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HEPATOLOGY Vol. 22, No. 4, Pt. 2, 1995 AASLD ABSTRACTS 459A 1409 LIVER HISTOLOGY AND SERUM HCV-RNA OF ANTI-HCV POSITIVE PATIENTS WITH PERSISTENT NORMAL AMINOTRANSFERASE LEVELS 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 We evaluated liver histology and serum HCV-RNA levels in 30 patients anti-HCV positive on ELISA H, 19 of 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 HBsAg or anfi-HIV or with autoimmune diseases or metabolic disorders. Testing for HCV-RNA was performecl by nested PCR using primers of 5' UT region. Liver biopsy was performed percutaneously with M~nghini's needle. 16 patients were serum HCV- RNA positive (53%); on RIBA 1] 13 of them (68%) were positive and 3 (27%) were indeterminate. HIstology of the HCV-RNA positive patients showed g cases of CPH, 6 cases of CAH, 1 case of CLH and 1 case of NL (normal liver). Histology of the HCV-RNA negative patients showed 4 cases of CPH, 1 case of CAH, 2 cases of CLH and 7 eases of NL These results indicate that subjects positive on RIBA II, but with persistently normal ALT values, have a high possibility of being serum HCV-RNA positive and that almost all these viremic subjects present histological signs of liver disease. In contrast, subjects with indeterminate RIBA II have a moderate probability' of being HCV-RNA positive, but a number of these may present signs of liver disease. Fluctuations in viremia may possibly lead to false negative results; in any case a close follow-up of these cases is necessary to detect evolution of liver disease. 1410 HETEROGENEITY IN THE INDUCTION OF ALPHA-FETOPROTEIN(AFP) GENE EXPRESSION IN AN EXPERIMENTAL ANIMAL MODEL OF CIRRHOSIS AND HEPATOCELLULAR CARCINOMA (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. Small amountsof AFP are often detected in the cirrhotic liver but it is increased during HCC. Since HCC is 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 CCl 4 three times per week during two months. To induce HCC previous to the chronic CCl 4 treatment, rats ware treated w~h diethyl nitrosamine (DEN), ecetylamine fluorene (AAF) and then with an acute intragaetricdose of CCI 4 (CC). Rats untreated (N), treated with DEN and AAF (CA) and only with the CCI 4 chronic treatment (CR) ware included. Samples were taken at 2, 6 and 10 weeks after finishing the chronic CCl 4 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:37 vs 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:0.04 vs 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. 1411 PLASMAPHERESIS IN PATIENTS WITH SEVERE PRIMARY DYSFUNCTION AFFER LIVER TRANSPLANTATION E. Mee, D. Skerrett t, S. Curtiss. P. Sheiner, S. Emre~ S. Guy. M. Schwartz, C. Miller. Dept. of Surgew and The Blood Bank t, 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 eleva- tious 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~Stparfusion biopsy (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 re- LTx. 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. 1412 EFFECTS OF LONG TERM ALCOHOL ADMINISTRATION ON NITKIC OXIDE PRODUCTION BY HEPATIC MACROPHAGES AND ENDOTHELIAL CELLS. L Morio, DE Heck, JD Laskin, C Gardner, and DL Laskin. Rutgers University/ UMDNJ Robert Wood Johnson Medical School, Piscataway, NJ 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 differential centri- fugation and elutriation. We found that MP and EC from both untreated and alcohol-treated rats 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. MP and EC 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.MP appeared to be more sensitive to the in vivo effectsof alcohol than were EC. Northern and western blot analysis demonstrated that nitricoxide production by MP and EC in response to LPS plus IFN7 was due to increased expression of an inducible form of nitricoxide synthase (iNOS) mRNA 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 MP and EC 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.

Plasmapheresis in patients with severe primary dysfunction after liver transplantation Dept. of Surgery and The Blood Bank1, The Mount Sinai Medical Center, New York

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HEPATOLOGY Vol . 22, N o . 4, P t . 2, 1995 A A S L D A B S T R A C T S 4 5 9 A

1409 LIVER HISTOLOGY AND SERUM HCV-RNA OF ANTI-HCV POSITIVE PATIENTS WITH PERSISTENT N O R M A L AMINOTRANSFERASE LEVELS

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

We evaluated liver histology and serum HCV-RNA 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 HBsAg or anfi-HIV or with autoimmune diseases or metabolic disorders. Testing for HCV-RNA was performecl by nested PCR using primers o f 5' UT region. Liver biopsy was performed percutaneously with M~nghini's needle. 16 patients were serum HCV- RNA positive (53%); on RIBA 1] 13 o f them (68%) were positive and 3 (27%) were indeterminate. HIstology of the HCV-RNA positive patients showed g cases o f CPH, 6 cases of CAH, 1 case o f CLH and 1 case o f N L (normal liver). Histology o f the HCV-RNA negative patients showed 4 cases o f CPH, 1 case o f CAH, 2 cases o f CLH 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 HCV-RNA 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 HCV-RNA 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 of these cases is necessary to detect evolution o f liver disease.

1 4 1 0 HETEROGENEITY IN THE INDUCTION OF ALPHA-FETOPROTEIN (AFP) GENE EXPRESSION IN AN EXPERIMENTAL ANIMAL MODEL OF CIRRHOSIS AND HEPATOCELLULAR CARCINOMA (HCC). Moutoya H. Rinc6n AFt and A Panduro. Basic Research Center, University of Aguascalientes and Institute of Molecular Biology in Medicine, CUCS. U.de G. and H.C. de Guadalajara. M6xico.

Small amounts of AFP are often detected in the cirrhotic liver but it is increased during HCC. Since HCC is closely associated with 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 CCl 4 three times per week during two months. To induce HCC previous to the chronic CCl 4 treatment, rats ware treated w~h diethyl nitrosamine (DEN), ecetylamine fluorene (AAF) and then with an acute intragaetric dose of CCI 4 (CC). Rats untreated (N), treated with DEN and AAF (CA) and only with the CCI 4 chronic treatment (CR) ware included. Samples were taken at 2, 6 and 10 weeks after finishing the chronic CCl 4 treatment, At 10th week the CC group presented a multinodular appearance liver, fibrosis and limited figures of pseudo ecinar structures with anaplastic hepetecytes (irregulare, ill¢ontorned, acidophilic and 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:37 vs 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:0.04 vs 0.62.t0.05 mg/10O ml]), whereas in the CR and CA 9roups LFT came back to values cicee to normal. No changes in serum albumin were detected in either experimental group. 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 group ware 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 established an experimental animal model of cirrhosis and HCC in a shorl period of time. The molecular findings 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.

1411 PLASMAPHERESIS IN PATIENTS WITH SEVERE PRIMARY DYSFUNCTION AFFER LIVER TRANSPLANTATION E. Mee, D. Skerrett t, S. Curtiss. P. Sheiner, S. Emre~ S. Guy. M. Schwartz, C. Miller. Dept. of Surgew and The Blood Bank t, 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 eleva- tious 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~Stparfusion biopsy (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 re- LTx. 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.

1412 E F F E C T S O F L O N G T E R M A L C O H O L A D M I N I S T R A T I O N O N N I T K I C O X I D E P R O D U C T I O N B Y H E P A T I C MACROPHAGES A N D E N D O T H E L I A L C E L L S . L Morio, DE Heck, J D Laskin , C Gardne r , a n d DL Laskin . Ru tgers Univers i ty / U M D N J Robert Wood Johnson Medical School, P i sca taway , N J

Hepatic macrophages (MP) and endothelial cells (EC) release an array of proinflammatory and cytotoxic mediators that have been implicated in the development of liver injury. In the present studies we analyzed the effects of chronic alcohol administration on production of nitric oxide by these cells. Rats were exposed to ethanol (10:4 g/kg) by intragastric feeding. Cells were isolated from the animals 3-6 weeks later by in situ perfusion of the liver with collagenase and pronase followed by differential centri- fugation and elutriation. We found that MP and EC from both untreated and alcohol-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-7 (IFNT) and lipopolysacoharide (LPS) alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitsrs, N'%monomethyl-l-arginine and l-canavanine. MP and EC produced similar amounts of nitric oxide in response to these inflammatory mediators. Nitric oxide production by both cell types in response to LPS plus IFN7 was decreased following treatment of rats with alcohol. MP appeared to be more sensitive to the in vivo effects of alcohol than were EC. Northern and western blot analysis demonstrated that nitric oxide production by MP and EC in response to LPS plus IFN7 was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Taken together, our data demonstrate that exposure of rats to alcohol decreases the capacity of both MP and EC to produce nitric oxide which is regulated distinctly in these cell types. Decreased sensitivity of nonparenchymal cells to inflammatory mediators may be important in the hepatic response to alcohol.