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Ethanol increases production of lipopolysaccharide binding protein (LBP) by hepatocytes
April 1998 DEX did not affect the cell viability up to llaM and suppressed PLA2-II secretion at 100nM, a maximum(50% of control, P<0.005). IL-6 induced PLA2-II secretion from HepG2 incubating with 1001aMof UDCA significantly decreased(68% of control, P<0.01). Under the condition with 100nM of DEX and 1001aM of UDCA, IL-6 induced PLA2-II expression was suppressed more than just DEX treatment (74% of only DEX, P<0.05). (3) The expression levels of IL-6Ro~ in membrane fraction were unchanged by incubation with 100}alVl of UDCA for 6hrs. In addition, the expression levels of gpl30 and STAT-3 immunoprecipitated by specific antibodies for gpl30 and STAT-3 were not changed by incubation with UDCA. Tyrosine phosphorylation of gpl30 and STAT-3 were occurred 2-5 minutes after IL-6 stimulation. However, in the ceils pre-incubated with 100}aM of UDCA, phosphorylation of gpl30 and STAT-3 following IL-6 stimulation were not altered. Conclusions: Effect of UDCA on suppression for IL-6 induced PLA2-II was additive for the effect of DEX. It is supposed that this additive effect is not caused from the attenuation of cytokine signal by modulation of IL-6 receptor-Jak/STAT pathway. It is speculated that this phenomenon could explain, in part, the ligand-independent activation of the GR by UDCA. • L0259 ETHANOL INCREASES PRODUCTION OF LIPOPOLYSACCHARIDE BINDING PROTEIN (LBP) BY HEPATOCYTES. K Ik~ima I, N Enomoto 2, A Ikejima 1, RG Thurman2 and DA BrennerL Div. of Digestive Diseases and Nutrition, Dept. of Medicine l, Lab. of Hepatobiology and Toxicology, Dept. of Pharmacology 2, Univ. of North Carolina at Chapel Hill, NC. Recent studies using a rat model of chronic enteral ethanol infusion (Tsukamoto-French) have revealed that activation of Kupffer cells by gutderived endotoxin (lipopolysaccharide, LPS) plays a pivotal role in development of alcohol induced liver injury. LPS binding protein (LBP), which is produced mainly by hepatocytes, is necessary for the activation of Kupffer cells via the LPS/LBP receptor (CD14). Acute and chronic ethanol treatment increases both CD14 and LBP mRNA in the liver (K Ikejima et al., Ale. Clin. Exp, Res.; 21, 53A, 1997). AIM: The purpose of this study was to clarify the mechanism by which ethanol increases production of LBP by hepatocytes. METHODS: Female Sprague Dawley rats weighing 200g were given a bolus intragastric injection of ethanol (5g/kg) and serum and liver samples were collected 2, 6, 12 and 24 hr later. Total liver RNA was analyzed by Northern blot hybridization for LBP. To evaluate whether serum LBP levels were increased by ethanol, LBP was detected in a bioassay using TNF-~t production by RAW 264.7 macrophages. RAW 264.7 cells were incubated with LPS (1 ng/ml) in 1% serum from ethanol-treated rats for 1 hr and TNF-~t mRNA was detected by Northern blotting. Rat hepatocytes isolated by collagenase digestion were cultured for 48 hr prior to experiments and direct effects of ethanol and LPS on LBP mRNA were evaluated by Northern blotting. RESULTS: LBP mRNA was increased in the liver 6 hr after ethanol injection followed by gradual decrease, with peak levels about 5-fold higher than controls. Twelve hr after ethanol, rat serum potentiated LPS-induced increases in TNF-a mRNA in RAW 264.7 cells about 4-fold more than controls, indicating that ethanol increased serum LBP. Further, the increase in LBP mRNA was partially blocked when rats were given intragastric injections of antibiotics (polymixin B and neomycin) for 4 days prior to ethanol, supporting the hypothesis that gut-derived endotoxin is involved in induction of LBP mRNA in the liver by ethanol. Both ethanol (100 mM) and LPS (I0 ng/ml) increased LBP mRNA in hepatocytes 2 to 2.5-fold in 6 hr. CONCLUSIONS: Both ethanol and gut-derived endotoxin directly increase LBP production by hepatocytes, thereby increasing Kupffer cell responsiveness to endotoxin. This may explain why gut-derived endotoxin causes extensive liver injury following chronic ethanol consumption (AA-03624). • L0260 A CRITICAL ROLE OF THE LIVER IN INDUCING ORAL TOLERANCE TO ADENOVIRAL VECTORS FOR LONG-TERM GENE THERAPY. Y Ilan 1,2, N Roy Chowdhury 1,2, BS Sauter 1,2, NR ThummalAL2, A Davidson 1,3, MS HorwitzL3, I Nakamura4, H Fox4 and J Roy Chowdhury 1,2. 1Marion Bessin Liver Research Center, and Depts of 2Medicine, 2Molecular Genetics, and 3Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY 10461; and 4Dept of Transplantation Surgery, University of Nebraska, Omaha, NE. We have shown previously, that oral administration of adenoviral proteins induces suppresser lymphocyte-mediated antigen-specific tolerance, permitting long-term gene therapy by repeated administration of adenoviral vectors. To determine whether the liver plays a role in inducing or maintaining oral tolerance, we administered the major proteins of a recombinant adenovirus type 5 (1 mg PO daily for 10 days) into adult Wistar rats, before or after performing an end-to-side portoeaval shunt (PCS). The rats were injected with 101° pfu of a recombinant adenovirus (Ad-LaeZ) expressing E. coli 13-galactosidase (J3gal) immediately after the tolerization and again, 40 days later. Five days after the first injection, >80% of the hepatocytes in liver biopsy specimens stained positive for I~gal, but only 2% remained positive at 35 days. When the PCS preceded antigen feeding, hightiter antibodies (1:213) and cytotoxie lymphocytes against the adenovirus developed, and the second Ad-LacZ injection failed to generate hepatic [3gal. In contrast, when the antigen feeding preceded PCS, both the humoral and CTL response were abrogated, and the second Ad-LacZ injection resulted in 13gal expression in >80% hepatoeytes. To evaluate whether Kupffer cells are involved in oral tolerization, Kupffer cell function was transiently blocked by
AASLD A1261
injecting 15 mg/kg GdCI 3 i.v. in bilirubin-UDP-glucuronosyltransferase (BUGT)-defieient jaundiced Gunn rats, before or after feeding adenoviral proteins. A recombinant adenovirus expressing human BUGT (ad-hBUGT) was injected (1010 pfu i.v.) immediately after the tolerization and again, 56 days later. After the first injection, BUGT expression was shown by reduction of mean serum bilirubin levels from 7.26 to 1.64 mg/dl in 14 days, followed by a gradual increase to pretreatment levels in 6 weeks. In rats receiving GdC13 before antigen feeding, oral tolerization did not occur, as shown by anti-adenoviral antibody and CTL response, and lack of bilirubin reduction after the second injection. However, injection of GdC13 after antigen feeding did not abolish the tolerance, as indicated by inhibition of humoral and CTL responses, and reduction of serum bilirubin levels after the second Ad-hBUGT injection. Conclusion: A first pass of portal blood through the liver and integrity of Kupffer cell function are essential for induction of oral tolerance, but not the continuation of established tolerance. L0261 PRENATAL TOLERIZATION TO RECOMBINANT ADENOVIRUS PERMITS LONG-TERlVl CORRECTION OF BILIRUBIN GLUCURONIDATION DEFECT IN GUNN RATS BY REPEATED ADENOVIRUS-MEDIATED GENRE THERAPY. Y. Ilanl, 2, BS Sauter 1,2, N Roy Chowdhury 1,2, NR Thummala1,2, A Davidson1.3, MS Horwitz 1,3, and J Roy Chowdhury 1,2. 1Marion Bessin Liver Research Center, and Departments of 2Medicine, 2Molecular Genetics, and 3Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY 10461. Many inherited metabolic diseases, including Crigler-Najjar syndrome type I (CN-I), can be diagnosed prenatally by genetic analysis. This could allow prenatal therapy for prevention of life-threatening perinatal complications. To evaluate the feasibility of prenatal adenovirus-mediated gene therapy, heterozygous female Gunn rats were bred with homozygous male Gunn rats. The pregnant Gunn rats were injected with a recombinant adenovirus (5 x 109 pfu/rat) capable of transferring the human BUGT 1 eDNA driven by a cytomegalovirus early promoter (Ad-hBUGT). As expected, genetic analysis showed that about 50% of the pups were homozygons for BUGT 1 deficiency. Serum bilirubin levels of 1 week old homozygous Gunn rats born of the Ad-hBUGT-treated or untreated (control) mothers were 3.47 +_0.87 mg/dl (mean _ S.D.) and 7.45 -+ 1.12 mg/dl, respectively. Bilirubin levels increased gradually after birth in rats reaching a level of 4.48 mg/dl on day 54 after birth. A marked decrease in bilirubin levels occurred after Ad-hBUGT injection in these rats, with levels reaching as low as 2.24 mg/dl. Similarly, injection of an adenoviral vector expressing E. coli 13-galactosidase (Ad-LacZ) into pregnant Sprague-Dawley rats caused 13-galactosidase expression both in fetuses in utero and in newborns. After injection of both Ad-hBUGT and Ad-LacZ in the pregnant mother, offspring did not develop anti-adenoviral antibodies or cytotoxic lymphocytes despite repeated postnatal injection of the recombinant viruses. Conclusion: Prenatal treatment of inherited liver diseases by recombinant adenovirus provides the dual advantage of early correction of the metabolic defect with prevention of intrauterine and perinatal complications, and induction of specific tolerance to the vector, permitting long-term gene therapy by repeated injection of the recombinant virus after birth. L0262 PERCUTANEOUS INFARCTION THERAPY (PIT) FOR HEPATOCELLULAR CARCINOMA (HCC). M, Imamura. S. Shiina, T. Teratani, S. Obi, S. Sato, K. Hamamura, Y. Koike, Y. Dan, H. Yoshida, T. Okudaira, F. Kanal, N. Kato, H. Yoshida, T. Kawabe, Y. Shiratori and M. Omata. Second Department of Internal Medicine, Univ. of Tokyo, Japan. OBJECTIVE: In order to treat large size of HCC effectively, we tried to induce massive hepatic infarction of tumor infested areas due to HCC by injection of ethanol percutaneously into feeding arteries to tumor nodules. M A T E R I A L S A N D METHODS: 17 patients (16 male, 1 female; mean age 65.6 + 9.8 years (range 52 to 87 years)) with HCC were used for this PIT treatment. The 17 HCC lesions were 2.1-8.4cm in the greatest dimension (mean+_SD, 5.1 +-1.6cm). During visualization of feeding arteries under power Doppler ultrasonography, the tip of a needle was placed into the vessels and a small amount of ethanol (l~4m) was injected. Hepatic infarction area was evaluated by dynamic CT 2 weeks later, and complete ablation of the afferent artery was checked by power Doppler US at 4 weeks and several months later. Blood examination were measured every other day after this procedure until the levels returned to the pretreatment levels. The levels of the tumor markers alpha-fetoprotein(AFP) and des-gamma-carboxy prothrombin (DCP) were measured every 2 weeks. RESULTS: Hepatic infarction was successful in the tumor-burdened lesions in 16 out of 17 patients. Marked increases in AST and bilirnbin levels were demonstrated 1 and 3 days after this procedure, respectively, but returned to the pretreatment levels within three weeks (peak AST:l,093 IU/L, peak bilirubin:3.2 mg/dl). The volume of the infarcted area ranged from 60 cm3 to 240 cm3, and was estimated as 5% to 21% of the total hepatic volume. Furthermore, serious adverse effects were not observed in association with this procedure except one case presenting cholangitis and cholangio-bronchial fistula, leading to sepsis and hepatic failure. Serum levels of AFP and DCP decreased to the level of normal values in 11 patients. Local recurrence of hepatocellular carcinoma at the infarction site was not at all observed in the follow up period of 13 -+ 11 months. CONCLUSION: Percutaneous infarction therapy may be a new method for the treatment of large hepatocellular carcinoma.
AASLD A1261
injecting 15 mg/kg GdCI 3 i.v. in bilirubin-UDP-glucuronosyltransferase (BUGT)-defieient jaundiced Gunn rats, before or after feeding adenoviral proteins. A recombinant adenovirus expressing human BUGT (ad-hBUGT) was injected (1010 pfu i.v.) immediately after the tolerization and again, 56 days later. After the first injection, BUGT expression was shown by reduction of mean serum bilirubin levels from 7.26 to 1.64 mg/dl in 14 days, followed by a gradual increase to pretreatment levels in 6 weeks. In rats receiving GdC13 before antigen feeding, oral tolerization did not occur, as shown by anti-adenoviral antibody and CTL response, and lack of bilirubin reduction after the second injection. However, injection of GdC13 after antigen feeding did not abolish the tolerance, as indicated by inhibition of humoral and CTL responses, and reduction of serum bilirubin levels after the second Ad-hBUGT injection. Conclusion: A first pass of portal blood through the liver and integrity of Kupffer cell function are essential for induction of oral tolerance, but not the continuation of established tolerance. L0261 PRENATAL TOLERIZATION TO RECOMBINANT ADENOVIRUS PERMITS LONG-TERlVl CORRECTION OF BILIRUBIN GLUCURONIDATION DEFECT IN GUNN RATS BY REPEATED ADENOVIRUS-MEDIATED GENRE THERAPY. Y. Ilanl, 2, BS Sauter 1,2, N Roy Chowdhury 1,2, NR Thummala1,2, A Davidson1.3, MS Horwitz 1,3, and J Roy Chowdhury 1,2. 1Marion Bessin Liver Research Center, and Departments of 2Medicine, 2Molecular Genetics, and 3Microbiology and Immunology, Albert Einstein College of Medicine, New York, NY 10461. Many inherited metabolic diseases, including Crigler-Najjar syndrome type I (CN-I), can be diagnosed prenatally by genetic analysis. This could allow prenatal therapy for prevention of life-threatening perinatal complications. To evaluate the feasibility of prenatal adenovirus-mediated gene therapy, heterozygous female Gunn rats were bred with homozygous male Gunn rats. The pregnant Gunn rats were injected with a recombinant adenovirus (5 x 109 pfu/rat) capable of transferring the human BUGT 1 eDNA driven by a cytomegalovirus early promoter (Ad-hBUGT). As expected, genetic analysis showed that about 50% of the pups were homozygons for BUGT 1 deficiency. Serum bilirubin levels of 1 week old homozygous Gunn rats born of the Ad-hBUGT-treated or untreated (control) mothers were 3.47 +_0.87 mg/dl (mean _ S.D.) and 7.45 -+ 1.12 mg/dl, respectively. Bilirubin levels increased gradually after birth in rats reaching a level of 4.48 mg/dl on day 54 after birth. A marked decrease in bilirubin levels occurred after Ad-hBUGT injection in these rats, with levels reaching as low as 2.24 mg/dl. Similarly, injection of an adenoviral vector expressing E. coli 13-galactosidase (Ad-LacZ) into pregnant Sprague-Dawley rats caused 13-galactosidase expression both in fetuses in utero and in newborns. After injection of both Ad-hBUGT and Ad-LacZ in the pregnant mother, offspring did not develop anti-adenoviral antibodies or cytotoxic lymphocytes despite repeated postnatal injection of the recombinant viruses. Conclusion: Prenatal treatment of inherited liver diseases by recombinant adenovirus provides the dual advantage of early correction of the metabolic defect with prevention of intrauterine and perinatal complications, and induction of specific tolerance to the vector, permitting long-term gene therapy by repeated injection of the recombinant virus after birth. L0262 PERCUTANEOUS INFARCTION THERAPY (PIT) FOR HEPATOCELLULAR CARCINOMA (HCC). M, Imamura. S. Shiina, T. Teratani, S. Obi, S. Sato, K. Hamamura, Y. Koike, Y. Dan, H. Yoshida, T. Okudaira, F. Kanal, N. Kato, H. Yoshida, T. Kawabe, Y. Shiratori and M. Omata. Second Department of Internal Medicine, Univ. of Tokyo, Japan. OBJECTIVE: In order to treat large size of HCC effectively, we tried to induce massive hepatic infarction of tumor infested areas due to HCC by injection of ethanol percutaneously into feeding arteries to tumor nodules. M A T E R I A L S A N D METHODS: 17 patients (16 male, 1 female; mean age 65.6 + 9.8 years (range 52 to 87 years)) with HCC were used for this PIT treatment. The 17 HCC lesions were 2.1-8.4cm in the greatest dimension (mean+_SD, 5.1 +-1.6cm). During visualization of feeding arteries under power Doppler ultrasonography, the tip of a needle was placed into the vessels and a small amount of ethanol (l~4m) was injected. Hepatic infarction area was evaluated by dynamic CT 2 weeks later, and complete ablation of the afferent artery was checked by power Doppler US at 4 weeks and several months later. Blood examination were measured every other day after this procedure until the levels returned to the pretreatment levels. The levels of the tumor markers alpha-fetoprotein(AFP) and des-gamma-carboxy prothrombin (DCP) were measured every 2 weeks. RESULTS: Hepatic infarction was successful in the tumor-burdened lesions in 16 out of 17 patients. Marked increases in AST and bilirnbin levels were demonstrated 1 and 3 days after this procedure, respectively, but returned to the pretreatment levels within three weeks (peak AST:l,093 IU/L, peak bilirubin:3.2 mg/dl). The volume of the infarcted area ranged from 60 cm3 to 240 cm3, and was estimated as 5% to 21% of the total hepatic volume. Furthermore, serious adverse effects were not observed in association with this procedure except one case presenting cholangitis and cholangio-bronchial fistula, leading to sepsis and hepatic failure. Serum levels of AFP and DCP decreased to the level of normal values in 11 patients. Local recurrence of hepatocellular carcinoma at the infarction site was not at all observed in the follow up period of 13 -+ 11 months. CONCLUSION: Percutaneous infarction therapy may be a new method for the treatment of large hepatocellular carcinoma.