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Detection of hepatitis C virus replication by in situ hybridization: A cautionary note
HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995
AASLD ABSTRACTS
1425 APOPTOSIS AND CLONALGROWTH INHIBITION OF HBV-AND SV-40
1426
VIRUS TRANSFECTEDHEPATOMACELLS BY INTERFERON-eAND TUMOR NECROSIS FACTOR-a. PH Naylor, JM Moshier. G Yared. J Dosescu; MB Nathani, S Reddy, K Hussain. and MGMutchnick. Div. Gastroenterology, Dept. of Internal Medicine, Wayne State Univ. Sch. of Med., Detroit, MI 48201 Cytokines d i r e c t l y
SEROPREVALENCE OF HEPATITIS A IN AMIENS UNIVERSITY HOSPITAL: EPIDEMIOLOGY AND HOSPITAL O C C U P A T I O N A L H A Z A R D . E Nguyen-Khacl, R Delcenserie 1, G Duverlie 2, J Li~nard 3, D Capronl,JP Capron l. 1Service ~ d'Htpato-gastroenttrologie, '-Service de Virologic, 3Service de Mtdecine Prtventive. CHU d'Amiens 80054 Amiens Cedex France. A seroepidemiologie study was carried out beetwen February and June 94 in Amiens University Hospital. The aims of this study were a better description of the epidemiology of hepatitis A, and an estimation of hospital occupational hazard: Methods: Seroepidemiologieal data were collected for each of 525 people ~'~ff~i~"~spital staff) working in the units of Gastroenterology, Pediatry, Internal Medicine, Digestive Radiology, laboratories and in the kitchens and maintenance departments. In these units the participation rate was 88%. Results-" Seroprevalenee of 50:10% was similar to that observed in other but significantly lower than that mesured in French general population (p<0.001) after standardization. High age, low study level, country of birth oF military service in an endemic region and number of siblings more than 2 were significantly associated with anti-hepatitis A antibody. Seroprevalence was not higher in departments exposed to stools (pediatry, digestive endoscopy and laboratory). Any seroprevalence difference was observed between clinical services and no clinical services. High rate of antibody was observed in internal medicine, kitchens, maintenance department and laboratories compared with radiology. However, after adjustment on extra professional parameters, the difference disappeared and hazard is similar to that observed in general population. C o n c l u s i o n s : 1) This study confirms the decrease of the seroprevalence of hepatitis A in France. 2) Age and socioeconomic factors are very important in the epidemiology of hepatitis A. These factors must be taken into account for the evaluation of occupational risk. 3) In thi s study, the hospital occupational hazard seems to be lower than that measured in general population.
DETECTION OF HEPATITIS C VIRUS REPLICATION BY IN SITU HYBRIDIZATION: A CAUTIONARY NOTE F Negro, E Giostra, ~L. Ru.u_b_b_ia_:_B_r_~dk_~_G_~__M_ en_tl3_a~A__H~__d__eng_u_e_.Gastroenterology Division, Depts. of IPathology and 2Surgery, University Hospital, Geneva, Switzerland. A sensitive and specific procedure for detecting hepatitis C virus (HCV) RNA by in situ hybridization(lSH) would be of paramount importance in view of its molecular virology and clinical applications. Although several ISH protocols for detecting HCV RNA have been published, many doubts remain as to their dependability, since 1) none has been independently reproduced, and 2) results are widely diverging in terms of viral tropism and level of expression. We critically reviewed most published protocols in an attempt to assess their validity. Liver tissues from chronic hepatitis C patients were fixed either in formalin or in paraformaldehyde and stat frozen under cryopreserving conditions. Probes were: a mixture of oligonucleotides, double- and single-stranded PCR-generated DNA probes (core and 5'-noncoding region), or a mixture of 12 short (200 to 450 bases) single-stranded RNA probes covering up to 40% of the H c v genome (portions of the 5'-noncoding, core, NS2, NS3, NS4 and NS5 regions). Labels were digoxigenin, aSS or 33p. Hybrids were revealed by alkaline phosphatase (AP)-conjugated anti-digoxigenin antibodies with or without the AP/anti-AP amplification, or by autoradiography: Specificity experiments included; use of an extensive series of bona fide HCV RNA-negative tissues (by RT-PCR), predigestion of sections with RNase, use of unrelated probes, crnss-matching of the signal localization obtained with digoxigenin-labeled probes with that obtained with the same probe radioactively, and analysis of the melting curve of hybrids detected in HCV-pnsitive and -negative tissues. Although a cytoplasmic signal could be easily detected in hepatocytes, irrespectively of the~label, findings were clearly due to a cross-hybridization with a host RNA (i.e. not specific), as they did not satisfy all the above specificity criteria. In detail, the analysis of signal localization obtained with different labels and the behaviour of the hybrids meking curves proved to be crucially discriminating. The other specificity experiments (universally employed to validate most ISH procedures) were often insufficient. These results underscore the importance of stringent conditions to optimize ISH protocols for detecting HCV RNA and the need for a critical reappraisal of the concept of specificity of ISH findings published so far.
i n f l u e n c e t h e c l e a r a n c e of v i r a l
infection by decreasing viral gene expression and/or by destroying virus infected cells. HepG2 hepatoblastoma cells transfected w i t h e i t h e r HBV or SV-40 virus (HBV-HepG2 or SV-40-HepG2, respectively) were used to define the direct effects of cytokines on cell growth by 14 days and death by apoptosis at 24 hrs. Tumor Necrosis Factor-a (TNF-a; 50 ng/ml) significantly inhibited the clonal growth of the HepG2 (89% of the media growth) and the HBV-HepG2 (56%). However, TNF-a enhancedthe growth of the SV-40HepG2 cells (500%). Interferon-e (IFN-a) at 100 ng/ml inhibited the growth of all cells: HepG2cells (12%), HBVHepG2 cells (23%), and SV-40 HepG2cells (44%). Cell death by apoptosis (MTT assay and DNA ladders) was induced by TNF-a in HepG2 (20%) and HBV-HepG2(40%) cells. In contrast to the clonogenlc assay, TNF-a had no effect on SV-40-HepG2 cells. IFN-a had no cytotoxic effect even at concentrations up to 10 ug/ml. Preincubation of the HBVHepG2 ceils with IFN-a, IFN-y, or TNF-B had no effect on subsequent TNF-a induced apoptosis. These studies are consistent with the anti-viral a c t i v i t y described for both TNF-a and IFN-m Furthermore, the HepG2 viral transfected cells are useful in v i t r o models for defining the mechanism(s) of the anti-viral actions of TNF-a and IFN-a. The contrast between the effects of the two cytokines on cells transfected with two different viruses emphasizesthe complexity of the roles of TNF-e and IFN-a in controlling viral infection.
1427
463A
1428
EFFECTS OF INTRACELLULAR PH AND NUTRITIONAL STATE OF DONOR ON TRANSPLANTED LIVER FUNCTIONS. T Nishida. Y Morimoto. W Kamiike. T Huan,,. H Kazuo. E Hamada, and H Matsuda. First Dept. Surgery, Osaka 13niversity Medical School, Osaka, Japan [INTRODUCTION] To improve viability of the liver after transplantation, optimal nutritional conditions of donor livers and pH values of the solution, both of which affect intracellular pH (pHi), are important but not established. In this study, changes in pHi during preservation and donor's nutritional conditions were evaluated. [MATERIALS & METHODS] Inbred Lewis rats were divided into fed and fasted (24h) groups. Two different UW solutions, adjusted to pH 7.45 (UW7.45) or 6.70 (UW6.70), Were used for the preservation. Livers were harvested by the technique of Kamada and Calne, and were stored in each solution at 4°C. pHi of preserved livers was measured using 31P-NMR (Bruker AM 400). Changes in energy status (ATP, energy charge) and lactate production were examined during preservation. Orthotupic liver transplantation was performed after g h preservation. Bile flow rate, and plasma AST and LDH were determined after reperfusion. [RESULTS] In fasted rats, pHi, which was rapidly equilibrated with pH values of UW solutions, was not significantly changed throughout preservation. In fed rats, pHi gradually decreased to 6.6, but pHi of UW7.45 was maintained relatively high compared with that of UW6.70. Lactate production, which was scarcely observed in fasted livers, was higher in fed livers of UW7.45 than in those of UW6.70. Hepatic ATP level and energy charge of fed livers with UW7.45 were maintained higher than those of fed livers with UW6.70 and fasted livers with UW7.45 or UW6.70. Bile flow rate after transplantation of fed livers with UW7.45 was higher than that of the other three groups. Plasma AST and LDH levels after transplantation were not significantly different among four groups. [CONCLUSION] These results suggested that pHi was rapidly equilibrated with flush solution in fasted donors but p h i of fed donors was regulated by lactate production during preservation and that graft functions might be affected by donor's nutritional conditions, but not by pH values of UW solutions.
AASLD ABSTRACTS
1425 APOPTOSIS AND CLONALGROWTH INHIBITION OF HBV-AND SV-40
1426
VIRUS TRANSFECTEDHEPATOMACELLS BY INTERFERON-eAND TUMOR NECROSIS FACTOR-a. PH Naylor, JM Moshier. G Yared. J Dosescu; MB Nathani, S Reddy, K Hussain. and MGMutchnick. Div. Gastroenterology, Dept. of Internal Medicine, Wayne State Univ. Sch. of Med., Detroit, MI 48201 Cytokines d i r e c t l y
SEROPREVALENCE OF HEPATITIS A IN AMIENS UNIVERSITY HOSPITAL: EPIDEMIOLOGY AND HOSPITAL O C C U P A T I O N A L H A Z A R D . E Nguyen-Khacl, R Delcenserie 1, G Duverlie 2, J Li~nard 3, D Capronl,JP Capron l. 1Service ~ d'Htpato-gastroenttrologie, '-Service de Virologic, 3Service de Mtdecine Prtventive. CHU d'Amiens 80054 Amiens Cedex France. A seroepidemiologie study was carried out beetwen February and June 94 in Amiens University Hospital. The aims of this study were a better description of the epidemiology of hepatitis A, and an estimation of hospital occupational hazard: Methods: Seroepidemiologieal data were collected for each of 525 people ~'~ff~i~"~spital staff) working in the units of Gastroenterology, Pediatry, Internal Medicine, Digestive Radiology, laboratories and in the kitchens and maintenance departments. In these units the participation rate was 88%. Results-" Seroprevalenee of 50:10% was similar to that observed in other but significantly lower than that mesured in French general population (p<0.001) after standardization. High age, low study level, country of birth oF military service in an endemic region and number of siblings more than 2 were significantly associated with anti-hepatitis A antibody. Seroprevalence was not higher in departments exposed to stools (pediatry, digestive endoscopy and laboratory). Any seroprevalence difference was observed between clinical services and no clinical services. High rate of antibody was observed in internal medicine, kitchens, maintenance department and laboratories compared with radiology. However, after adjustment on extra professional parameters, the difference disappeared and hazard is similar to that observed in general population. C o n c l u s i o n s : 1) This study confirms the decrease of the seroprevalence of hepatitis A in France. 2) Age and socioeconomic factors are very important in the epidemiology of hepatitis A. These factors must be taken into account for the evaluation of occupational risk. 3) In thi s study, the hospital occupational hazard seems to be lower than that measured in general population.
DETECTION OF HEPATITIS C VIRUS REPLICATION BY IN SITU HYBRIDIZATION: A CAUTIONARY NOTE F Negro, E Giostra, ~L. Ru.u_b_b_ia_:_B_r_~dk_~_G_~__M_ en_tl3_a~A__H~__d__eng_u_e_.Gastroenterology Division, Depts. of IPathology and 2Surgery, University Hospital, Geneva, Switzerland. A sensitive and specific procedure for detecting hepatitis C virus (HCV) RNA by in situ hybridization(lSH) would be of paramount importance in view of its molecular virology and clinical applications. Although several ISH protocols for detecting HCV RNA have been published, many doubts remain as to their dependability, since 1) none has been independently reproduced, and 2) results are widely diverging in terms of viral tropism and level of expression. We critically reviewed most published protocols in an attempt to assess their validity. Liver tissues from chronic hepatitis C patients were fixed either in formalin or in paraformaldehyde and stat frozen under cryopreserving conditions. Probes were: a mixture of oligonucleotides, double- and single-stranded PCR-generated DNA probes (core and 5'-noncoding region), or a mixture of 12 short (200 to 450 bases) single-stranded RNA probes covering up to 40% of the H c v genome (portions of the 5'-noncoding, core, NS2, NS3, NS4 and NS5 regions). Labels were digoxigenin, aSS or 33p. Hybrids were revealed by alkaline phosphatase (AP)-conjugated anti-digoxigenin antibodies with or without the AP/anti-AP amplification, or by autoradiography: Specificity experiments included; use of an extensive series of bona fide HCV RNA-negative tissues (by RT-PCR), predigestion of sections with RNase, use of unrelated probes, crnss-matching of the signal localization obtained with digoxigenin-labeled probes with that obtained with the same probe radioactively, and analysis of the melting curve of hybrids detected in HCV-pnsitive and -negative tissues. Although a cytoplasmic signal could be easily detected in hepatocytes, irrespectively of the~label, findings were clearly due to a cross-hybridization with a host RNA (i.e. not specific), as they did not satisfy all the above specificity criteria. In detail, the analysis of signal localization obtained with different labels and the behaviour of the hybrids meking curves proved to be crucially discriminating. The other specificity experiments (universally employed to validate most ISH procedures) were often insufficient. These results underscore the importance of stringent conditions to optimize ISH protocols for detecting HCV RNA and the need for a critical reappraisal of the concept of specificity of ISH findings published so far.
i n f l u e n c e t h e c l e a r a n c e of v i r a l
infection by decreasing viral gene expression and/or by destroying virus infected cells. HepG2 hepatoblastoma cells transfected w i t h e i t h e r HBV or SV-40 virus (HBV-HepG2 or SV-40-HepG2, respectively) were used to define the direct effects of cytokines on cell growth by 14 days and death by apoptosis at 24 hrs. Tumor Necrosis Factor-a (TNF-a; 50 ng/ml) significantly inhibited the clonal growth of the HepG2 (89% of the media growth) and the HBV-HepG2 (56%). However, TNF-a enhancedthe growth of the SV-40HepG2 cells (500%). Interferon-e (IFN-a) at 100 ng/ml inhibited the growth of all cells: HepG2cells (12%), HBVHepG2 cells (23%), and SV-40 HepG2cells (44%). Cell death by apoptosis (MTT assay and DNA ladders) was induced by TNF-a in HepG2 (20%) and HBV-HepG2(40%) cells. In contrast to the clonogenlc assay, TNF-a had no effect on SV-40-HepG2 cells. IFN-a had no cytotoxic effect even at concentrations up to 10 ug/ml. Preincubation of the HBVHepG2 ceils with IFN-a, IFN-y, or TNF-B had no effect on subsequent TNF-a induced apoptosis. These studies are consistent with the anti-viral a c t i v i t y described for both TNF-a and IFN-m Furthermore, the HepG2 viral transfected cells are useful in v i t r o models for defining the mechanism(s) of the anti-viral actions of TNF-a and IFN-a. The contrast between the effects of the two cytokines on cells transfected with two different viruses emphasizesthe complexity of the roles of TNF-e and IFN-a in controlling viral infection.
1427
463A
1428
EFFECTS OF INTRACELLULAR PH AND NUTRITIONAL STATE OF DONOR ON TRANSPLANTED LIVER FUNCTIONS. T Nishida. Y Morimoto. W Kamiike. T Huan,,. H Kazuo. E Hamada, and H Matsuda. First Dept. Surgery, Osaka 13niversity Medical School, Osaka, Japan [INTRODUCTION] To improve viability of the liver after transplantation, optimal nutritional conditions of donor livers and pH values of the solution, both of which affect intracellular pH (pHi), are important but not established. In this study, changes in pHi during preservation and donor's nutritional conditions were evaluated. [MATERIALS & METHODS] Inbred Lewis rats were divided into fed and fasted (24h) groups. Two different UW solutions, adjusted to pH 7.45 (UW7.45) or 6.70 (UW6.70), Were used for the preservation. Livers were harvested by the technique of Kamada and Calne, and were stored in each solution at 4°C. pHi of preserved livers was measured using 31P-NMR (Bruker AM 400). Changes in energy status (ATP, energy charge) and lactate production were examined during preservation. Orthotupic liver transplantation was performed after g h preservation. Bile flow rate, and plasma AST and LDH were determined after reperfusion. [RESULTS] In fasted rats, pHi, which was rapidly equilibrated with pH values of UW solutions, was not significantly changed throughout preservation. In fed rats, pHi gradually decreased to 6.6, but pHi of UW7.45 was maintained relatively high compared with that of UW6.70. Lactate production, which was scarcely observed in fasted livers, was higher in fed livers of UW7.45 than in those of UW6.70. Hepatic ATP level and energy charge of fed livers with UW7.45 were maintained higher than those of fed livers with UW6.70 and fasted livers with UW7.45 or UW6.70. Bile flow rate after transplantation of fed livers with UW7.45 was higher than that of the other three groups. Plasma AST and LDH levels after transplantation were not significantly different among four groups. [CONCLUSION] These results suggested that pHi was rapidly equilibrated with flush solution in fasted donors but p h i of fed donors was regulated by lactate production during preservation and that graft functions might be affected by donor's nutritional conditions, but not by pH values of UW solutions.