212A A A S L D A B S T R A C T S HEPATOLOGY O c t o b e r 1995

421 99m-Tc-LABELED HUMAN SERUM ALBUMIN MICROSPHERES ARE EFFECTIVE AND DEGRADABLE CELL SURROGATES FOR ANALYZING SAFETY OF HEPATOCYTE TRANSPLANTATION. P Rajvanshl, __K Bhar~ava ' , S Gaeandeen, _C Palestro* and S Gupta. Liver Research Center and Department of *Nuclear Medielne at Long Island Jewish Hospital, Albert Einstein College of Medicine, Bronx, NY.

Liver repopulatlon would greatly facilitate hepatic gene therapy and novel therapies in hepatle failure. To develop nontoxic, biodegradable and effective cell surrogates for easing clinical safety analysis of hepatocyte transplantation, we tested dislribution of 99m-Tc-labeled human serum albumin microspheres, size 10/t to 45# (99m-Tc-HAM), against rat hepa- tocytes laheled with either 99m-Tc-pertechnetate or ln-l l l -oxine. When 99m-Tc-hepatocytes were intrasplenical ly injected into normal rats, dynamic gamma imaging showed that cells instantaneously migrated to the liver. 99m-Tc activity was primarily in liver (85% + 3%) or spleen (13% +3%) and much less in lungs (i .2 +__ 0.5%). In contrast, when in- jected via femoral v., cells went to lungs (62% +13%), although activity was redistributed in additional organs, such as liver, spleen or kidneys, due to cell destruction and uptake of fragments. Upon intrasplenic injec- tion of In-! l l-hepatocytes and 99m-Tc-HAM mixed together, dual isotope imaging showed similar biodistributions. Injection of 99m-Te-HAM into portal v. deposited "100% activity into liver and exclusion of posterior liver lobes from circulation resulted in deposition of 99m-Tc-HAM into only nonexeluded lobes. In contrast , injection of 99m-Tc-HAM into femoral v. deposited activity mainly into lungs (84% +_ 2%) and less so in liver (15% +__2%) or spleen (0.8% + 0.05%). Upon intraportal injection of 99m-Tc-HAM into rats with portal v. constriction, 99m-Tc-HAM local- ized in liver (75% + 7%), as well as lungs (25% + 7%, p<0.00t ) , con- sistent with portasystemic shunting. To next analyze whether intrahepatic shunts developed during acute liver failure, we injected 0.7 g/kg D- galactosamine i.p. into F344 rats, which produced significant liver injury and activation of GGT + ve cells. The biodistr ibution of intraportal ly injected 99m-Tc-HAM was similar in rats either 24 hrs or 48 hrs post-D- gal, with no significant difference from normal controls. CONCLU- SIO.NS: Human serum albumin microspheres are effective cell surrogates fur analyzing the safety of hepatucyte transplantation. Ahsenea of signifi- cant pnrtasystemic shunting in acute liver injury indicates that cell trans- plantation into hepatic sinusoids should he safe in this situation.

422 AGE-DEPENDENT REPRESSION OF ALPHA 2 URINARY-GLOBULIN IN RAT HEPATOCYTES IS REVERSIBLE AND INFLUENCED BY THE AGE OF THE ORGANISM RATHER THAN THE AGE OF THE TRANSPLANTED HEPATOCYTES. Bahri M. Bilir. Tracev Hanks. Alina Ostrowska. Frederick Karrer. Section of Hepatology and Transplantation Surgery, University of Colorado School of Medicine, Denver.

The study of aging in liver may be possible by identification of certain genes that are up or down regulated as the organism ages. Alpha 2 U-globulin which encodes a 18KD protein is an example of these marker genes. It is expressed in young male rats in the perivenular region and repressed in the senile rats. The mechanism and reversibility of age-dependent repression of alpha 2 U- globulin gene has not been investigated. In this study, we first defined the age- dependent expression of alpha 2U-globulin in rat livers of different ages at the protein and the mRNA level. This gene was expressed by the perivenular hepatocytes of young (3-4 months) and intermediate-age (12-14 months) but not the old (24-26 months) rats. Our control gene was albumin which did not have quantifiable change at the message level in animals of all three age groups. We then isolated the hepatocytes from young animals and transplanted into the spleens of old animals and vice versa. Old to old and young to young hepatocyte transplantations were also performed as controls. Three months after transplantation, the recipients were sacrificed and the alpha 2U-globulin expression in their livers and spleens were studied by Northern Blots and immunohistochemistry. We found out that the expression of alpha 2U-globulin in transplanted hepatocytes adapted to the pattern predicted by the age of the recipient: alpha 2U-globulin gene was repressed in "young hepatocytes" transplanted into "old-spleens" and there was a de-repression of this gene in "old hepatocytes" transplanted into the "young spleens." To rule out the possibility that this dynamic regulation is due to factor(s) specific to splenic environment, the gone expression pattern of hepatocytes from DPPIV(+) rats transplanted into the livers of DPPIV(-) rats were examined. The staining of DPPIV and alpha 2U-globulin helped to identify the donor hepatocytes which adopted the gene expression pattern of the recipient in the liver. We conclude that, age-dependent repression alpha 2U-globulin seems to be mainly influenced by the age of the organism rather than the actual age of the transplanted hepatocyte.

423 PRELIMINARY EVALUATION OF A NEW EXTRACORPOREAL LIVER SUPPORT SYSTEM IN FULMINANT HEPATIC FAILURE RATS. V Dixit.* S Bi~,t, ins.* D Friedlich.* M Arthur.* S Chen.** J Roz~a.** P Martin.* A Dcmetriou.** G Gitnick*. *Department of Medicine, University of California, Los Angeles and **Cedars Sinai Medical Center, Los Angeles.

We evaluated a novel, hemoperfusion-based, hepatocyte bioreaetor using microeneapsulated porcine hepatocytes (MPH). Microencapsulated hepatocytes have previously been shown to provide significant liver function following transplantation in animal models of liver disease. The proposed bioreactor design involves the direct hemoperfusion of arterial blood through a packed-bed column of MPH enclosed in a cylindrical plastic chamber. Blood exiting the bioreactor is returned to the venous circulation. A peristaltic pump is used to drive the system. Since microcapsules have a very high surface area to volume ratio, such a system can provide both a large diffusion surface area for mass-transfer and a high capacity for hepatocyte mass. Methods: Hepatocytes, isolated from 10kg Babcock pigs by the portal vein collagenase perfusion technique, Were microencapsulated with alginate-polylysine mieroeapsules prepared by the standard droplet generation technique. Fulminant hepatic failure (FHF) was induced in 275+25g male Wistar rats (N=12) by a single intraperitoneal injection (3g/kg body wt) of galaetosamine (GAIN). Vascular access was obtained by eannulating the femoral artery and vein. Hemoperfusion was performed 24 hrs. after GaIN injection in conscious animals for 2 hours at maximum flow rates of 1.5-2.0 ml/min. Hemodynamics were evaluated using a computerized physiological monitoring system. Plasma ALT and total bilimbin levels were measured pre and post bioreactor treatment. FHF animals were grouped as: Group A: untreated controls; Group B: control bioreactor treated; Group C: MPH bioreaetor treated; Results: Survival times (mean_-LSEM) in the experimental groups were: Group A: 84±7 hrs.; Group B: 123+60 hrs.; Group C: 220+78 hrs. There were no significant differences in survival time between groups A & B, and B & C. A significant difference (P<O.03) was observed between groups A & C. Also, one animal in group C recovered completely. No significant changes in hemodynamics, ALT, and bilimbin levels were observed in control animals. FHF animals had ALT levels decrease by an average of 37% post bioreactor treatment (P<O.08). All animals tolerated bioreactor treatment without discomfort. No detrimental effects were observed during or after treatment. Conclusions: The proposed hepatocyte bioreactor: (i) Improved the survival time of animals with FHF. (it) Did not adversely affect the animal's hemodynamics during treatment. (iii) Was convenient, easy to operate and is well tolerated by the animals.

424 HEPATOCYTE TRANSPLANT COMBINED WITH NONPAREN- CHYMAL CELLS IN THE MESENTERIC FAT PAD IN MOUSE $ Narumi. L Zhon~hua. JP Roberts. and NL Ascher. Liver Transplant Division, Dept. of Surgery, University of California, San Francisco.

Experimental hepatocyte(HC) transplantation has relied on the use of spleen or portal vein as sites of injection of HC. Theses sites make subsequent study of the immune response to HC difficult. The mesenteric fat pad(MFP) on the other hand can support cell growth and provide easy access to the local immune response. The purpose of this study was to determine the optimal condition for HC and nonparenchymal eel1 (NPCs) for HC growth and function in the MFP. M a t e r i a l s and methods: C3H/Hen (H-2 K) female mice were used as recipients and donors of syngeneic NPCs or HC. 129/B6(H-2 b) wild type and ~2-knockout (-/-) female mice were used as donors of allogeneic cells. HC isolation was done by two-step collagenase(0.05%) method. NPCs were isolated with 0.2% protease peffusion and DNAase. HC and NPCs were mixed and were transplanted into MFP. The recipient mouse underwent 30% hepatectomy prior to the transplant.The MFP and the spleen were harvested at POD 7, 28 or later. MFP was histologically examined and graded for inf lammatory response. Cytotoxici ty assay(CTL) was performed using spleen. RT-PCR was used to detect an albumin mRNA in the MFP for the functional assessment of transplanted HC. Resul ts : Syngeneic HC mixed with syngeneic NPCs along with 30% hepatectomy demonstrated survival of HC and albumin mRNA in the MFP at POD 90 in contrast to hepatocyte alone. Even allogeneic HC could be detected with some inflammation despite of no an albumin mRNA at POD 60. Wild type NPCs induced more CTL response and inflammation than wild type HC. In contrast, (-/-) NPC and (4-) HCs did not induce little, if any, CTL. Antigen\Target EL4 tumor (H-2 b) Balb/C ConA (H-2 d) AlloHC (100:1,12.5:1) 39 .5+4 .0 ,14.5+2.5 4 .3+2 .0 , 0 .7+0 .1 AlloNPCs(100:I,12.5:I) 66.5 + 4.0*,44.0 + 2.0* 15.0 + 7.9, 2.3 + 1.5

Conclus ion : NPCs are necessary for HC survival. However, NPCs elicited more immunogenicity than HC transplanted in the MFP. The optimal combination of HC/NPC must be determined to maximize survival without prohibitively increasing immunogenicity.