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Stability and detection of HCV RNA by RT-PCR in plasma and peripheral blood mononuclear cells (PBMCs)
466A 1437
AASLD ABSTRACTS STABILITY AND DETECTION OF HCV RNA BY R T - P C R IN PLASMA AND PERIPHERAL BLOOD M O N O N U C L E A R CELLS (PBMCs). CB O'Brien 1, B Henzel 1, L Wolfe 2 and A Yoder2. ILiver Center, University of Pennsylvania Medical Center, Philadelphia, PA 19104 and 2Roche Molecular Systems, Branchburg, NJ 08876. OBJECTIVES: 1) To determine if HCV RNA could be detected by RTPCR in plasma and PBMCs from blood samples collected in Vacutainer CPT tubes. 2) To determine the stability of HCV RNA present in mononuclear cells and plasma over time. METHODS: Five 8 ml VACUTAINER CPT tubes (Becton Dickinson VACUTA/NER Systems) of whole blood were collected from five patients with chroriic HCV. All tubes were centrifuged for 20 minutes at 1800 xg within 2 hours of blood draw to separate the PBMCs and plasma from whole blood. One tube from each patient was set aside to be processed immediately. Three 1 ml aliquots of plasma were removed and stored at -70°C. The cells were pelleted and divided into 4 aliquots and stored at -70°C. The remaining tubes were inverted 5 to 8 times to mix cell layer with plasma and placed at 4°C. One tube from each patient was processed at 3, 24, 48 and 72 hours from the time of centrifugation. The samples were extracted using GuSCN lysis buffer reconstituted with carrier and 2-mercaptoethanol. Two amplifications were run per extraction. A prototype HCV RNA assay (Roche Molecular Systems) which involves a single primer pair (5'untranslated region of HCV, biotinylated downstream primer) with a single enzyme (rTth DNA polymerase) was used to carry out both RT and DNA polymerase functions. Uracil,N-glycosylase was incorporated to prevent amplicon contamination. Detection was carried out in a microwell (AvidinHRP) format. RESULTS: The following contains the mean HCV RNA titers of the PBMCs and the plasma at the 5 time points. Timefhrs) PBMC's Plasma
0 5.12E+03 9.86E+05
3 2.83E+03 9.56E+05
24 5.17E+03 1.97E+06
48 1.30E+04 1.18E+06
1438
CONCLUSION: The VACUTAINER CPT tube can be used to measure HCV RNA in plasma or mononuclear cells. This tube provides an easy, one-step method for collection and processing of mononuclear cells and plasma. The results show the HCV RNA is stable for up to 72 hours in both plasma and cells stored at 4°C provided whole blood is spun within 2 hours of collection. The cell titers are lower than the plasma titers possibly due to the small size of the pellets.
FACTOR (EGF) RECEPTOR IS IMPAIRED AFTER CHRONIC ETHANOL ADMINISTRATION. MF O~,ourke. DJ Tuma and CA Casev, Liver Study Unit, VA Medical Center and the University of Nebraska Medical Center, Omaha NE 68105 We have previously shown that chronic ethanol feeding decreases binding of epidermal growth factor (EGF) by 40-50% to isolated rat hepatocytes. This decreased ligand binding was observed at both high affinity and low affinity binding states. In contrast, binding of anti-EGF receptor antibody was not altered. Thus, chronic ethanol feeding decreases EGF binding to its receptor without corresponding changes in receptor number or affinity, suggesting that receptor inactivation may be the primary factor involved in impaired EGF binding. The EGF receptor consists of an extracellular binding domain, a short transmembrane region, and intracellular tyrosine kinase and regulatory domains. Binding of EGF to its receptor is known to induce autophosphorylation of the EGF receptor at a tyrosine residue, initiating the signal transduction cascade ultimately leading to the stimulation of DNA synthesis, cell growth and cell division. The purpose of the present study was to determine whether the ethanolassociated decreased ability of the EGF receptor to bind ligand affected the ability of the receptor to become phosphorylated. For these studies, male, Wistar rats were fed ethanol-containing or control diets for 5-7 weeks. Isolated hepatocytes were then incubated with 5 nM EGF at room temperature for 2 min. Ligand binding was determined in one set of cell samples. In a parallel set, cells were lysed and the EGF receptor immunoprecipitated, subjected to SDS-PAGE, and transferred to Hybond membranes. The receptor was then probed with anti-phoshotyrosine antibody conjugated to horseradish peroxidase, and detected with an ECL detection kit. Results from 9 pairs of animals showed that ligand binding after 2 min was decreased by 40-45% (p<0.05) in the ethanol cells. In addition, the ability of the EGF receptor in ethanol-fed animals to become phosphorylated was decreased by 30-35% (p<0.005) compared to controls. These decreases in ligand binding and autophosphorylation of the EGF receptor a~er ethanol treatment may impair proper functioning of the signalling cascade associated with EGF-stimulation in these cells.
VARIABILITY IN HCV RNA QUANTITATION: THE NEED FOR REPEAT DETERMINATIONS. J. O'Connel4 M. McCarthy, E. Kenny-Walsh, J.K. Collins~ M.J. Whelton. Departments of Hepatology and Medicine, University Hospital, Cork, Ireland. Quantitative HCV RNA measurement appears an ideal monitor of HCV viral load. Repeat testing of specimens from a unique cohort of Irish women infected with HCV contaminated Anti-D immunoglobulin using the Roche Amplicor MonitorTM Quantitative PCR kit indicated limited reproducibility. A direct assessment was performed to establish and define the variability of the test. Methods 15 aliquots (5 x 0.1 ml) were obtained from sera of three (5 each) HCV RNA positive patients and frozen at -86oc. Each specimen was then tested independently by the Roche MonitorTM procedure exactly according to the manufacturer's protocol. Statistical analysis was performed on each of the 3 sets of 5 results. Results The three sera had mean titres of 5.2 ± 2.1 x 103; 2.6 ± 0.55 x 105 and 1.3 ± 0.49 x 106 eq ml"1, with percentage coefficients of variation of 40, 21 and 38 respectively (n=5). The viral titres showed 4-fold, 2-fold and 3-fold discrepancies respectively between the highest and lowest determinations in each set of 5 measurements. Conclusion Reproducibility of quantitative HCV RNA measurements with the Roche Amplicor MonitorTM HCV test is limited. Percentage coefficients of variation range from 21 to 40 in 15 specimens tested (5 each from 3 patients). Repeat tests were however consistently within 40% C.V. Results of quantitative testing by this technique must take account of these limitations especially where quantitative data from a single measurement only is recorded.
72 2.00E+04 5.94E+05
1439 AUTOPHOSPHORYLATION OF THE EPIDERMAL GROWTH
HEPATOLOGY October 1995
1440
EFFECTS OF SIMVASTATIN, PENTOXYFYLLINE AND SPIRONOLACTONE ON HEPATIC FIBROSIS AND PORTAL H Y P E R T E N S I O N IN B I L E DUCT L I G A T E D RATS. F. Oberti. C. P i l e t t e . H. Rifflet. M. M a i ~ a . A. M o r e a u . y, Gallois. A. Girault. JJ. Le Jeune. G. Feldmann, P, Cal~s. CHU A n g e r s and I N S E R M U327 Paris, France. Simvastatin (SVT) and p e n t o x y f y l l i n e (PTX) are inhibitors of Ito cell proliferation. Spironolactone (SPN) p r o b a b l y d e c r e a s e s m y o c a r d i a l fibrosis. Moreover, two of these drugs have vasoactive properties. O u r a i m was to s t u d y the antifibrotic and hemodynamic effects of these d r u g s in a m o d e l of b i l i a r y cirrhosis in rats. B l i n d s t u d y was p e r f o r m e d in 4 g r o u p s of S p r a g u e D a w l e y rats : placebo (PL) (n~9), SVT (n=9, 2.5 m g / k g / j ) , PTX (n=9, 50 mg/kg/j) a n d SPN (n=ll, i00 m g / k g / j ) . D r u g s w e r e a d m i n i s t e r e d by d a i l y g a v a g e over a 4 week p e r i o d as soon as bile duct l i g a t i o n was p e r f o r m e d . At day 28, the f o l l o w i n g p a r a m e t e r s were evaluated : liver fibrosis area by image analysis after picrosirius staining and serum levels of h y a l u r o n a t e , splanchnio and systemic hemodynamics (radiolabelled microspheres). R e s u l t s : p o r t a l v e n o u s p r e s s u r e (PL : 16±2, SVT : 16±2, P T X : 15±1, SPN : 14±2 mmHg, P<0.01) and p o r t o - s y s t e m i c shunts (PL : 25±32, SVT : 19±28, PTX : 22±22, SPN : 7±2 %, p < 0 . 0 5 ) w e r e s i g n i f i c a n t l y reduced in t h e SPN group ; other hemodynamic parameters were not significantly altered. H y a l u r o n a t e level was i0 t i m e s h i g h e r in bile duct ligated rats than in normal rats and was correlated with fibrosis area (r=0.48, p<0.05). The liver fibrosis area was not significantly d i f f e r e n t a m o n g the 4 g r o u p s of f i b r o t i c rats (PL : 8±4, SVT : 7~4, PTX : 9±3, SPN : 7±4 %). Conclusion in t h i s m o d e l , none of the drugs p r e v e n t e d h e p a t i c fibrosis. On the o t h e r hand, SPN decreased portal pressure and prevented portosystemic shunts. Therefore, this drug may have b e n e f i c i a l e f f e c t s in early p o r t a l h y p e r t e n s i o n .
AASLD ABSTRACTS STABILITY AND DETECTION OF HCV RNA BY R T - P C R IN PLASMA AND PERIPHERAL BLOOD M O N O N U C L E A R CELLS (PBMCs). CB O'Brien 1, B Henzel 1, L Wolfe 2 and A Yoder2. ILiver Center, University of Pennsylvania Medical Center, Philadelphia, PA 19104 and 2Roche Molecular Systems, Branchburg, NJ 08876. OBJECTIVES: 1) To determine if HCV RNA could be detected by RTPCR in plasma and PBMCs from blood samples collected in Vacutainer CPT tubes. 2) To determine the stability of HCV RNA present in mononuclear cells and plasma over time. METHODS: Five 8 ml VACUTAINER CPT tubes (Becton Dickinson VACUTA/NER Systems) of whole blood were collected from five patients with chroriic HCV. All tubes were centrifuged for 20 minutes at 1800 xg within 2 hours of blood draw to separate the PBMCs and plasma from whole blood. One tube from each patient was set aside to be processed immediately. Three 1 ml aliquots of plasma were removed and stored at -70°C. The cells were pelleted and divided into 4 aliquots and stored at -70°C. The remaining tubes were inverted 5 to 8 times to mix cell layer with plasma and placed at 4°C. One tube from each patient was processed at 3, 24, 48 and 72 hours from the time of centrifugation. The samples were extracted using GuSCN lysis buffer reconstituted with carrier and 2-mercaptoethanol. Two amplifications were run per extraction. A prototype HCV RNA assay (Roche Molecular Systems) which involves a single primer pair (5'untranslated region of HCV, biotinylated downstream primer) with a single enzyme (rTth DNA polymerase) was used to carry out both RT and DNA polymerase functions. Uracil,N-glycosylase was incorporated to prevent amplicon contamination. Detection was carried out in a microwell (AvidinHRP) format. RESULTS: The following contains the mean HCV RNA titers of the PBMCs and the plasma at the 5 time points. Timefhrs) PBMC's Plasma
0 5.12E+03 9.86E+05
3 2.83E+03 9.56E+05
24 5.17E+03 1.97E+06
48 1.30E+04 1.18E+06
1438
CONCLUSION: The VACUTAINER CPT tube can be used to measure HCV RNA in plasma or mononuclear cells. This tube provides an easy, one-step method for collection and processing of mononuclear cells and plasma. The results show the HCV RNA is stable for up to 72 hours in both plasma and cells stored at 4°C provided whole blood is spun within 2 hours of collection. The cell titers are lower than the plasma titers possibly due to the small size of the pellets.
FACTOR (EGF) RECEPTOR IS IMPAIRED AFTER CHRONIC ETHANOL ADMINISTRATION. MF O~,ourke. DJ Tuma and CA Casev, Liver Study Unit, VA Medical Center and the University of Nebraska Medical Center, Omaha NE 68105 We have previously shown that chronic ethanol feeding decreases binding of epidermal growth factor (EGF) by 40-50% to isolated rat hepatocytes. This decreased ligand binding was observed at both high affinity and low affinity binding states. In contrast, binding of anti-EGF receptor antibody was not altered. Thus, chronic ethanol feeding decreases EGF binding to its receptor without corresponding changes in receptor number or affinity, suggesting that receptor inactivation may be the primary factor involved in impaired EGF binding. The EGF receptor consists of an extracellular binding domain, a short transmembrane region, and intracellular tyrosine kinase and regulatory domains. Binding of EGF to its receptor is known to induce autophosphorylation of the EGF receptor at a tyrosine residue, initiating the signal transduction cascade ultimately leading to the stimulation of DNA synthesis, cell growth and cell division. The purpose of the present study was to determine whether the ethanolassociated decreased ability of the EGF receptor to bind ligand affected the ability of the receptor to become phosphorylated. For these studies, male, Wistar rats were fed ethanol-containing or control diets for 5-7 weeks. Isolated hepatocytes were then incubated with 5 nM EGF at room temperature for 2 min. Ligand binding was determined in one set of cell samples. In a parallel set, cells were lysed and the EGF receptor immunoprecipitated, subjected to SDS-PAGE, and transferred to Hybond membranes. The receptor was then probed with anti-phoshotyrosine antibody conjugated to horseradish peroxidase, and detected with an ECL detection kit. Results from 9 pairs of animals showed that ligand binding after 2 min was decreased by 40-45% (p<0.05) in the ethanol cells. In addition, the ability of the EGF receptor in ethanol-fed animals to become phosphorylated was decreased by 30-35% (p<0.005) compared to controls. These decreases in ligand binding and autophosphorylation of the EGF receptor a~er ethanol treatment may impair proper functioning of the signalling cascade associated with EGF-stimulation in these cells.
VARIABILITY IN HCV RNA QUANTITATION: THE NEED FOR REPEAT DETERMINATIONS. J. O'Connel4 M. McCarthy, E. Kenny-Walsh, J.K. Collins~ M.J. Whelton. Departments of Hepatology and Medicine, University Hospital, Cork, Ireland. Quantitative HCV RNA measurement appears an ideal monitor of HCV viral load. Repeat testing of specimens from a unique cohort of Irish women infected with HCV contaminated Anti-D immunoglobulin using the Roche Amplicor MonitorTM Quantitative PCR kit indicated limited reproducibility. A direct assessment was performed to establish and define the variability of the test. Methods 15 aliquots (5 x 0.1 ml) were obtained from sera of three (5 each) HCV RNA positive patients and frozen at -86oc. Each specimen was then tested independently by the Roche MonitorTM procedure exactly according to the manufacturer's protocol. Statistical analysis was performed on each of the 3 sets of 5 results. Results The three sera had mean titres of 5.2 ± 2.1 x 103; 2.6 ± 0.55 x 105 and 1.3 ± 0.49 x 106 eq ml"1, with percentage coefficients of variation of 40, 21 and 38 respectively (n=5). The viral titres showed 4-fold, 2-fold and 3-fold discrepancies respectively between the highest and lowest determinations in each set of 5 measurements. Conclusion Reproducibility of quantitative HCV RNA measurements with the Roche Amplicor MonitorTM HCV test is limited. Percentage coefficients of variation range from 21 to 40 in 15 specimens tested (5 each from 3 patients). Repeat tests were however consistently within 40% C.V. Results of quantitative testing by this technique must take account of these limitations especially where quantitative data from a single measurement only is recorded.
72 2.00E+04 5.94E+05
1439 AUTOPHOSPHORYLATION OF THE EPIDERMAL GROWTH
HEPATOLOGY October 1995
1440
EFFECTS OF SIMVASTATIN, PENTOXYFYLLINE AND SPIRONOLACTONE ON HEPATIC FIBROSIS AND PORTAL H Y P E R T E N S I O N IN B I L E DUCT L I G A T E D RATS. F. Oberti. C. P i l e t t e . H. Rifflet. M. M a i ~ a . A. M o r e a u . y, Gallois. A. Girault. JJ. Le Jeune. G. Feldmann, P, Cal~s. CHU A n g e r s and I N S E R M U327 Paris, France. Simvastatin (SVT) and p e n t o x y f y l l i n e (PTX) are inhibitors of Ito cell proliferation. Spironolactone (SPN) p r o b a b l y d e c r e a s e s m y o c a r d i a l fibrosis. Moreover, two of these drugs have vasoactive properties. O u r a i m was to s t u d y the antifibrotic and hemodynamic effects of these d r u g s in a m o d e l of b i l i a r y cirrhosis in rats. B l i n d s t u d y was p e r f o r m e d in 4 g r o u p s of S p r a g u e D a w l e y rats : placebo (PL) (n~9), SVT (n=9, 2.5 m g / k g / j ) , PTX (n=9, 50 mg/kg/j) a n d SPN (n=ll, i00 m g / k g / j ) . D r u g s w e r e a d m i n i s t e r e d by d a i l y g a v a g e over a 4 week p e r i o d as soon as bile duct l i g a t i o n was p e r f o r m e d . At day 28, the f o l l o w i n g p a r a m e t e r s were evaluated : liver fibrosis area by image analysis after picrosirius staining and serum levels of h y a l u r o n a t e , splanchnio and systemic hemodynamics (radiolabelled microspheres). R e s u l t s : p o r t a l v e n o u s p r e s s u r e (PL : 16±2, SVT : 16±2, P T X : 15±1, SPN : 14±2 mmHg, P<0.01) and p o r t o - s y s t e m i c shunts (PL : 25±32, SVT : 19±28, PTX : 22±22, SPN : 7±2 %, p < 0 . 0 5 ) w e r e s i g n i f i c a n t l y reduced in t h e SPN group ; other hemodynamic parameters were not significantly altered. H y a l u r o n a t e level was i0 t i m e s h i g h e r in bile duct ligated rats than in normal rats and was correlated with fibrosis area (r=0.48, p<0.05). The liver fibrosis area was not significantly d i f f e r e n t a m o n g the 4 g r o u p s of f i b r o t i c rats (PL : 8±4, SVT : 7~4, PTX : 9±3, SPN : 7±4 %). Conclusion in t h i s m o d e l , none of the drugs p r e v e n t e d h e p a t i c fibrosis. On the o t h e r hand, SPN decreased portal pressure and prevented portosystemic shunts. Therefore, this drug may have b e n e f i c i a l e f f e c t s in early p o r t a l h y p e r t e n s i o n .