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Does autoimmunity affect the outcome of liver transplantation?
422A
AASLD ABSTRACTS
1261 IMMUNIZATION OF MICE WITH CARBONIC ANHYDRASE II ELICITS HIGH TITER ANTIBODIES WITHOUT HISTOLOGICAL EVIDENCE OF AUTOIMMUNE CHOLANGITIS. S.C. Gordon. J.S. Neill. A.L. Silverman & I.M. Zitron. GI-Hepatology Division, William Beaumont Hospital, Royal Oak, ML
Serum antibodies specific for carbonic anhydrase II (CAll)have been demonstrated in patients with autoimmune cholangitis (AC) [Gastroenterology 1995;108:1802]. We investigated whether an AC-like syndrome maybe induced in inbred mice by deliberate immunization with CAll. Female mice were immunized with human CAll emulsified in Freunds' complete adjuvant (FCA); control animal animals received Ovalbumin (OVA) in FCA. Serum alkaline ph0sphatase (AP) and antibody titer were monitored and sections of liver and kidney examined for indications of patholo0ical changes. C57BL/6 mice were followed for 30 days post immunization (p.i.), during which time serum antibodies were demonstrated from day 10 p.i., but no significant changes in serum AP were observed. Kidney sections showed no pathological changes, but occasional Iobular andportal infiltrates were noted. A second series of C57BL/6 mice were immunized using antigen in a smaller dose of FCA and were observed over a longer time period. At days 32, 39 and 46 days p.i., the serum AP levels o f the CAll-immune mice were higher than those of the OVA-immune animals (p<0.01), although the absolute levels were only about 50% higher. Subsequent time points Showed no differences. Liver sections obtained by partial hepatectomy at 90 days p.i. showed no differences in liver histology between the two groups. No differences were seen between the CAll- and OVA-immune mice, in either serum AP levels or liver histology, after secondary challenge. Five other inbred strains of mice (A/J, AKR, BALB/c, C3H and SJL) were tested by a similar approach. All strains responded to CAll, as judged by serum antibody, but no differences were observed, in either serum AP or liver histology, when CAll- and OVA-immune animals were compared. We conclude that anti-CAll antibodies are not the principal pathogenic mechanism in AC. While type II hypersensitivity reactions do not appear to play an important role in this disease, the results do not compromise the importance of anti-CAll antibodies as a distinguishing feature of AC. The results suggest that, in order to evaluate the pathogenic significance of autoreactivity to CAll in an animal model, immunization conditions must be employed which will favor an inflammatory, Thl cell-mediated response over a Th2dominated humoral response.
HEPATOLOGYOctober 1995
1262 DOES AUTOIMMUNITY AFFECT THE OUTCOME OF LIVER
TRANSPLANTATION? J Gournav. P Bacchetti. JP Roberts. NL Ascher. JR Lake. Liver Transplant Program and Department of Epidemiology and Biostatistics, University 0fCaiifomia San Francisco.
Autoimmune liver diseases are frequent indication for orthotopic liver transplantation (LT). However, autoimmune markers are frequently found in patients with chronic hepatitis C. This study aimed to determine whether the presence of autoimmune markers, with or without HCV infection, affects the outcome of LT. Methods: 700 LT were performed in UCSF between February 1988 and April 1995. We retrospectively studied 358 liver transplant recipients (51.8%) for whom complete viral and immunological data were available. The mean age was 47+14 years (range: 0-71). Patients with HBsAg prior to LT were excluded from the study. Re-transplantations were not included in the study, A titer of antinuclear antibodies (ANA) greater than or equal to 1/80 pdor to LT was consideredpositive. Patients with anti-HCV positive by ELISA or RIBA prior to LTwere considered HCV-infected. Survival analyses were performed using Cox regression, Results: ANA was positive in 36% of the patients. Anti:mitochonddai antibodies and anti-smooth muscle antibodies were detected in 4.7% and 45.2% of the patients respectively. There was a rank correlation between ANA titer and age (r=0.24, p=.0001). The relative risk for death increased significantly with age (p<.O004). Pre-transp antation HCV infection was found in 32.1% Of the patients. 30.4% of the HCV (+) patients and 38.7% of the HCV (-) patients were ANA positive (p=.16). There was no association between diagnosis and presence of ANA. For ANA (+) patients, the relative risk for death, controlled for age, was 0.615 (CI: 0.34-1.1, p=.10). For HCV (+) patients, the relative risk for death, controlled for age, was 0.78 (CI: 0.43-1.41, p=.4). When compared to ANA (-) HCV (-) patients, the relative risk for death for ANA (+) HCV (-) patients was 0.57 (CI: 0.29-1.1, p=.10) and the relative risk for death for ANA (+) HCV (+) patients was 0.51 (CI: 0,18.1.16, p=.21). Summary: Markers of autoimmunity are common in liver transplant candidates, regardless of the liver disease. Our results suggest that the presence of ANA prior to LT tends to be associated with better survival after LT. Although the number of patients was too small to conclude, this effect seems to be present also when ANA are associated with HCV infection. These preliminary data suggest that autoimmunity can affect the outcome of LT.
1263 THE NUCLEOSIDE ANALOGUES 2-CHLORODEOXYADENOSINE AND
1264 INFLUENCE OF ALL-TRANS AND 9-CIS RETINOIC ACID ON
GEMCITABINE INHIBIT GROWTH OF HUMAN HEPATOMA CELLS IN VITRO. l.Graziadei, A Propst, M Sehirmer, G Konwalinka. JR Patseh: and W Vogel. DepL of Internal Medicine, University of Innsbruck, Austria
CONNECTIVE TISSUE SYNTHESIS BY CULTURED FAT-STORING CELLS. I Grinko, T Niki, E Wisse, and A Geerts. Laboratory for Cell Biology and Histology, Free University of Brussels (V.U.B.), Brussels, Belgium.
Hepatocellular carcmoma (HCC) is one of the most common malignant tumors in the world with a very poor prognosis. The median survival for patient with unreseetable HCC is less than half a year. At present only resection and chemoembolisation have shown to prolong life. This treatment modalities are superior to any chemotherapy. Therefore. newer antineoplastie substances are needed. 2-Chlorodeoxyadenosine (2-CDA) and Gemcitabine are synthetic antimetabolites of the cellular pyrimidine nucleotidc metabolism and have been used effectively m the treatment of leukemia and solid tumors with no hepaUc side effects. In this study we investigated the effect of these two antitumor agents on the growth of hepatoma cells in vitro. For the experiments we used the human hepatoma cell lines HepG2 and Chang ceils. HepG2 cells were cultured in RPMl1640 + 10% FCS and Chang cells m BME Hanks + 10% FCS at 37°C and 5% CO 2. 2-CDA and Gemeitabine were added to the culture dishes in increasing concentrations and cell number was counted after incubation of 96h. Cell viability was determined by exclusion of 0.1% eosin The growth of the hepatoma cell lines HepG2 and Chang cells were significantly inhibited by 2-CDA as well as gemeitabine. The EC50 of 2-CDA on HepG2 was 44.58~-0.24 nM and on Chang cells 32.29~-1.57 nM, Gemeitabine yielded EC50-vnlues of 3.91±0.02 nM (HepG2) and 5.60±0.80 nM (Chang). Complete iithibition of both cell lines occurred at concentrations of 150nM of 2-CDA and 50nM of gemcitabine. Cell viability was decreased to maximal 25% of control ]'he inhibitory potency of 2-CDA and gcmeitabine was completely reversed by the addition of the natural analogue deoxyeytidine. These results suggest that 2-CDA and gemeitnhine are promising antitumor agents for human hepatoma cells and should be fmther evaluated in its efficiency in the treatment of H C C
Fat-storing cells (FSC) were isolated from normal male Wistar ruts by the pronase/collagvaase method. Cells were plated in culture dishes and flasks, passagcd a t d a y 5-7 and examined at day 14-16. Cells were exposed to 10-6, 10-7, and 10-s M all-trans or 9-cis retinoic acid (RA) for 48 h. Cells were metabolically labeled with Trans asS-label during the last 24 h of culture. Conditioned medium and cell layer were collected, extracted and immunoprecipitated with antibodies to collagen types I, III, IV, laminin and fibrunectin, Immunoprecipitates were analyzed by SDS-PAGE. Gels were examined on a Bio-Rad Molecular Imager System. In a second series of experiments total RNA was extracted from FSC exposed to 10-6M all-trans or 9-cis RA. Total RNA were fractioned in agarose/paraformaldehyde gels and transferred onto a nylon membrane by capillary blotting. Filters were hybridized with the following probes labeled with 32P-dCTP: rat procollagen al(I), rat procollagen al(llI), mouse procollagen al(IV), rat fibronectin, and mouse laminin BI. At the protein level, we found a significant suppressive effect (up to 50%) of 10-6M and 10-7M all-trans RA on collagen type I, III and laminin in the medium and cell layer. For fibronectin the effect was restricted to the medium. These changes were confirmed by downregulation of mRNAs. For 9-cis RA the suppressive effect was less outspoken; it oecared on collagen type I in medium at lO"6M and in ceil layer at 10-6 and 10-TM; on collagen type III, in the medium and cell lysate at 10"6M and on collagen type IV in the medium at 10-6M. 9-cis RA did not have any effect on laminin or fibronectin synthesis. At the mRNA level we did not find significant changes with 9-cis RA. We conclude that both isoforms of retinnic acid have a suppressive effect on the synthesis and secretion of the main connective tissue components by cultured FSC. This effect is more prominent for all-trans RA.
AASLD ABSTRACTS
1261 IMMUNIZATION OF MICE WITH CARBONIC ANHYDRASE II ELICITS HIGH TITER ANTIBODIES WITHOUT HISTOLOGICAL EVIDENCE OF AUTOIMMUNE CHOLANGITIS. S.C. Gordon. J.S. Neill. A.L. Silverman & I.M. Zitron. GI-Hepatology Division, William Beaumont Hospital, Royal Oak, ML
Serum antibodies specific for carbonic anhydrase II (CAll)have been demonstrated in patients with autoimmune cholangitis (AC) [Gastroenterology 1995;108:1802]. We investigated whether an AC-like syndrome maybe induced in inbred mice by deliberate immunization with CAll. Female mice were immunized with human CAll emulsified in Freunds' complete adjuvant (FCA); control animal animals received Ovalbumin (OVA) in FCA. Serum alkaline ph0sphatase (AP) and antibody titer were monitored and sections of liver and kidney examined for indications of patholo0ical changes. C57BL/6 mice were followed for 30 days post immunization (p.i.), during which time serum antibodies were demonstrated from day 10 p.i., but no significant changes in serum AP were observed. Kidney sections showed no pathological changes, but occasional Iobular andportal infiltrates were noted. A second series of C57BL/6 mice were immunized using antigen in a smaller dose of FCA and were observed over a longer time period. At days 32, 39 and 46 days p.i., the serum AP levels o f the CAll-immune mice were higher than those of the OVA-immune animals (p<0.01), although the absolute levels were only about 50% higher. Subsequent time points Showed no differences. Liver sections obtained by partial hepatectomy at 90 days p.i. showed no differences in liver histology between the two groups. No differences were seen between the CAll- and OVA-immune mice, in either serum AP levels or liver histology, after secondary challenge. Five other inbred strains of mice (A/J, AKR, BALB/c, C3H and SJL) were tested by a similar approach. All strains responded to CAll, as judged by serum antibody, but no differences were observed, in either serum AP or liver histology, when CAll- and OVA-immune animals were compared. We conclude that anti-CAll antibodies are not the principal pathogenic mechanism in AC. While type II hypersensitivity reactions do not appear to play an important role in this disease, the results do not compromise the importance of anti-CAll antibodies as a distinguishing feature of AC. The results suggest that, in order to evaluate the pathogenic significance of autoreactivity to CAll in an animal model, immunization conditions must be employed which will favor an inflammatory, Thl cell-mediated response over a Th2dominated humoral response.
HEPATOLOGYOctober 1995
1262 DOES AUTOIMMUNITY AFFECT THE OUTCOME OF LIVER
TRANSPLANTATION? J Gournav. P Bacchetti. JP Roberts. NL Ascher. JR Lake. Liver Transplant Program and Department of Epidemiology and Biostatistics, University 0fCaiifomia San Francisco.
Autoimmune liver diseases are frequent indication for orthotopic liver transplantation (LT). However, autoimmune markers are frequently found in patients with chronic hepatitis C. This study aimed to determine whether the presence of autoimmune markers, with or without HCV infection, affects the outcome of LT. Methods: 700 LT were performed in UCSF between February 1988 and April 1995. We retrospectively studied 358 liver transplant recipients (51.8%) for whom complete viral and immunological data were available. The mean age was 47+14 years (range: 0-71). Patients with HBsAg prior to LT were excluded from the study. Re-transplantations were not included in the study, A titer of antinuclear antibodies (ANA) greater than or equal to 1/80 pdor to LT was consideredpositive. Patients with anti-HCV positive by ELISA or RIBA prior to LTwere considered HCV-infected. Survival analyses were performed using Cox regression, Results: ANA was positive in 36% of the patients. Anti:mitochonddai antibodies and anti-smooth muscle antibodies were detected in 4.7% and 45.2% of the patients respectively. There was a rank correlation between ANA titer and age (r=0.24, p=.0001). The relative risk for death increased significantly with age (p<.O004). Pre-transp antation HCV infection was found in 32.1% Of the patients. 30.4% of the HCV (+) patients and 38.7% of the HCV (-) patients were ANA positive (p=.16). There was no association between diagnosis and presence of ANA. For ANA (+) patients, the relative risk for death, controlled for age, was 0.615 (CI: 0.34-1.1, p=.10). For HCV (+) patients, the relative risk for death, controlled for age, was 0.78 (CI: 0.43-1.41, p=.4). When compared to ANA (-) HCV (-) patients, the relative risk for death for ANA (+) HCV (-) patients was 0.57 (CI: 0.29-1.1, p=.10) and the relative risk for death for ANA (+) HCV (+) patients was 0.51 (CI: 0,18.1.16, p=.21). Summary: Markers of autoimmunity are common in liver transplant candidates, regardless of the liver disease. Our results suggest that the presence of ANA prior to LT tends to be associated with better survival after LT. Although the number of patients was too small to conclude, this effect seems to be present also when ANA are associated with HCV infection. These preliminary data suggest that autoimmunity can affect the outcome of LT.
1263 THE NUCLEOSIDE ANALOGUES 2-CHLORODEOXYADENOSINE AND
1264 INFLUENCE OF ALL-TRANS AND 9-CIS RETINOIC ACID ON
GEMCITABINE INHIBIT GROWTH OF HUMAN HEPATOMA CELLS IN VITRO. l.Graziadei, A Propst, M Sehirmer, G Konwalinka. JR Patseh: and W Vogel. DepL of Internal Medicine, University of Innsbruck, Austria
CONNECTIVE TISSUE SYNTHESIS BY CULTURED FAT-STORING CELLS. I Grinko, T Niki, E Wisse, and A Geerts. Laboratory for Cell Biology and Histology, Free University of Brussels (V.U.B.), Brussels, Belgium.
Hepatocellular carcmoma (HCC) is one of the most common malignant tumors in the world with a very poor prognosis. The median survival for patient with unreseetable HCC is less than half a year. At present only resection and chemoembolisation have shown to prolong life. This treatment modalities are superior to any chemotherapy. Therefore. newer antineoplastie substances are needed. 2-Chlorodeoxyadenosine (2-CDA) and Gemcitabine are synthetic antimetabolites of the cellular pyrimidine nucleotidc metabolism and have been used effectively m the treatment of leukemia and solid tumors with no hepaUc side effects. In this study we investigated the effect of these two antitumor agents on the growth of hepatoma cells in vitro. For the experiments we used the human hepatoma cell lines HepG2 and Chang ceils. HepG2 cells were cultured in RPMl1640 + 10% FCS and Chang cells m BME Hanks + 10% FCS at 37°C and 5% CO 2. 2-CDA and Gemeitabine were added to the culture dishes in increasing concentrations and cell number was counted after incubation of 96h. Cell viability was determined by exclusion of 0.1% eosin The growth of the hepatoma cell lines HepG2 and Chang cells were significantly inhibited by 2-CDA as well as gemeitabine. The EC50 of 2-CDA on HepG2 was 44.58~-0.24 nM and on Chang cells 32.29~-1.57 nM, Gemeitabine yielded EC50-vnlues of 3.91±0.02 nM (HepG2) and 5.60±0.80 nM (Chang). Complete iithibition of both cell lines occurred at concentrations of 150nM of 2-CDA and 50nM of gemcitabine. Cell viability was decreased to maximal 25% of control ]'he inhibitory potency of 2-CDA and gcmeitabine was completely reversed by the addition of the natural analogue deoxyeytidine. These results suggest that 2-CDA and gemeitnhine are promising antitumor agents for human hepatoma cells and should be fmther evaluated in its efficiency in the treatment of H C C
Fat-storing cells (FSC) were isolated from normal male Wistar ruts by the pronase/collagvaase method. Cells were plated in culture dishes and flasks, passagcd a t d a y 5-7 and examined at day 14-16. Cells were exposed to 10-6, 10-7, and 10-s M all-trans or 9-cis retinoic acid (RA) for 48 h. Cells were metabolically labeled with Trans asS-label during the last 24 h of culture. Conditioned medium and cell layer were collected, extracted and immunoprecipitated with antibodies to collagen types I, III, IV, laminin and fibrunectin, Immunoprecipitates were analyzed by SDS-PAGE. Gels were examined on a Bio-Rad Molecular Imager System. In a second series of experiments total RNA was extracted from FSC exposed to 10-6M all-trans or 9-cis RA. Total RNA were fractioned in agarose/paraformaldehyde gels and transferred onto a nylon membrane by capillary blotting. Filters were hybridized with the following probes labeled with 32P-dCTP: rat procollagen al(I), rat procollagen al(llI), mouse procollagen al(IV), rat fibronectin, and mouse laminin BI. At the protein level, we found a significant suppressive effect (up to 50%) of 10-6M and 10-7M all-trans RA on collagen type I, III and laminin in the medium and cell layer. For fibronectin the effect was restricted to the medium. These changes were confirmed by downregulation of mRNAs. For 9-cis RA the suppressive effect was less outspoken; it oecared on collagen type I in medium at lO"6M and in ceil layer at 10-6 and 10-TM; on collagen type III, in the medium and cell lysate at 10"6M and on collagen type IV in the medium at 10-6M. 9-cis RA did not have any effect on laminin or fibronectin synthesis. At the mRNA level we did not find significant changes with 9-cis RA. We conclude that both isoforms of retinnic acid have a suppressive effect on the synthesis and secretion of the main connective tissue components by cultured FSC. This effect is more prominent for all-trans RA.