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P-387 COld storage-induced alteration of hepatic energy metabolizm in isolated and perfused rat liver: Influence of leukocyte accumulation
Posters~International Hepatology Communications 3 Suppl. (1995) $37-S169
P-384
R e l a t i o n s h i p b e t w e e n O_uantitation of l t e p a t i t i s C Virus RNA b y C o m p e t i t i v e PCR, a n d Its G e n o t y p e a n d R e g i o n a l S p e c i f i c Ab R e s p o n s e s Seung Kew Yoon, Young Min Park, Ksxt Sik Shim, Boo Sung Kim Depr. of Internal Medicine, Catholic University Medical College, Seoul, Korea O uantitation of I tCV RNA b~, competitive polymerase chain reaetion(PCR) has been used to investigate the factors that influence disease severir} and therapeutic effectiveness in hepatitis C virus (IICV) infection. This study was conducted to define the correlation between serum ilCV RNA levels, and HCV genotypes and antibody responses toward II('V core(Cl2-3), NS3(C33c) and NS4(5-1-1, C100-3). Serum samples from forty-fl~ e patients with chronic I ICV infection v, ho were positive lot anti-IICV, were tested for antibody to regional specific I ICV proteins by a second generation recombinant immunoblot assay(FdBA-2), and lor ltCV RNA detection, itCV genotyping and quantitation of I ICV RNA by re~ erse transcription(RT)-nested PCR. Forty-one of 45 patients(91.1%) had antibodies to ttCV C22-3 and C33C protein irrespective of serum i lCV RNA le~ els. No significant difl'erence was seen in the IICV RNA levels between chronic hepatitis and hepatocellular carcinoma. The lesels of the antibodies reactivity r~l II('V 4;22-3 and C33C proteins correlated significantly with the concentr-ation of serum I1CV RNA(r=0.gb, P<0.05; r=0.33,P<0.05, respecti',ely). More than one antibody to the lout antigens in RIBA-Z ,has detected in 31 of 45 patientsi68.9%) in the grc,up with high viremic levels of >10f'copies/ml of serum, and in 14 of 45 patienrs(31,1%) in the group with low "~iremic le'~els of < l~)copies/ml of serum. Antibodies to I ICV NS4(5-1-1 and C I O0-3)prorein v, ere detected less frequent]3 than those to I1CV core(C22-31 and NS3(C33C) proteins, but not to a statistically significant extent. These results suggest firstly: that low ",iremic IlCV carriers m a y ha'~e poor antibod3 responses to lt('V protein compared to highly siremic IICV carriers; se,.'undly thai IICV core and NS3 protein m a y be more immunogenic than tlCV NS4 protein; and thirdly that IICV genot>pes m a y influem'e the i m m u n e response of antibody to IICV protein regardless of ItCV replication.
P-386
THE F(ab')2 F R A G M E N T O F AN ANTI-ICAM-1 M O N O C L O N A L ANTIBODY ATTENUATES LIVER INJURY AFTER ORTHOTOPIC LIVER TRANSPLANTATION Y. Takei, Y. Nishimara, S. Kawano, M. Goto, K. Nagano, S. Tsuji, I-L Nagai, A. Ohmae, H. Fusamoto, T. Kamada 1st Dept. of Medicine, Osaka Univ.Sch. of Medicine, Suita, Japan Extended storage of liver grafts results in increased adhesion of leukocytes onto the sinusoidal walls. This study was designed to examine whether intracellular adhesion molecule-1 (ICAM-1) is involved in attachment of leukocytes after liver transplantation. Inbred Lewis rats were used as both donors and recipients. Livers were stored for either 1 or 6 hr in ice-cold Euro-Collins solution and subsequently implanted. The expression of ICAM-1 was examined immunohistochemically. In some rats that received livers stored for 6 hr, the intact IgG (1.0 mg/kg) or the F(ab')2 fragment (0.5 mg/kg) of an anti-ICAM-1 mAb (1A29) was administered via the tail vein immediately after reperfusion of portal blood. In the 6 hrstored group, ICAM-1 began to be expressed on the sinusoidal endothelial cells as early as 15 rain after reperfusion. Strong ICAM-1 expression was observed from 2 up to 24 hr after reperfusion. In contrast, expression of ICAM-1 was not evident at any time point postoperatively in the 1-hr storage. Serum A L T levels were significantly higher in the 6-hr storage group (1-hr: 171 + 9 IU/1; 6-hr: 825 + 109 IU/1, p<0.05; mean S.E.M.) 24 hr after transplantation. Serum ALT levels were markedly reduced by treatment with the F(ab'):z fragment of 1A29 (247 + 34 IUfl, p<0.05 vs. 6hr storage group), whereas treatment with the intact IgG of 1A29 increased serum A L T levels dramatically (5297 + 634 IU/1, p<0.05 vs. 6-hr storage group) and reduced serum complement. Treatment with the F(ab')2 fragment decreased the liver damage; in marked contrast, treaanent with intact IgG strikingly aggravated the injury characterized by massive necrosis throughout the liver. Liver damage caused by the intact IgG might be related to activation of the complement system by the Fc portion of the antibody. Taken together, these results indicate that ICAM-1 is involved in the mechanism of postoperative liver injury following liver transplantation.
S133
P-385 o:xr~~ i c ACID s i n = ~ A OF WARM l ~ C INJURY IN THE (t~A~'l'.~;u LIVI~R? J.Kimura, Y.Nakajima, K.Ito, M.Tamura, H.Kamachi, H.Kon, M.Nishibe, J.Uchino First department of Surgery, Hokkaido University School of Medicine, Sapporo, Japan The aim of this study was to investigate whether serum hyaluronic acid (HA) could serve as a marker of hepatic warm ischemic injury in porcine liver transplantation (LTX). Methods: Twelve LTX were performed using temporary portal arterialization (PA) technique. An insult of warm ischemia was not given to the graft in group A(n=7). In group B (n=5), the donor livers were transplanted after 60 rain of warm ischemia before harvesting. Arterial and hepatic venous HA (ng/ml) values were measured during operation. Statistical analysis was performed by Mann-Whitney U test. Results: In group A, all pigs survived more than 4 days, but in group B, all died within 30 hrs due to graft failure. Arterial HA values showed no differences between 2 groups (laparotomy: 102.3_+18.1 vs. 78.2_+ 10.2, during PA: 458.6_+194.6 vs. 462.4_+174.0, 60 min after portal revascularization: 272.6_+91.8 vs. 235.6 _+57.4). Hepatic venous HA values also showed no differences between the groups. Clearance of HA by the liver was calculated frc~ the arterial and the hepatic venous values, and there were no significant differences between the groups as well. Conclusions: Serum HA and its hepatic clearance were not influenced at all by 60 min warm ischemia. Thus, it might be said that HA could not serve as a marker of warm ischemic injury in the grafted liver.
P-387 ('OLI) STORAGE-INDUCED ALTERATION OF H E P A T I C E N E R G Y M E T A B O L I Z M IN I S O L A T E D A N D PERFI.SED RAT LIVER: INFLUENCE OF LEUKOCYTE %('CI,%'IUI,ATION II iliguchl. 1 Kulo,',c. S Mnlra, S Kato. and H lshii. I)~ t,v ~,/ htlcr, at Med, School o/Med., KeicJ Umv. Tokr~, 160. Japan. '1 he present ~,tudy was designed to investigate the temporal and spatial relationship between leukocyte accumulation and hepatic energy metabolism after the cold storage followed by reperfusion. The liver of male Wistar rat~, was isolated and perfitsed with 95% 0 2 babbled-EuroCohns solution (37 C. pt17.4) from the catheter inserted into the portal vcm '1 he NADll auloflt~orescence in the surface of liver was monitored ¢e\citation 330-380 nm. emission 460 nm) and analyzed by the computera~',lsted thlorescence microscopic system. During the cold storage, the NADII autoflnore~,cence significantly increased, and decreased to the hasehnc level iuamediately aider the reperfusion. At 60 minutes after the ~eperfu~nm. lhe NADIt autofluorescence increased again in perivenular area ol file liver ",ul!iected to 120 minutes cold storage, but not 60 mitulle~ Ill another set of experiment. CFSE-labeled lymphocytes or tleutrnphil~, t l x 10~' cells) were iqiected from portal vein at the beginning ~+1 teperlu~,l~m alter 120 minutes cold storage. Just after the admini'~tratmn of ctther IL-2 (1000 units/roll-treated lymphocytes or uetllrol+hils, tile ('l:gE-assocmted fluorescence was accnmulated in the peliportal area. dud retamed during entire course of observation. The NADtt autoflnorescence significantly increased in the periportal area not only in the pert\ entdar area. The accumulation of the it!iected leukocytes and an increase ill NADIt in the periportal area were significantly attenuated by the sl multaneons injection of monoclonal antibody directed against clthel ('DI~ or ICAM-I. The present study suggests that the cold 'qorage t'olhn.~.ed I',y repcrfusion causes alteration of redox state ill the perlvenular area ol rat hver. and that the leukocyte accumulation in the perlportal area enhances the postiscbemic mitochondrial dysfnnction.
P-384
R e l a t i o n s h i p b e t w e e n O_uantitation of l t e p a t i t i s C Virus RNA b y C o m p e t i t i v e PCR, a n d Its G e n o t y p e a n d R e g i o n a l S p e c i f i c Ab R e s p o n s e s Seung Kew Yoon, Young Min Park, Ksxt Sik Shim, Boo Sung Kim Depr. of Internal Medicine, Catholic University Medical College, Seoul, Korea O uantitation of I tCV RNA b~, competitive polymerase chain reaetion(PCR) has been used to investigate the factors that influence disease severir} and therapeutic effectiveness in hepatitis C virus (IICV) infection. This study was conducted to define the correlation between serum ilCV RNA levels, and HCV genotypes and antibody responses toward II('V core(Cl2-3), NS3(C33c) and NS4(5-1-1, C100-3). Serum samples from forty-fl~ e patients with chronic I ICV infection v, ho were positive lot anti-IICV, were tested for antibody to regional specific I ICV proteins by a second generation recombinant immunoblot assay(FdBA-2), and lor ltCV RNA detection, itCV genotyping and quantitation of I ICV RNA by re~ erse transcription(RT)-nested PCR. Forty-one of 45 patients(91.1%) had antibodies to ttCV C22-3 and C33C protein irrespective of serum i lCV RNA le~ els. No significant difl'erence was seen in the IICV RNA levels between chronic hepatitis and hepatocellular carcinoma. The lesels of the antibodies reactivity r~l II('V 4;22-3 and C33C proteins correlated significantly with the concentr-ation of serum I1CV RNA(r=0.gb, P<0.05; r=0.33,P<0.05, respecti',ely). More than one antibody to the lout antigens in RIBA-Z ,has detected in 31 of 45 patientsi68.9%) in the grc,up with high viremic levels of >10f'copies/ml of serum, and in 14 of 45 patienrs(31,1%) in the group with low "~iremic le'~els of < l~)copies/ml of serum. Antibodies to I ICV NS4(5-1-1 and C I O0-3)prorein v, ere detected less frequent]3 than those to I1CV core(C22-31 and NS3(C33C) proteins, but not to a statistically significant extent. These results suggest firstly: that low ",iremic IlCV carriers m a y ha'~e poor antibod3 responses to lt('V protein compared to highly siremic IICV carriers; se,.'undly thai IICV core and NS3 protein m a y be more immunogenic than tlCV NS4 protein; and thirdly that IICV genot>pes m a y influem'e the i m m u n e response of antibody to IICV protein regardless of ItCV replication.
P-386
THE F(ab')2 F R A G M E N T O F AN ANTI-ICAM-1 M O N O C L O N A L ANTIBODY ATTENUATES LIVER INJURY AFTER ORTHOTOPIC LIVER TRANSPLANTATION Y. Takei, Y. Nishimara, S. Kawano, M. Goto, K. Nagano, S. Tsuji, I-L Nagai, A. Ohmae, H. Fusamoto, T. Kamada 1st Dept. of Medicine, Osaka Univ.Sch. of Medicine, Suita, Japan Extended storage of liver grafts results in increased adhesion of leukocytes onto the sinusoidal walls. This study was designed to examine whether intracellular adhesion molecule-1 (ICAM-1) is involved in attachment of leukocytes after liver transplantation. Inbred Lewis rats were used as both donors and recipients. Livers were stored for either 1 or 6 hr in ice-cold Euro-Collins solution and subsequently implanted. The expression of ICAM-1 was examined immunohistochemically. In some rats that received livers stored for 6 hr, the intact IgG (1.0 mg/kg) or the F(ab')2 fragment (0.5 mg/kg) of an anti-ICAM-1 mAb (1A29) was administered via the tail vein immediately after reperfusion of portal blood. In the 6 hrstored group, ICAM-1 began to be expressed on the sinusoidal endothelial cells as early as 15 rain after reperfusion. Strong ICAM-1 expression was observed from 2 up to 24 hr after reperfusion. In contrast, expression of ICAM-1 was not evident at any time point postoperatively in the 1-hr storage. Serum A L T levels were significantly higher in the 6-hr storage group (1-hr: 171 + 9 IU/1; 6-hr: 825 + 109 IU/1, p<0.05; mean S.E.M.) 24 hr after transplantation. Serum ALT levels were markedly reduced by treatment with the F(ab'):z fragment of 1A29 (247 + 34 IUfl, p<0.05 vs. 6hr storage group), whereas treatment with the intact IgG of 1A29 increased serum A L T levels dramatically (5297 + 634 IU/1, p<0.05 vs. 6-hr storage group) and reduced serum complement. Treatment with the F(ab')2 fragment decreased the liver damage; in marked contrast, treaanent with intact IgG strikingly aggravated the injury characterized by massive necrosis throughout the liver. Liver damage caused by the intact IgG might be related to activation of the complement system by the Fc portion of the antibody. Taken together, these results indicate that ICAM-1 is involved in the mechanism of postoperative liver injury following liver transplantation.
S133
P-385 o:xr~~ i c ACID s i n = ~ A OF WARM l ~ C INJURY IN THE (t~A~'l'.~;u LIVI~R? J.Kimura, Y.Nakajima, K.Ito, M.Tamura, H.Kamachi, H.Kon, M.Nishibe, J.Uchino First department of Surgery, Hokkaido University School of Medicine, Sapporo, Japan The aim of this study was to investigate whether serum hyaluronic acid (HA) could serve as a marker of hepatic warm ischemic injury in porcine liver transplantation (LTX). Methods: Twelve LTX were performed using temporary portal arterialization (PA) technique. An insult of warm ischemia was not given to the graft in group A(n=7). In group B (n=5), the donor livers were transplanted after 60 rain of warm ischemia before harvesting. Arterial and hepatic venous HA (ng/ml) values were measured during operation. Statistical analysis was performed by Mann-Whitney U test. Results: In group A, all pigs survived more than 4 days, but in group B, all died within 30 hrs due to graft failure. Arterial HA values showed no differences between 2 groups (laparotomy: 102.3_+18.1 vs. 78.2_+ 10.2, during PA: 458.6_+194.6 vs. 462.4_+174.0, 60 min after portal revascularization: 272.6_+91.8 vs. 235.6 _+57.4). Hepatic venous HA values also showed no differences between the groups. Clearance of HA by the liver was calculated frc~ the arterial and the hepatic venous values, and there were no significant differences between the groups as well. Conclusions: Serum HA and its hepatic clearance were not influenced at all by 60 min warm ischemia. Thus, it might be said that HA could not serve as a marker of warm ischemic injury in the grafted liver.
P-387 ('OLI) STORAGE-INDUCED ALTERATION OF H E P A T I C E N E R G Y M E T A B O L I Z M IN I S O L A T E D A N D PERFI.SED RAT LIVER: INFLUENCE OF LEUKOCYTE %('CI,%'IUI,ATION II iliguchl. 1 Kulo,',c. S Mnlra, S Kato. and H lshii. I)~ t,v ~,/ htlcr, at Med, School o/Med., KeicJ Umv. Tokr~, 160. Japan. '1 he present ~,tudy was designed to investigate the temporal and spatial relationship between leukocyte accumulation and hepatic energy metabolism after the cold storage followed by reperfusion. The liver of male Wistar rat~, was isolated and perfitsed with 95% 0 2 babbled-EuroCohns solution (37 C. pt17.4) from the catheter inserted into the portal vcm '1 he NADll auloflt~orescence in the surface of liver was monitored ¢e\citation 330-380 nm. emission 460 nm) and analyzed by the computera~',lsted thlorescence microscopic system. During the cold storage, the NADII autoflnore~,cence significantly increased, and decreased to the hasehnc level iuamediately aider the reperfusion. At 60 minutes after the ~eperfu~nm. lhe NADIt autofluorescence increased again in perivenular area ol file liver ",ul!iected to 120 minutes cold storage, but not 60 mitulle~ Ill another set of experiment. CFSE-labeled lymphocytes or tleutrnphil~, t l x 10~' cells) were iqiected from portal vein at the beginning ~+1 teperlu~,l~m alter 120 minutes cold storage. Just after the admini'~tratmn of ctther IL-2 (1000 units/roll-treated lymphocytes or uetllrol+hils, tile ('l:gE-assocmted fluorescence was accnmulated in the peliportal area. dud retamed during entire course of observation. The NADtt autoflnorescence significantly increased in the periportal area not only in the pert\ entdar area. The accumulation of the it!iected leukocytes and an increase ill NADIt in the periportal area were significantly attenuated by the sl multaneons injection of monoclonal antibody directed against clthel ('DI~ or ICAM-I. The present study suggests that the cold 'qorage t'olhn.~.ed I',y repcrfusion causes alteration of redox state ill the perlvenular area ol rat hver. and that the leukocyte accumulation in the perlportal area enhances the postiscbemic mitochondrial dysfnnction.