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Plasmapheresis in patients with severe primary dysfunction after liver transplantation
HEPATOLOGY Vol. 22, N o . 4, P t . 2, 1 9 9 5
1409
AASLD
ABSTRACTS
LIVER H I S T O L O G Y AND S E R U M H C V - R N A OF ANTI-HCV POSITIVE PATIENTS WITH PERSISTENT NORMAL AMINOTRANSFERASE LEVELS
1 4 1 0 HETEROGENEITY IN THE INDUCTION OF ALPHA-FETOPROTEIN(AFP) GENE EXPRESSION IN AN EXPERIMENTAL ANIMAL MODEL OF CIRRHOSIS AND HEPATOCELLULARCARCINOMA(HCC). Moutoya H. Rinc6n AFt and A Panduro. Basic Research Center, University of Aguascalientesand Institute of Molecular Biology in Medicine, CUCS. U.de G. and H.C. de Guadalajara. M6xico.
Montalto G,Zi~nego AL, Ruggeri MI, Monfi M, Bascone F, Careccia G. Cattedra di Medicina Interna~ Universit~ di Palermo e Istituto di I~edicina Interna, Universit~ di Firenze, Italy
Small amountsof AFP are often detected in the cirrhotic liver but it is increased during HCC.Since HCCis closely associatedwith liver fibrosis, we analyzed the expression of AFP at the mRNA level in an experimental animal model of cirrhosis and HCC. Liver fibrosis was induced in rats by i.p. doses of CCl4 three times per week during two months. To induce HCC previous to the chronic CCl4 treatment, rats ware treated w~h diethyl nitrosamine(DEN), ecetylamine fluorene (AAF) and then with an acute intragaetricdose of CCI4 (CC). Rats untreated (N), treated with DEN and AAF (CA) and only with the CCI4 chronic treatment (CR) ware included. Samples were taken at 2, 6 and 10 weeks after finishing the chronic CCl4 treatment, At 10th week the CC group presented a multinodular appearance liver, fibrosis and limited figures of pseudo ecinar structures with anaplastichepetecytes(irregulare, ill¢ontorned, acidophilicand cariopycnotios), These findings ware not present in CA but only fibrosis in the CR group. Liver function test (LIT) ware altered dudng treatment in the CA, CR and CC groups, remained increased 10 weeks later in the CC group (IAST 440-J:37vs 74+10 IU/I. (N)], [ALl' 179~3 vs 705:11 IU/L], [ALP 193+16 vs 1095:13 IU/I.], [total bilirubin [2.6-J:0.04vs 0.62.t0.05 mg/10Oml]), whereas in the CR and CA 9roups LFT came back to values cicee to normal. No changes in serum albumin were detected in either experimentalgroup. In N rats, AFP mRNA was not detected in liver tissue by northern blot. However, in the CC group a strong induction in the expression of the AFP 9ene at the mRNA level was observed, such increase was 14 times higher than the CR and CA groupware analized densitometrically.Such increase of AFP mRNA was not always present in different samples of the same tissue, even when low levels (40-50 %) of albumin mRNA were detected. In this study we establishedan experimentalanimal model of cirrhosis and HCC in a shorl period of time. The molecularfindings showed heterogeneity in the induction of AFP gene expression at the mRNA level and indicate the feasibility of analyze the mechanisms that control geee expression at the molecular level.
We evaluated liver histology and serum H C V - R N A levels in 30 patients anti-HCV positive on ELISA H, 19 o f which were confirmed positive and 11 indetcaxninate on RIBA H, presenting persistently normal ALT values over 24 months, determined every month for the first 18 months and monthly in the 6 months prior to liver biopsy. We excluded from the study drug abusers, alcoholics and subjects positive for H B s A g or anfi-HIV or with autoimmune diseases or metabolic disorders. Testing for H C V - R N A was performecl by nested P C R using primers o f 5' UT region. Liver biopsy was performed percutaneously with M~nghini's needle. 16 patients were serum H C V R N A positive (53%); on R I B A 1] 13 o f them (68%) were positive and 3 (27%) were indeterminate. HIstology of the H C V - R N A positive patients showed g cases o f CPH, 6 cases of CAH, 1 case o f C L H and 1 case o f N L (normal liver). Histology o f the H C V - R N A negative patients showed 4 cases o f CPH, 1 case o f CAH, 2 cases o f C L H and 7 eases o f N L These results indicate that subjects positive on RIBA II, but with persistently normal ALT values, have a high possibility o f being serum H C V - R N A positive and that almost all these viremic subjects present histological signs o f liver disease. In contrast, subjects with indeterminate RIBA II have a moderate probability' o f being H C V - R N A positive, but a number o f these may present signs o f liver disease. Fluctuations in viremia may possibly lead to false negative results; in any case a close follow-up o f these cases is necessary to detect evolution o f liver disease.
1411
PLASMAPHERESIS IN PATIENTS WITH SEVERE PRIMARY DYSFUNCTION AFFER LIVER TRANSPLANTATION E. Mee,D. Skerrettt,S. Curtiss.P. Sheiner, S. Emre~ S. Guy. M. Schwartz, C. Miller. Dept. of Surgew and The Blood Bankt, The Mount Sinai Medical Center, New York Severe primary hepatic graft dysfunction (PGD) after liver transplant CLTx) is associated with high morbidity and mortality. In many cases, re-LTx is required. We postulated that plasmapberesis (PL) in patients with PGD may reduce cytokine elevatious that result from severe endothelial cell and bepatocellul~ damage and thus lower the rate of graft failure. Methods: Between 7]91-7/94, PL was initiated in patients with PGD within 24 hr after LTx. POD was diagnosed in the presence of at least 2 of the following in the 1st 24 hr pest- LTx: absence of bile production, prothrombin time > 16.5 see, ALT >2500 U/L, stage IH or IV coma, and oliguria. One-and-one-half volume exchange was performed daily for a total of 4 procedures unless the patient showed significant improvement or underwent re-LTx. Removed fluid was replaced with f~esh frozen plasma and saline at a ratio of 7:3. clinical outcomes (graft and patient survival, ICU stay, and need for dialysis) were compared in 18 patients who underwent PL and 22 patients in a historical control. Severity of preservation injury as defined by degree of ceaguiative necrosis and neutrophil infiltration on IX~Stparfusionbiopsy (Bx) was also compared. In 6 patients, serum eytokine levels (TNF, K-l, IL-6, and IL-8) were measured by ELISA before and after PL. Results: Groups did not differ by age, sex, primary disease, or baseline ereatinine. Peak ALT and PT in the 1st 24 hr Imst-LTx were not different between groups. Dialysis was required by 9118 patients in the PL group and 4/22 in the control (p<0.05). Mean c~eatinine level on POD 2 was 2.47 rag/ dL in the PL group and 1.62 mg/dL in the control (p<0.ff2). Post'perfusion Bx revealed +2 or higher grade of neerusis in 71% and 75% of PL and control patients, respectively. Ten patients completed the 4 treatment course; 2410 rexluired re-LTx. Eight patients discontinued PL because of improvement (4), re-LTx (3), c~ death (1). Overall patient and graft survival were comparable. Five PL patients and 4 in the control underwent reLTx. Three patients in the PL group and 2 in the control died <3 mes post-LTx~ Mean ICU stay was 15 days in the PL group and 10 days in the control (p=ns). In the 6 patients in whom cytokine levels were measured, a post-PL reduction in TNP of 66% and IL-6 of 55.2% was noted. There was no change in IL-1 or IL-8 levels after PL. Conclusions: Plasanapheresis did not improve outcome in patients with severe primary graft dysfunction, although the treatment group had a higher incidence of renal failure, suggesting more severe liver dysfunction. Although systemic TNF and IL-6 levels may be reduced by plasmapheresis, this reduction has no impact on graft and patient survival.
459A
1412
EFFECTS OF LONG TERM ALCOHOL ADMINISTRATION ON NITKIC OXIDE PRODUCTION BY HEPATIC MACROPHAGES A N D E N D O T H E L I A L C E L L S . L Morio, D E Heck, J D Laskin, C G a r d n e r , a n d D L Laskin. R u t g e r s U n i v e r s i t y / U M D N J Robert Wood J o h n s o n Medical School, P i s c a t a w a y , N J
Hepatic macrophages (MP) and endothelial cells(EC) release an array of proinflammatory and cytotoxicmediators that have been implicated in the development of liverinjury. In the present studies we analyzed the effectsof chronic alcohol administration on production of nitricoxide by these cells. Rats were exposed to ethanol (10:4 g/kg) by intragastricfeeding. Cells were isolated from the animals 3-6 weeks later by in situ perfusion of the liver with collagenase and pronase followed by differentialcentrifugation and elutriation. W e found that M P and E C from both untreated and alcohol-treatedrats readily synthesized nitricoxide following in vitro stimulation with interferon-7(IFNT) and lipopolysacoharide (LPS) alone and in combination. This response was dependent on l-arginineand was blocked by two nitricoxide synthase inhibitsrs,N'%monomethyl-l-arginine and l-canavanine. M P and E C produced similar amounts of nitricoxide in response to these inflammatory mediators. Nitric oxide production by both celltypes in response to LPS plus IFN7 was decreased following treatment of rats with alcohol.M P appeared to be more sensitive to the in vivo effectsof alcohol than were EC. Northern and western blot analysis demonstrated that nitricoxide production by M P and E C in response to LPS plus IFN7 was due to increased expression of an inducible form of nitric oxide synthase (iNOS) m R N A and protein. Using fluorescence image analysis,iNOS protein was found to be localizedin the cytoplasm of the cells. Taken together, our data demonstrate that exposure of rats to alcohol decreases the capacity of both M P and E C to produce nitric oxide which is regulated distinctlyin these celltypes. Decreased sensitivityof nonparenchymal cellsto inflammatory mediators may be important in the hepatic response to alcohol.
1409
AASLD
ABSTRACTS
LIVER H I S T O L O G Y AND S E R U M H C V - R N A OF ANTI-HCV POSITIVE PATIENTS WITH PERSISTENT NORMAL AMINOTRANSFERASE LEVELS
1 4 1 0 HETEROGENEITY IN THE INDUCTION OF ALPHA-FETOPROTEIN(AFP) GENE EXPRESSION IN AN EXPERIMENTAL ANIMAL MODEL OF CIRRHOSIS AND HEPATOCELLULARCARCINOMA(HCC). Moutoya H. Rinc6n AFt and A Panduro. Basic Research Center, University of Aguascalientesand Institute of Molecular Biology in Medicine, CUCS. U.de G. and H.C. de Guadalajara. M6xico.
Montalto G,Zi~nego AL, Ruggeri MI, Monfi M, Bascone F, Careccia G. Cattedra di Medicina Interna~ Universit~ di Palermo e Istituto di I~edicina Interna, Universit~ di Firenze, Italy
Small amountsof AFP are often detected in the cirrhotic liver but it is increased during HCC.Since HCCis closely associatedwith liver fibrosis, we analyzed the expression of AFP at the mRNA level in an experimental animal model of cirrhosis and HCC. Liver fibrosis was induced in rats by i.p. doses of CCl4 three times per week during two months. To induce HCC previous to the chronic CCl4 treatment, rats ware treated w~h diethyl nitrosamine(DEN), ecetylamine fluorene (AAF) and then with an acute intragaetricdose of CCI4 (CC). Rats untreated (N), treated with DEN and AAF (CA) and only with the CCI4 chronic treatment (CR) ware included. Samples were taken at 2, 6 and 10 weeks after finishing the chronic CCl4 treatment, At 10th week the CC group presented a multinodular appearance liver, fibrosis and limited figures of pseudo ecinar structures with anaplastichepetecytes(irregulare, ill¢ontorned, acidophilicand cariopycnotios), These findings ware not present in CA but only fibrosis in the CR group. Liver function test (LIT) ware altered dudng treatment in the CA, CR and CC groups, remained increased 10 weeks later in the CC group (IAST 440-J:37vs 74+10 IU/I. (N)], [ALl' 179~3 vs 705:11 IU/L], [ALP 193+16 vs 1095:13 IU/I.], [total bilirubin [2.6-J:0.04vs 0.62.t0.05 mg/10Oml]), whereas in the CR and CA 9roups LFT came back to values cicee to normal. No changes in serum albumin were detected in either experimentalgroup. In N rats, AFP mRNA was not detected in liver tissue by northern blot. However, in the CC group a strong induction in the expression of the AFP 9ene at the mRNA level was observed, such increase was 14 times higher than the CR and CA groupware analized densitometrically.Such increase of AFP mRNA was not always present in different samples of the same tissue, even when low levels (40-50 %) of albumin mRNA were detected. In this study we establishedan experimentalanimal model of cirrhosis and HCC in a shorl period of time. The molecularfindings showed heterogeneity in the induction of AFP gene expression at the mRNA level and indicate the feasibility of analyze the mechanisms that control geee expression at the molecular level.
We evaluated liver histology and serum H C V - R N A levels in 30 patients anti-HCV positive on ELISA H, 19 o f which were confirmed positive and 11 indetcaxninate on RIBA H, presenting persistently normal ALT values over 24 months, determined every month for the first 18 months and monthly in the 6 months prior to liver biopsy. We excluded from the study drug abusers, alcoholics and subjects positive for H B s A g or anfi-HIV or with autoimmune diseases or metabolic disorders. Testing for H C V - R N A was performecl by nested P C R using primers o f 5' UT region. Liver biopsy was performed percutaneously with M~nghini's needle. 16 patients were serum H C V R N A positive (53%); on R I B A 1] 13 o f them (68%) were positive and 3 (27%) were indeterminate. HIstology of the H C V - R N A positive patients showed g cases o f CPH, 6 cases of CAH, 1 case o f C L H and 1 case o f N L (normal liver). Histology o f the H C V - R N A negative patients showed 4 cases o f CPH, 1 case o f CAH, 2 cases o f C L H and 7 eases o f N L These results indicate that subjects positive on RIBA II, but with persistently normal ALT values, have a high possibility o f being serum H C V - R N A positive and that almost all these viremic subjects present histological signs o f liver disease. In contrast, subjects with indeterminate RIBA II have a moderate probability' o f being H C V - R N A positive, but a number o f these may present signs o f liver disease. Fluctuations in viremia may possibly lead to false negative results; in any case a close follow-up o f these cases is necessary to detect evolution o f liver disease.
1411
PLASMAPHERESIS IN PATIENTS WITH SEVERE PRIMARY DYSFUNCTION AFFER LIVER TRANSPLANTATION E. Mee,D. Skerrettt,S. Curtiss.P. Sheiner, S. Emre~ S. Guy. M. Schwartz, C. Miller. Dept. of Surgew and The Blood Bankt, The Mount Sinai Medical Center, New York Severe primary hepatic graft dysfunction (PGD) after liver transplant CLTx) is associated with high morbidity and mortality. In many cases, re-LTx is required. We postulated that plasmapberesis (PL) in patients with PGD may reduce cytokine elevatious that result from severe endothelial cell and bepatocellul~ damage and thus lower the rate of graft failure. Methods: Between 7]91-7/94, PL was initiated in patients with PGD within 24 hr after LTx. POD was diagnosed in the presence of at least 2 of the following in the 1st 24 hr pest- LTx: absence of bile production, prothrombin time > 16.5 see, ALT >2500 U/L, stage IH or IV coma, and oliguria. One-and-one-half volume exchange was performed daily for a total of 4 procedures unless the patient showed significant improvement or underwent re-LTx. Removed fluid was replaced with f~esh frozen plasma and saline at a ratio of 7:3. clinical outcomes (graft and patient survival, ICU stay, and need for dialysis) were compared in 18 patients who underwent PL and 22 patients in a historical control. Severity of preservation injury as defined by degree of ceaguiative necrosis and neutrophil infiltration on IX~Stparfusionbiopsy (Bx) was also compared. In 6 patients, serum eytokine levels (TNF, K-l, IL-6, and IL-8) were measured by ELISA before and after PL. Results: Groups did not differ by age, sex, primary disease, or baseline ereatinine. Peak ALT and PT in the 1st 24 hr Imst-LTx were not different between groups. Dialysis was required by 9118 patients in the PL group and 4/22 in the control (p<0.05). Mean c~eatinine level on POD 2 was 2.47 rag/ dL in the PL group and 1.62 mg/dL in the control (p<0.ff2). Post'perfusion Bx revealed +2 or higher grade of neerusis in 71% and 75% of PL and control patients, respectively. Ten patients completed the 4 treatment course; 2410 rexluired re-LTx. Eight patients discontinued PL because of improvement (4), re-LTx (3), c~ death (1). Overall patient and graft survival were comparable. Five PL patients and 4 in the control underwent reLTx. Three patients in the PL group and 2 in the control died <3 mes post-LTx~ Mean ICU stay was 15 days in the PL group and 10 days in the control (p=ns). In the 6 patients in whom cytokine levels were measured, a post-PL reduction in TNP of 66% and IL-6 of 55.2% was noted. There was no change in IL-1 or IL-8 levels after PL. Conclusions: Plasanapheresis did not improve outcome in patients with severe primary graft dysfunction, although the treatment group had a higher incidence of renal failure, suggesting more severe liver dysfunction. Although systemic TNF and IL-6 levels may be reduced by plasmapheresis, this reduction has no impact on graft and patient survival.
459A
1412
EFFECTS OF LONG TERM ALCOHOL ADMINISTRATION ON NITKIC OXIDE PRODUCTION BY HEPATIC MACROPHAGES A N D E N D O T H E L I A L C E L L S . L Morio, D E Heck, J D Laskin, C G a r d n e r , a n d D L Laskin. R u t g e r s U n i v e r s i t y / U M D N J Robert Wood J o h n s o n Medical School, P i s c a t a w a y , N J
Hepatic macrophages (MP) and endothelial cells(EC) release an array of proinflammatory and cytotoxicmediators that have been implicated in the development of liverinjury. In the present studies we analyzed the effectsof chronic alcohol administration on production of nitricoxide by these cells. Rats were exposed to ethanol (10:4 g/kg) by intragastricfeeding. Cells were isolated from the animals 3-6 weeks later by in situ perfusion of the liver with collagenase and pronase followed by differentialcentrifugation and elutriation. W e found that M P and E C from both untreated and alcohol-treatedrats readily synthesized nitricoxide following in vitro stimulation with interferon-7(IFNT) and lipopolysacoharide (LPS) alone and in combination. This response was dependent on l-arginineand was blocked by two nitricoxide synthase inhibitsrs,N'%monomethyl-l-arginine and l-canavanine. M P and E C produced similar amounts of nitricoxide in response to these inflammatory mediators. Nitric oxide production by both celltypes in response to LPS plus IFN7 was decreased following treatment of rats with alcohol.M P appeared to be more sensitive to the in vivo effectsof alcohol than were EC. Northern and western blot analysis demonstrated that nitricoxide production by M P and E C in response to LPS plus IFN7 was due to increased expression of an inducible form of nitric oxide synthase (iNOS) m R N A and protein. Using fluorescence image analysis,iNOS protein was found to be localizedin the cytoplasm of the cells. Taken together, our data demonstrate that exposure of rats to alcohol decreases the capacity of both M P and E C to produce nitric oxide which is regulated distinctlyin these celltypes. Decreased sensitivityof nonparenchymal cellsto inflammatory mediators may be important in the hepatic response to alcohol.