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Regulation of hepatitis B virus enhancer activity by Phyllanthus niruri is involved in its antiviral effects
Al140
AASLD ABSTRACTS
GASTROENTEROLOGY, Vol. 1 0 8 , NO. 4
• REGULATION OF HEPATITIS B VIRUS ENHANCER ACTIVITY BY PHYLLANTHUS NIRURI IS INVOLVED IN ITS ANTIVIRAL EFFECTS.
I N T E R E S T O F H Y A L U R O N I C A C I D (HA) IN ASSESSMENT OF LIVER FIBROSIS IN CHRONIC VIRAL HEPATITIS C H. OUI, H. TROUETTE, B. LE BAIL, G. LACAPE, M. FONCK, F. DUMAS, P. COUZIGOU Serv. d'Hdpatogastroont~rologia H6pital Haut-Lev6que 33604 PESSAC France
M. Ott, S.P. Thyagarajan" and S. Gupta. Liver Research Center, Albert Einstein College of Medicine, NY and "Department of Microbiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, India.
• In liver fibrosis, the basement menbranes appear belong the sinusmdal walls (capillarizafion), so the function of the endothelial cells are impaired. More than 90 % of circulating hyaluronic Acide (HA) is degraded in hepatic endothelial cells. The blood HA level is known to increase markedly in patients with viral cirrhosis and in primary biliary cirrhosis. • The a i m o f the study was to assess HA as a marker of fibrosis in chronic hepatitis C • Methods : 141 patients (74 males and 67 females, mean : 49 years range 19-74) with chronic viral hepatitis C (43 cirrhosis, 41 child A) underwent a liver biopsy. All samples were examined by one anatomopathologist, and the degree of fibrosis was graded semiquantitatively according to 2 scoring systems (Kn6dell- Cbevallier). At that time, blood samples were collected to determine HA and amino peptide procollagen Ill (PIIINP) with commercial RIA kits (Behring- Pharmacia) - Kurskall-Wallis analysis of variance was used to compare serum PIIINP and HA levels according to the histological score o f fibrosis.
An extract from Phyllanthus niruri was recently shown to inhibit HBV DNA polymerase, as well as to transcriptionally downregulate HBV mRNA expression. To define mechanisms in transcriptional control of HBV after exposure to R niruri, we investigated whether R niruri affected the HBV enhancer I (HBEn) or an HBsAg promoter (HBsP). The HBV sequences were derived from the pCP10 plasmid (HBEn, map positions (rap) 490-1402; HBsP, mp 2425-2839) and cloned into luciferase (luc) expression plasmids. The plasmid pHBEn/HBsP-luc contained HBV regulatory elements, pHBEn/SV40P-luc contained the HBEn driving the SV40 promoter and pSV40En/HBsP-luc contained the SV40 enhancer and HBsP. The ptasmids pGL2-control, containing the SV40 enhancer and promoter, and pGL2-basic with no regulatory elements (Promega Corp.), served as positive or negative controls, respectively. When various plasmids were introduced via Lipcfectin T M into cultured HepG2 cells,/uc was expressed by all plasmids except pGL2-basic. A DMSO-soluble R niruri extract was added to the culture medium 6 hrs after gene transfer and cells lysed at 48 hrs to measure luc activity. In contrast with vehicle alone, R niruridose-dependently (up to : 2 0 0 pg/ml crude extract) suppressed/uc maximally in cells transfected with pHBEn/HBsP-luc, to 4 8 % + 1 0 % (p<0.001), followed by pHBEn/SV40P-luc, to 79%__+14% (p<0.03), of control cells. In plasmids pSV40En/HBsP-luc, pGL2-control and pGL2basic, R niruri did not alter luc expression: R niruri had no effect on either cell viability as demonstrated by utilization of MTT (Thiazolyl blue) or expression of an E. coil lacZ gene driven by the SV40 enhancer/promoter elements. CONCLUSIONS: R niruri downregulated HBEn activity in a specific manner with no impairment in cellular gene expression. Whether R niruri binds to specific HBEn domains or alters interactions between HBEn and cellular transactivators via additional mechanisms requires further analysis.
CARBOHYDRATE-DEFICIENT TRANSFERRIN (CDT) AND LIVER DISEASES. F Ouyahva. Y Bacq, F Schellenberg, E-H Metman, J Weiil. Service d'h~patogastroent~rologie et laboratoire de biochimie, HSpital Trousseau, Tours, France. CDT has been proposed as a marker of alcohol consumption. The aim of this study was to evaluate the accuracy of CDT for the diagnosis of excessive alcohol intake in patients with liver diseases. Patients and methods: 94 patients (68 men, 26 women, mean age 46+14 years) with alcoholic liver disease (31 cirrhosis, 2 alcohol hepatitis, 9 fatty liver) or with non-alcoholic liver disease (38 chronic viral hepatitis C, 8 chronic viral hepatitis B, and 6 other liver diseases) were studied. Alcohol consumption was evaluated with a quantitative questionnaire. 26 patients had a consumption of > 40 g alcohol per day during the month preceding the present investigation (mean alcohol intake 84+52 g per day) and were considered to be excessive drinkers. CDT serum level was measured by ion exchange chromatography followed by radioimmunoassay (Kit CDTect TM, Kabi Pharmacia, Uppsala, Sweden), N_<20 U/I i n men, N<_26 U/I in women: AST, GGT, glutamate dehydrogenase (GDH), and mean corpuscular volume (MCV) were measured by routine methods. Results: Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of these markers for the diagnosis of excessive alcohol intake are as follows: Marker Se (%) Sp (%/ PPV (%) NPV/%) CDT (>N) 35(17-53)* 91 (84-98) 60 (35-85) 78 (69-87) AST (>30UI/I) 88(76-100) 43 (31-55)37 (25-49) 91(81-100) GGT (>40UI/I) 85 (71-99) 44 (32-56) 37 (25-49) 88 (77-99) GDH (>3.5Ul/I) 77 (61-93) 40 (28-52) 33 (21-45) 82 (69-95) MCV (>98 fl) 38 119-57) 79 (69-89) 42 /22-62) 77 (67-87) * 95 per cent confidence interval; N: upper normal limit Conclusion: In patients with liver diseases, C D T i s a very specific marker of excessive alcohol intake but lack of sensitivity may limit its use.
• Results
:
All patients with HA !evels higher than 200 ng/ml had It-ear cirrhosis - fibrosis according to Knodell svstem :
Fibrosis n= , I HA (ng/ml) means + SD ran[}e NPIIIP (UlYrr~) means + SD range
II
0
I 53
3 34
4 43
33 +_ 32 12- 124
48 _+ 33 12 - 140
85_+ 54 18- 198
498 _+593 64 - 2 9 0 0
p < 0,01
0,81 ~+ll.18 0 , 5 - I,I
0.95 _+0.25 0,5-2,13
LI2_+ 0.31 0 , 6 - 1,8
1.32+038 0,6-2,35
p < 0,0t
Significant differences in HA (p<0,01) and in PIIINP levels (p<0,01) were also found between "fibrosis l" and fibrosis Y - HA was a better discriminant for fibrosis when compared to PIIINP - fibrosis according to Chevallier system : AH r = 0,54 PIIINP : r 0.49 (Spearman correlation) HA levels were significantly different according to Disse spaces's fibrosis. ,i C o n c l u s i o n
:
In this study, HA higher than 200 ng/ml always reflected cirrhosis. There was a significant difference in HA levels between "fibrosis 1" and fibrosis 3", but also in PIIINP levels. Discrimination between "fibrosis 0-1" and "fibrosis 3-4" was difficult = cut off values had low sensitivity and low Negative Predictive Value - HA seemed to be a better marker of fibrosis than PIIlNP - The interest of HA in following patients with chronic hepatitis C might be assessed:
•
THE CULTIVATION OF HEPATITIS C VIRUS AND THE EFFECT ON HCV CULTURE OF SOME DRUGS AND ULTRAVIOLET T.Ozeki&K.Funaknshi*The 3rd Dept of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyusyu,*The Dept of Pathology, Kyusyu Dental College,Ritakyasyu,Japaa. (Aim) interferoo(IFN) has been used for the medical treatment of patients with hepatitis(C). However, patients with complete remission were only about 305 in Japan. Therefore new drug for hepatitis C virus(HCV) should he discovered. We must need many chimpanzees for the sake of this study, However, a chimpanzee is very expensive. It is tho~ht that HCV culture using cultured cell could be the most useful for this study. We discoverd that the Indwelling HCV is already present in the Chang cell sold ha Japan. Therefore, We studied the usltum methods of the indwelling HCV and the effect of IFN a 2a and steroid hormone on it. Materials and Methods. Chang cells were obtained from Tsukuba cell Bank(Tsukuba,Japan) and Dainippon Pharmaceutical Co. ,LTD(Ouska,Japan). Chang cells stored at -70"(:: were resolved at room temperature. After trypeination, the cells were suspended in Eagle's MEM solution. Containing 10~ FBS, 100 units of penicillin G and streptomycin and adjusted to 4×10~celis/a bottle(250ml). The cell suspension were incubated at 37~C in 5~ CO~ and 95f, air for 4 weeks.After the culture was finished, the HCV in supernatant(SUP) and trypeinated cell layer were studied. Furthermore, the cultures were carried out adding dexamethasone(10~mM) betamethasoas (l~mM), DMSO and [FN a2a(1200u/ml) to the culture medium. The culture after the irradiation of Ultravinlet(UV) also was done. Reverse transeriptaee and polymerase chain reactinn(RT-PCR) was carried out using 3 primers refferring to 5'-noncoding region of HCV. Competitive RT-PCR(CRT-PCR) also was done. The RT-PCR for subtype of HCV also was dons. Analysis of base sequences was carried oat using dye terminator cycle sequencing method. The staining of HCV using in situ hybridisation(ISH) and indirect immusopemxidase method(IP) (using NS5 IgG RCV antibody by Tsutsumi;Repatology 19,265,1994) were done. Results. The indwelling HCV surely was recognized by RT-PCR, ISH and IP in the Chang cell obtained from two companies in Japan(in Japan, only 2 companies are selling it). The target band in the sup and cell fraction was recognized. There was no target band in RT-PCR in the culture medium, in both staining positive signals were found in cytoplasm. Both pattern was almost same. In IP, prior absorption of anti-HCVNSSlgG with its immunogen completely abobished specifis immusostaining. In ISH, positive staining of HCV RNA was abobished by pretreatment with RNase. The subtype of the indwelling HCV was like type Ill. The identity of the region of typeM(nt 148 to 321 in core region) was 94~. The HCV RNA in the sup increased best in the culture with adding dexamethasone and betamethasone(10' coppies/ml) compared with DMSO and nothing(10 ~ coppiee/m]). However, RCV RNA increased most in the irradiation of UV(105 topples). In addition of IFN to the culture medium, the target band abobisbsd bat in addition of IFN and dexamethasose the target band appeard. We tried the RT-PCR of the other cultured ceUs(HeLa. Hep G2 etc). However, there were no target band in these cells. We also are studing to certify HCV RNA in the chang cell in American type culture coneetion(ATCC) in USA. Conclusion. It turned out using RT-PCR analysis of base sequence, ISH, and IP that the Chang cells in Japan have HCV. At present, we also are studing the HCV in the Chang cell in ATCC. It was recognized that the Chang ten could increase using steroid hormone and eepicially UV. The cell with already indwelling HCV is very useful for studing the effect of various drugs on HCV. The effect of IFNa 2a on HCV is abobieksd by the addition of dexamethasoas even In a single cell without immunological System. It is thought until now that IFN inhihits virus from invadIng into cells. However, it was recognized in this study that IFN also can seam away the indwelling HCV.
AASLD ABSTRACTS
GASTROENTEROLOGY, Vol. 1 0 8 , NO. 4
• REGULATION OF HEPATITIS B VIRUS ENHANCER ACTIVITY BY PHYLLANTHUS NIRURI IS INVOLVED IN ITS ANTIVIRAL EFFECTS.
I N T E R E S T O F H Y A L U R O N I C A C I D (HA) IN ASSESSMENT OF LIVER FIBROSIS IN CHRONIC VIRAL HEPATITIS C H. OUI, H. TROUETTE, B. LE BAIL, G. LACAPE, M. FONCK, F. DUMAS, P. COUZIGOU Serv. d'Hdpatogastroont~rologia H6pital Haut-Lev6que 33604 PESSAC France
M. Ott, S.P. Thyagarajan" and S. Gupta. Liver Research Center, Albert Einstein College of Medicine, NY and "Department of Microbiology, Dr. A.L.M. Postgraduate Institute of Basic Medical Sciences, University of Madras, India.
• In liver fibrosis, the basement menbranes appear belong the sinusmdal walls (capillarizafion), so the function of the endothelial cells are impaired. More than 90 % of circulating hyaluronic Acide (HA) is degraded in hepatic endothelial cells. The blood HA level is known to increase markedly in patients with viral cirrhosis and in primary biliary cirrhosis. • The a i m o f the study was to assess HA as a marker of fibrosis in chronic hepatitis C • Methods : 141 patients (74 males and 67 females, mean : 49 years range 19-74) with chronic viral hepatitis C (43 cirrhosis, 41 child A) underwent a liver biopsy. All samples were examined by one anatomopathologist, and the degree of fibrosis was graded semiquantitatively according to 2 scoring systems (Kn6dell- Cbevallier). At that time, blood samples were collected to determine HA and amino peptide procollagen Ill (PIIINP) with commercial RIA kits (Behring- Pharmacia) - Kurskall-Wallis analysis of variance was used to compare serum PIIINP and HA levels according to the histological score o f fibrosis.
An extract from Phyllanthus niruri was recently shown to inhibit HBV DNA polymerase, as well as to transcriptionally downregulate HBV mRNA expression. To define mechanisms in transcriptional control of HBV after exposure to R niruri, we investigated whether R niruri affected the HBV enhancer I (HBEn) or an HBsAg promoter (HBsP). The HBV sequences were derived from the pCP10 plasmid (HBEn, map positions (rap) 490-1402; HBsP, mp 2425-2839) and cloned into luciferase (luc) expression plasmids. The plasmid pHBEn/HBsP-luc contained HBV regulatory elements, pHBEn/SV40P-luc contained the HBEn driving the SV40 promoter and pSV40En/HBsP-luc contained the SV40 enhancer and HBsP. The ptasmids pGL2-control, containing the SV40 enhancer and promoter, and pGL2-basic with no regulatory elements (Promega Corp.), served as positive or negative controls, respectively. When various plasmids were introduced via Lipcfectin T M into cultured HepG2 cells,/uc was expressed by all plasmids except pGL2-basic. A DMSO-soluble R niruri extract was added to the culture medium 6 hrs after gene transfer and cells lysed at 48 hrs to measure luc activity. In contrast with vehicle alone, R niruridose-dependently (up to : 2 0 0 pg/ml crude extract) suppressed/uc maximally in cells transfected with pHBEn/HBsP-luc, to 4 8 % + 1 0 % (p<0.001), followed by pHBEn/SV40P-luc, to 79%__+14% (p<0.03), of control cells. In plasmids pSV40En/HBsP-luc, pGL2-control and pGL2basic, R niruri did not alter luc expression: R niruri had no effect on either cell viability as demonstrated by utilization of MTT (Thiazolyl blue) or expression of an E. coil lacZ gene driven by the SV40 enhancer/promoter elements. CONCLUSIONS: R niruri downregulated HBEn activity in a specific manner with no impairment in cellular gene expression. Whether R niruri binds to specific HBEn domains or alters interactions between HBEn and cellular transactivators via additional mechanisms requires further analysis.
CARBOHYDRATE-DEFICIENT TRANSFERRIN (CDT) AND LIVER DISEASES. F Ouyahva. Y Bacq, F Schellenberg, E-H Metman, J Weiil. Service d'h~patogastroent~rologie et laboratoire de biochimie, HSpital Trousseau, Tours, France. CDT has been proposed as a marker of alcohol consumption. The aim of this study was to evaluate the accuracy of CDT for the diagnosis of excessive alcohol intake in patients with liver diseases. Patients and methods: 94 patients (68 men, 26 women, mean age 46+14 years) with alcoholic liver disease (31 cirrhosis, 2 alcohol hepatitis, 9 fatty liver) or with non-alcoholic liver disease (38 chronic viral hepatitis C, 8 chronic viral hepatitis B, and 6 other liver diseases) were studied. Alcohol consumption was evaluated with a quantitative questionnaire. 26 patients had a consumption of > 40 g alcohol per day during the month preceding the present investigation (mean alcohol intake 84+52 g per day) and were considered to be excessive drinkers. CDT serum level was measured by ion exchange chromatography followed by radioimmunoassay (Kit CDTect TM, Kabi Pharmacia, Uppsala, Sweden), N_<20 U/I i n men, N<_26 U/I in women: AST, GGT, glutamate dehydrogenase (GDH), and mean corpuscular volume (MCV) were measured by routine methods. Results: Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of these markers for the diagnosis of excessive alcohol intake are as follows: Marker Se (%) Sp (%/ PPV (%) NPV/%) CDT (>N) 35(17-53)* 91 (84-98) 60 (35-85) 78 (69-87) AST (>30UI/I) 88(76-100) 43 (31-55)37 (25-49) 91(81-100) GGT (>40UI/I) 85 (71-99) 44 (32-56) 37 (25-49) 88 (77-99) GDH (>3.5Ul/I) 77 (61-93) 40 (28-52) 33 (21-45) 82 (69-95) MCV (>98 fl) 38 119-57) 79 (69-89) 42 /22-62) 77 (67-87) * 95 per cent confidence interval; N: upper normal limit Conclusion: In patients with liver diseases, C D T i s a very specific marker of excessive alcohol intake but lack of sensitivity may limit its use.
• Results
:
All patients with HA !evels higher than 200 ng/ml had It-ear cirrhosis - fibrosis according to Knodell svstem :
Fibrosis n= , I HA (ng/ml) means + SD ran[}e NPIIIP (UlYrr~) means + SD range
II
0
I 53
3 34
4 43
33 +_ 32 12- 124
48 _+ 33 12 - 140
85_+ 54 18- 198
498 _+593 64 - 2 9 0 0
p < 0,01
0,81 ~+ll.18 0 , 5 - I,I
0.95 _+0.25 0,5-2,13
LI2_+ 0.31 0 , 6 - 1,8
1.32+038 0,6-2,35
p < 0,0t
Significant differences in HA (p<0,01) and in PIIINP levels (p<0,01) were also found between "fibrosis l" and fibrosis Y - HA was a better discriminant for fibrosis when compared to PIIINP - fibrosis according to Chevallier system : AH r = 0,54 PIIINP : r 0.49 (Spearman correlation) HA levels were significantly different according to Disse spaces's fibrosis. ,i C o n c l u s i o n
:
In this study, HA higher than 200 ng/ml always reflected cirrhosis. There was a significant difference in HA levels between "fibrosis 1" and fibrosis 3", but also in PIIINP levels. Discrimination between "fibrosis 0-1" and "fibrosis 3-4" was difficult = cut off values had low sensitivity and low Negative Predictive Value - HA seemed to be a better marker of fibrosis than PIIlNP - The interest of HA in following patients with chronic hepatitis C might be assessed:
•
THE CULTIVATION OF HEPATITIS C VIRUS AND THE EFFECT ON HCV CULTURE OF SOME DRUGS AND ULTRAVIOLET T.Ozeki&K.Funaknshi*The 3rd Dept of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyusyu,*The Dept of Pathology, Kyusyu Dental College,Ritakyasyu,Japaa. (Aim) interferoo(IFN) has been used for the medical treatment of patients with hepatitis(C). However, patients with complete remission were only about 305 in Japan. Therefore new drug for hepatitis C virus(HCV) should he discovered. We must need many chimpanzees for the sake of this study, However, a chimpanzee is very expensive. It is tho~ht that HCV culture using cultured cell could be the most useful for this study. We discoverd that the Indwelling HCV is already present in the Chang cell sold ha Japan. Therefore, We studied the usltum methods of the indwelling HCV and the effect of IFN a 2a and steroid hormone on it. Materials and Methods. Chang cells were obtained from Tsukuba cell Bank(Tsukuba,Japan) and Dainippon Pharmaceutical Co. ,LTD(Ouska,Japan). Chang cells stored at -70"(:: were resolved at room temperature. After trypeination, the cells were suspended in Eagle's MEM solution. Containing 10~ FBS, 100 units of penicillin G and streptomycin and adjusted to 4×10~celis/a bottle(250ml). The cell suspension were incubated at 37~C in 5~ CO~ and 95f, air for 4 weeks.After the culture was finished, the HCV in supernatant(SUP) and trypeinated cell layer were studied. Furthermore, the cultures were carried out adding dexamethasone(10~mM) betamethasoas (l~mM), DMSO and [FN a2a(1200u/ml) to the culture medium. The culture after the irradiation of Ultravinlet(UV) also was done. Reverse transeriptaee and polymerase chain reactinn(RT-PCR) was carried out using 3 primers refferring to 5'-noncoding region of HCV. Competitive RT-PCR(CRT-PCR) also was done. The RT-PCR for subtype of HCV also was dons. Analysis of base sequences was carried oat using dye terminator cycle sequencing method. The staining of HCV using in situ hybridisation(ISH) and indirect immusopemxidase method(IP) (using NS5 IgG RCV antibody by Tsutsumi;Repatology 19,265,1994) were done. Results. The indwelling HCV surely was recognized by RT-PCR, ISH and IP in the Chang cell obtained from two companies in Japan(in Japan, only 2 companies are selling it). The target band in the sup and cell fraction was recognized. There was no target band in RT-PCR in the culture medium, in both staining positive signals were found in cytoplasm. Both pattern was almost same. In IP, prior absorption of anti-HCVNSSlgG with its immunogen completely abobished specifis immusostaining. In ISH, positive staining of HCV RNA was abobished by pretreatment with RNase. The subtype of the indwelling HCV was like type Ill. The identity of the region of typeM(nt 148 to 321 in core region) was 94~. The HCV RNA in the sup increased best in the culture with adding dexamethasone and betamethasone(10' coppies/ml) compared with DMSO and nothing(10 ~ coppiee/m]). However, RCV RNA increased most in the irradiation of UV(105 topples). In addition of IFN to the culture medium, the target band abobisbsd bat in addition of IFN and dexamethasose the target band appeard. We tried the RT-PCR of the other cultured ceUs(HeLa. Hep G2 etc). However, there were no target band in these cells. We also are studing to certify HCV RNA in the chang cell in American type culture coneetion(ATCC) in USA. Conclusion. It turned out using RT-PCR analysis of base sequence, ISH, and IP that the Chang cells in Japan have HCV. At present, we also are studing the HCV in the Chang cell in ATCC. It was recognized that the Chang ten could increase using steroid hormone and eepicially UV. The cell with already indwelling HCV is very useful for studing the effect of various drugs on HCV. The effect of IFNa 2a on HCV is abobieksd by the addition of dexamethasoas even In a single cell without immunological System. It is thought until now that IFN inhihits virus from invadIng into cells. However, it was recognized in this study that IFN also can seam away the indwelling HCV.