively), wh!le sTNF-R55 levels were significantly correlated only with AST levels (p = 0.04). sTNF-R55 and sTNF-R75 were both slgnlflcantly correlated with necroticolnflammatory Index (p = 0 OOl), and their levels were significantly higher in patients with vs wlthout cirrhosis (3.2 f 0 2 vs. 2.5 f 0 2 ng/ml, (~~0.02) and 11.6 f 0.9 vs. 7 5 f 0.5 ng/ml, (p c 0.001) respectively) sTNF-R55. sTNF-R75 and TNF levels were not correlated wtth viral load, genotype or response to Interferon therapy Conclusion: Levels of soluble TNF receptors are strongly correlated with the severity of the disease but not with virological parameters such as quanbtative vlremia and genotype. High TNF-R production could thus suggest that HCV-related liver disease involves immunological mechanisms Including acttvation of the TNF system.
Posters
( P/CO5/017 1 RELATIVE QUANTIFICATION AND MAPPING OF HEPATITIS C VlRUS BY ‘in situ’ HYBRIDIZATION AND DlGlfAL IMAGE ANALYSIS E. Rodriauez-lfiiao’. J. GosBlvez2. J Bartolom~‘. JL. Ramiro-Diaz2, JF. Tom&?. and V. Carrefio’. ‘Dept. of Hepatology, Fundacibn imbnez Dlaz and Fundaci6n para el estudio de las Hepatiti virales; P’ GenetIc Unit, Dept of Biology, Universidad Autbnoma, 3Dept of Hematology, Hospital La Princesa. Madrid, Spain. Although several reports concerning the detection of hepatitis C virus (HCV) by “in situ” hybridization have been published, there are no data concerning the relative viral load in infected hepatocytes nor about its relation with serum viremia levels. So, we have developed a simple method based on digital image analysis of “in situ” hybridization signals that allows: 1) the estimation of the relative number of HCV genome copies per infected cell, 2) The analysis of the relationship between the relative amount of HCV in the biopsies and the serum viremia levels. “In situ” hybridization was performed in paraffin embedded liver biopsies from 4 patients with chronic hepatitis C. As probe, a 340 bp cDNA corresponding to the B’non-coding region of the HCV genome was used. The probe was labelled with digoxigenin-ll-ddUTP. The hybridization signals were analyzed by digital image analysis. Hepatitis C virus RNA was detected by “in situ” hybridization in the hepatocytes of the four liver samples. The hybridization signals were found in the cytoplasm although in some hepatocytes nuclear and nucleolar signals were also observed. The relative viral load per infected cell fitted second order polynomial curves in the four cases. The minimum and maximum relative viral load per infected hepatocyte differed in the four cases. However, the mean relative viral load was similar in the four liver samples analyzed (31295, 31090, 31854 and 37996 standard units). The percentage of HCV infected cells ranged from 4.8% to 65.6% in the four cases. The percentage of positive cells for the viral RNA correlates with the serum viremia levels tested by the Amplicor Monitor test (Roche). Our data suggest that HCV viremia in serum does not depend on the relative viral load per infected cell but on the number of infected hepatocytes.
1 PICO5/018
‘. N. A. Pa ’ GO1 -h4asm’ Li& Unit’ and Dept. OfPathology’, St V&at’s
IN HEPATITIS
E. H Hwpital,
C
CO F rrellv’, ERCI, Dublin, Jreland
Hepatitis C virus (HCV) infection is associated with autoimmunity and lymphoprolifemtion. Both CD5 B lymphocytes which produce naturally occurring polyreactive antibodies and @TCR T lymphocytes have been implicated in the pathogenesis of autoimmune diseases. In addition, increased numbers of CD5 B cells in chronic lymphocytic leukaemia, infectious mononucleosis and rheumatoid arthritis are associated with an increase in y8TCR T lymphocytes. Ail: To determine the peripheral blood levels of $TCR T lymphocytes and CD5 B cells in HCV infected individuals and age matched controls. Methods:Peripheral blood mononuclear cells from 13 HCV infected patients and age/sex matched controls were analysed by 3 colour flow cytometry for CD3, CD4, CD8, CD56, y6 and ap TCR. In addition 4 HCV infected individuals, 4 age/sex matched controls and 4 individuals with resolved infection were analysed for CD5 and CD19. Serum from all 12 individuals was analysed by indirect immunofluomsence for anti-nuclear, anti-smooth muscle, anti-parietal cell and antimitochondrial antibodies and by nephelometry for rheumatoid factor. Results: CDS B cells were significantly increased (1~0.02) in HCV infected individuals when compared to HCV RNA negative and normal controls. There was no significant di&rence in T lymphocytes subsets between HCV infected individuals and controls. Of the 4 HCV RNA positive individuals 3 had positive rheumatoid factor, 2 anti-nuclear antibody and 3 anti-smooth muscle antibody while none of the controls had positive auto-antibodies.
~~* .d P-l”.
I
A relatiohshi between iron status and the activity of chronic hepatitis C (H 6 ) has been proposed. Iron overload is frequent in patients with HC, and iron depletion may induce a reduction of transaminases that is not related to a modification of the viral load. A ene in the iron overload of HC
1
CDS B LYMPHOCYTES ARE INCREASED VIRUS INFECTION gM. P.Cu
LIVER IRON DISTRIBUTION, HISTOLOGICAL AClWlTY AND MUTATIONS IN THE HEMOCHROMATOSIS GENE (HFE) IN CHRONIC HEPATITIS C M. Sampietro, R. Boldorini*, N. Corbetta. M. Amato, A. Casatta,
4279
,013
6342
3074
148
4%
3 52
4 76
94w
2.461
1023
64 64
313,
189
291
2 26
419
9563
a02
100
074
0’4
057
006
OL2
OS0
035
Conch~sions: CD5 B cells are increased in chronic HCV infection independently of $iTCR T cells. This increase in CD5 B cells is associated with auto-antibody production and may contribute to the autoimmune phenomenon of HCV infection.
SOLUBLE TUMOR NECROSIS FACTOR RECEPTORS IN CHRONIC HEPATITIS C: A CORRELATION WITH SEVERITY OF LIVER DISEASE. H. Zylberberg’, AC. Rimaniol’, S. Pal’, D. De Groote’, P. Berthelot’, J-F Bach’, C. Br60hot’.3, F. Zavala’. ’ Unlt6 d’HBpatologie, ’ INSERM U-25, 3 INSERM U-370 HBpltal Necker. Tumor necrosis factor-alpha (TNF) IS a mediator of Background: inflammation and cellular Immune response Soluble TNF receptors (sTNFR) sTNF-R55 and sTNF-R75, which compete with cellular receptors for the bindlng of TNF, have been detected at high levels In various mfectlous diseases including HIV and HBV infection Aim:In order to invesbgate the acbvatlon of the TNF system In HCV infecbon, we have analyzed the balance between TNF and sTNF-R I” 60 HCV-Infected subjects according to their clinical. biological, vlrologlcal and histological characteristics. Methods Serum TNF, sTNF-R55 and sTNF-R75 levels were determlned before any therapy and were compared to a control group of 60 healthy subjects. Results: Mean TNF levels were 50 5 + 4.5 pglml In patlents and undetectable (~5 pg/ml) in the control sublects. sTNF-R55 and sTNF-R75 levels were si&A&&tly’higher in HCV-infected patients than in the controls: 2.9? 0.1 nglmlvs 1.3k0.1, (p=O.OOOl), and9.5r0.6 nglmlvs 4.2? 02, (p = 0.0001) respectively. In contrast to other Infectious diseases, there was no correlation between levels of sTNF-R and TNF. sTNF-R75 but not TNF levels were correlated with aminotransferases levels (p = 0.0006 and p = 0.02 for AST and ALT respectively), wh!le sTNF-R55 levels were significantly correlated only with AST levels (p = 0.04). sTNF-R55 and sTNF-R75 were both slgnlflcantly correlated with necroticolnflammatory Index (p = 0 OOl), and their levels were significantly higher in patients with vs wlthout cirrhosis (3.2 f 0 2 vs. 2.5 f 0 2 ng/ml, (~~0.02) and 11.6 f 0.9 vs. 7 5 f 0.5 ng/ml, (p c 0.001) respectively) sTNF-R55. sTNF-R75 and TNF levels were not correlated wtth viral load, genotype or response to Interferon therapy Conclusion: Levels of soluble TNF receptors are strongly correlated with the severity of the disease but not with virological parameters such as quanbtative vlremia and genotype. High TNF-R production could thus suggest that HCV-related liver disease involves immunological mechanisms Including acttvation of the TNF system.