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DB-cAMP treatment of experimental alcoholic liver disease
HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995
1625
1627
AASLD
EVALUATION O F T H E A M P L I C O R TM H C V R N A QUALITATIVE ASSAY IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF HEMODIALYSIS PATIENTS. P Zawadzki, G Duverlie, H Dobremel, C Sayada*, S Castelain, and F Eb. University Hospital of Amiens, *Roche Diagnostics Systems, France. Thirty-four HCV seropositive hemodialysis patients (Murex antiHCV and Ortho 3.0) were tested for the presence of HCV-RNA both in their plasma and PBMC by the Amplicor TM HCV RNA qualitative assay (EDTA treated blood). All of the patients had normal serum ALT values. For PBMC, HCV RNA was tested following the protocol of Anita Yoder, Roche Molecular Systems. Briefly, PBMC were separated by FieoU-Hypaque density centrifugation and collected at 5.106 cells/ml. Finally, RNA extracted from 50,000 cells was tested in each PCR tube following the standard procedure. Plasmas from 27 patients ('79%) and PBMC from 21 (62%) were positive for HCV RNA detection. All positive patients in PBMC were also positive in their plasma. Nineteen plasmas ('70%) and only 13 samples of PBMC (62%) gave a strong signal (O.D.>2). In conclnsion, the HCV RNA detection in PBMC seemed not to be very interesting in comparison of the plasma detection as we did not find any patient HCV RNA negative in plasma and positive in PBMC. The level of detection was lower in PBMC than in plasma, but we calculate that 50,000 cells was equivalent to a volume of 0.025ul (500~um3/PBMC) instead of 5/zl of plasma equivalent tested in the PCR tube. Then, It seems possible that the number of extracted cells was insufficient in the negative samples of PBMC. A quantitative approach will be valuable to know exactly if the sensitivity of the detection conld be optimized and potentially better in PBMC than in plasma.
COMPARATIVE STUDY OF CHRONIC HEPATITIS C AND B WITH A NEW CLASSIFICATION. K. ZHOUI, Y. HU I, WR. ZHA1~ AND GB. YAO' 'Clinical Immunology Research Center, .Jingan Central Hospital, Shanghai. 2Departmetuof Pathology, Shanghai Medical University. The object of this study is to investigatepathologicalcharacteristicsof chronic hepatitis C and B, and evaluate the usefullncss of the new Desmet classification. 49 HCV and 45 HBV liver biopsy specimenswere studied histologically. All the patients had proven HBV or HCV by relavent serological and virological parameters, and specimens were stained with HE and scored according to the Desmet grading method. Most of the HCV specimens showed mild to moderate hepatitis. Them are four features more likely to be seen in HCV than HBV infection : fatty degeneration (59% vs 22%, p<0.01), bile duct damage (55 % vs 24%, p<0.01), lymphocyte aggregation / follicles (48 % vs 22 %, p < 0.01) and increase of mononuclearcells in sinssoids (47% vs 22%, p<0.05). Liver cell ground-glass appearance and piecemeal necrosis were seen to be more severe in HBV. These features are useful pathologicalparameters in the diagnosis of chronic hepatitis C. Recently, a new system of classifying chronic hepatitis has been introduced instead of the previous one of CAH, CLH, CPH. In this study we used the new system to grade all specimens. The results show that grade GO and G1 often include mild CAH, CLH and CPH (with mild activity), G2 and G3 include moderate and severe CAN (with severe activity). When compared with the old nomenclature, the new classification was more accurate in determination of disease activity and the fibrotic process. It could give more information to clinical doctors to judge the clinical course and prognosis, and assist in making decisions in the choice of suitable treatment. Conclusion: 1. Fatty degeneration, bile duct damage, lymphocyteaggregation / follicles and increase of mononuclear cells in sinnsoids are major histological features of chronic hepatitis C. 2. The grading system has good relation with verbal assessment, it is valuable for clinical diagnosis and research.
ABSTRACTS
1626
513A
D~c,~IP MA~IE~I" OF E X I ~ t I I ~ N T A L ALCOHOLIC LIV~ DI~.~, Z-Q ~ , K Miyamom*, H T a k a ~ h i * , P Fu, R Sinew and S l~ench. Dept. of P~holoay and Radiology, Harbcf-U~.A Medical Center, California and *Nippon Kokan Hospital, I~an. The pre~mt study was performed to inv~tigate the effect of dibutyryl cyclic AMP (DB-cAMP) on ethanol-induced liv~ disvaN iu the rat to test the theory that cAMP influcmces liver blood flow to ameliorate ethanol-induced hypoxta. Both saline (i.p.) and DB-cAMP (10 mg/kg, i.p.) treated animals were fed ethanol and liquid diet for 2 months through permanently implanted gastric ¢~auulas. Urine alcohol level, which is about 1.25 : 1 proportional to the blood alcohol level, was measared weekly in order to adjust the amount of ethanol intake to m~nta!~ the blood alcohol level above 2 0 0 ~ . Ultrasound method, was performed to measure the diameter of portal vein and its blood flow velcclty after completing the study in anesthetized rats. Liver fimction was determined monthly by measuring serum ALl" and AST levels. The re#uka showed that ethanol caus¢cl focal necrosis and fibrosis only in ethanol-saline rats. The serum ALT level measured by 2 months of ethanol tre.~mant r~nded to he lower in DB-cAMP treated group, indicating that liver d!m=~e was ameliorated by DB-cAMP. Chronic DB.~AMP administration resulted in a tendency to increase the metabolic tolerance of ethanol in rats that a higher dose of alcohol/]r~/day was required to achieve the same level of urine as well as blood alcohol level. Doppler measurement in vivo of the portal vein diameter and flow velocity in ethanol-saline rats showed portal vein dilatation (est. 200%) and decreased peak velec~y (est. 50%), Hepatic vein waveform was blunted with absence of flow reversal during atrial contraction. Thus these results may indicate that chronic ethanol ingestion indt~¢s a low flow state of tl~ liver and supports the hypoxia hypofl~is of ethanol-induced liver injury, The eflmao!-induc~l h~oxi¢ effect may be ameliorated by DB-cAMP treatment. "This research was supported in part by the Dali~hi Pharm., in Japan".
1628
The Relationship between Pre-eere/core HBV Mutants and Clinical Signif ieenees.
Li Min Zhu M.D Tianjin Infectious Dis.Hosp.Tianjin,300192 P. R .of China Background:Recently the cl~-taring of mutations in a segment of Pre -core/core region of HBV DNA was found in H]~Ag- negative chronic hepatitis B (CI-IB) patients.However,the as~miation of the mutations in this region with the clinical significance remains unclear. Patients and methyls:57 patients with CHB were examined. HBeAg was negative in all patients.HBV DNA was extracted from the sera ,amplified by PCR with primers spanning the extire pre- core and core region.The second-round PCR-amplified Products were anlyT~l by means of electrophoresis.THis amplified DNA was purified and then used for direct sequencing. Didcoxynucleotied termination sequencing was performed with the sequonase kit. Results:We reported two mutations:Ml(G-A change at nucleetied 1898)M2(G -A change at nuclcotied 1898 and 1901). 57 patients of HBeAg-negative were examined.HBV DNA was detected in 31 patients (54%).Me(nO mutation)was detected in 16.M1]M= was detected in 8.Me and M1]M2 were detected in 5.Me and M~/M~ were not edtected in 2 .15 patients were infected with these kinds of mutant,poaltive rate was 480: Our studiea demonstrated.(1)There is no association between sex ,age and infection with mutation.(2)The postive rate of mutation in patients with CPH,CAH,CAA and Lc/Lc, necrosis,Hco are 0[1,9/16, 4/16,2[4 and 1[4.( 3)Thcre is relationship between the positive rate of mutation and the eeuse of disease.(4)The postitive rate of mutation increased with the inf]smmAtion of liver.The extent of the infl-r-,-stion in liver are alight,mid,serious,the rate are 50°/0,60%,83.3%](5)HBe~g positive,HBeAb postive or beth negative ,the lx~itiv ate of mutation are 27.5%,68.9% ,41%. Cor~lusion:In this group,the positive rate of mutations in pre ~core/core region of HBV DNA is 48%,this rate increased with the extent of inflammation in liver and this mutation was more frequently detected in the patients with HBeAb( + ) chronic hepatitis.
1625
1627
AASLD
EVALUATION O F T H E A M P L I C O R TM H C V R N A QUALITATIVE ASSAY IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF HEMODIALYSIS PATIENTS. P Zawadzki, G Duverlie, H Dobremel, C Sayada*, S Castelain, and F Eb. University Hospital of Amiens, *Roche Diagnostics Systems, France. Thirty-four HCV seropositive hemodialysis patients (Murex antiHCV and Ortho 3.0) were tested for the presence of HCV-RNA both in their plasma and PBMC by the Amplicor TM HCV RNA qualitative assay (EDTA treated blood). All of the patients had normal serum ALT values. For PBMC, HCV RNA was tested following the protocol of Anita Yoder, Roche Molecular Systems. Briefly, PBMC were separated by FieoU-Hypaque density centrifugation and collected at 5.106 cells/ml. Finally, RNA extracted from 50,000 cells was tested in each PCR tube following the standard procedure. Plasmas from 27 patients ('79%) and PBMC from 21 (62%) were positive for HCV RNA detection. All positive patients in PBMC were also positive in their plasma. Nineteen plasmas ('70%) and only 13 samples of PBMC (62%) gave a strong signal (O.D.>2). In conclnsion, the HCV RNA detection in PBMC seemed not to be very interesting in comparison of the plasma detection as we did not find any patient HCV RNA negative in plasma and positive in PBMC. The level of detection was lower in PBMC than in plasma, but we calculate that 50,000 cells was equivalent to a volume of 0.025ul (500~um3/PBMC) instead of 5/zl of plasma equivalent tested in the PCR tube. Then, It seems possible that the number of extracted cells was insufficient in the negative samples of PBMC. A quantitative approach will be valuable to know exactly if the sensitivity of the detection conld be optimized and potentially better in PBMC than in plasma.
COMPARATIVE STUDY OF CHRONIC HEPATITIS C AND B WITH A NEW CLASSIFICATION. K. ZHOUI, Y. HU I, WR. ZHA1~ AND GB. YAO' 'Clinical Immunology Research Center, .Jingan Central Hospital, Shanghai. 2Departmetuof Pathology, Shanghai Medical University. The object of this study is to investigatepathologicalcharacteristicsof chronic hepatitis C and B, and evaluate the usefullncss of the new Desmet classification. 49 HCV and 45 HBV liver biopsy specimenswere studied histologically. All the patients had proven HBV or HCV by relavent serological and virological parameters, and specimens were stained with HE and scored according to the Desmet grading method. Most of the HCV specimens showed mild to moderate hepatitis. Them are four features more likely to be seen in HCV than HBV infection : fatty degeneration (59% vs 22%, p<0.01), bile duct damage (55 % vs 24%, p<0.01), lymphocyte aggregation / follicles (48 % vs 22 %, p < 0.01) and increase of mononuclearcells in sinssoids (47% vs 22%, p<0.05). Liver cell ground-glass appearance and piecemeal necrosis were seen to be more severe in HBV. These features are useful pathologicalparameters in the diagnosis of chronic hepatitis C. Recently, a new system of classifying chronic hepatitis has been introduced instead of the previous one of CAH, CLH, CPH. In this study we used the new system to grade all specimens. The results show that grade GO and G1 often include mild CAH, CLH and CPH (with mild activity), G2 and G3 include moderate and severe CAN (with severe activity). When compared with the old nomenclature, the new classification was more accurate in determination of disease activity and the fibrotic process. It could give more information to clinical doctors to judge the clinical course and prognosis, and assist in making decisions in the choice of suitable treatment. Conclusion: 1. Fatty degeneration, bile duct damage, lymphocyteaggregation / follicles and increase of mononuclear cells in sinnsoids are major histological features of chronic hepatitis C. 2. The grading system has good relation with verbal assessment, it is valuable for clinical diagnosis and research.
ABSTRACTS
1626
513A
D~c,~IP MA~IE~I" OF E X I ~ t I I ~ N T A L ALCOHOLIC LIV~ DI~.~, Z-Q ~ , K Miyamom*, H T a k a ~ h i * , P Fu, R Sinew and S l~ench. Dept. of P~holoay and Radiology, Harbcf-U~.A Medical Center, California and *Nippon Kokan Hospital, I~an. The pre~mt study was performed to inv~tigate the effect of dibutyryl cyclic AMP (DB-cAMP) on ethanol-induced liv~ disvaN iu the rat to test the theory that cAMP influcmces liver blood flow to ameliorate ethanol-induced hypoxta. Both saline (i.p.) and DB-cAMP (10 mg/kg, i.p.) treated animals were fed ethanol and liquid diet for 2 months through permanently implanted gastric ¢~auulas. Urine alcohol level, which is about 1.25 : 1 proportional to the blood alcohol level, was measared weekly in order to adjust the amount of ethanol intake to m~nta!~ the blood alcohol level above 2 0 0 ~ . Ultrasound method, was performed to measure the diameter of portal vein and its blood flow velcclty after completing the study in anesthetized rats. Liver fimction was determined monthly by measuring serum ALl" and AST levels. The re#uka showed that ethanol caus¢cl focal necrosis and fibrosis only in ethanol-saline rats. The serum ALT level measured by 2 months of ethanol tre.~mant r~nded to he lower in DB-cAMP treated group, indicating that liver d!m=~e was ameliorated by DB-cAMP. Chronic DB.~AMP administration resulted in a tendency to increase the metabolic tolerance of ethanol in rats that a higher dose of alcohol/]r~/day was required to achieve the same level of urine as well as blood alcohol level. Doppler measurement in vivo of the portal vein diameter and flow velocity in ethanol-saline rats showed portal vein dilatation (est. 200%) and decreased peak velec~y (est. 50%), Hepatic vein waveform was blunted with absence of flow reversal during atrial contraction. Thus these results may indicate that chronic ethanol ingestion indt~¢s a low flow state of tl~ liver and supports the hypoxia hypofl~is of ethanol-induced liver injury, The eflmao!-induc~l h~oxi¢ effect may be ameliorated by DB-cAMP treatment. "This research was supported in part by the Dali~hi Pharm., in Japan".
1628
The Relationship between Pre-eere/core HBV Mutants and Clinical Signif ieenees.
Li Min Zhu M.D Tianjin Infectious Dis.Hosp.Tianjin,300192 P. R .of China Background:Recently the cl~-taring of mutations in a segment of Pre -core/core region of HBV DNA was found in H]~Ag- negative chronic hepatitis B (CI-IB) patients.However,the as~miation of the mutations in this region with the clinical significance remains unclear. Patients and methyls:57 patients with CHB were examined. HBeAg was negative in all patients.HBV DNA was extracted from the sera ,amplified by PCR with primers spanning the extire pre- core and core region.The second-round PCR-amplified Products were anlyT~l by means of electrophoresis.THis amplified DNA was purified and then used for direct sequencing. Didcoxynucleotied termination sequencing was performed with the sequonase kit. Results:We reported two mutations:Ml(G-A change at nucleetied 1898)M2(G -A change at nuclcotied 1898 and 1901). 57 patients of HBeAg-negative were examined.HBV DNA was detected in 31 patients (54%).Me(nO mutation)was detected in 16.M1]M= was detected in 8.Me and M1]M2 were detected in 5.Me and M~/M~ were not edtected in 2 .15 patients were infected with these kinds of mutant,poaltive rate was 480: Our studiea demonstrated.(1)There is no association between sex ,age and infection with mutation.(2)The postive rate of mutation in patients with CPH,CAH,CAA and Lc/Lc, necrosis,Hco are 0[1,9/16, 4/16,2[4 and 1[4.( 3)Thcre is relationship between the positive rate of mutation and the eeuse of disease.(4)The postitive rate of mutation increased with the inf]smmAtion of liver.The extent of the infl-r-,-stion in liver are alight,mid,serious,the rate are 50°/0,60%,83.3%](5)HBe~g positive,HBeAb postive or beth negative ,the lx~itiv ate of mutation are 27.5%,68.9% ,41%. Cor~lusion:In this group,the positive rate of mutations in pre ~core/core region of HBV DNA is 48%,this rate increased with the extent of inflammation in liver and this mutation was more frequently detected in the patients with HBeAb( + ) chronic hepatitis.