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Hepatocyte cell lines are beginning to be developed as universal donors for isolated liver cell transplantation, which is a less invasive method than orthotopic liver transplantation for treatment of metabolic liver disease. The immune response to isolated liver cell transplantation and its modification by costimulatory blockade are as yet not well delineated.Adenovirus expressing CTLA4Ig was used to study blockade of the costimulatory CD28/B7 pathway in murine models of hepatocyte transplantation, and the effects on alloreactive T and B cells were studied.CTLA4Ig delayed rejection of subcutaneously administered C57L-derived murine hepatoma cells in CBA/J recipients for50 days. Activation and cytokine secretion by allospecific CD4+ and CD8+ T cells were initially blocked by CTLA4Ig; delayed rejection was associated with tumor infiltration by CD8+ T cells that did not secrete interferon-gamma. CTLA4Ig failed to block transplant rejection in primed mice, indicating that memory effector T cells were resistant to its action. In contrast, CTLA4Ig suppressed both naive and memory alloreactive B cells. High levels of CTLA4Ig mediated acceptance of hepatoma cells delivered directly into the spleen. However, isolated primary C57BL/6 mouse hepatocytes delivered into the spleen were rejected with only moderately delayed kinetics.Transplant antigenicity, transplant site, and CTLA4Ig dose all affected the survival of transplanted liver cells. CD8+ T cells are significant mediators of hepatocyte transplant rejection and are relatively resistant to costimulatory blockade with CTLA4Ig. Strategies to specifically antagonize CD8+ T cells or to modulate MHC class I expression in association with costimulatory blockade by CTLA4Ig may enhance the clinical feasibility of transplanting allogeneic hepatocytes.

The shortage of human livers available for hepatocyte isolation limits its clinical application. The availability of cloned, conditionally immortalized hepatocytes that could be grown in culture but would lose their transformed phenotype and provide metabolic support upon transplantation would greatly facilitate the treatment of acute liver failure. Toward this goal, we transduced isolated Lewis rat hepatocytes using a replication-defective recombinant retrovirus capable of transferring a gene encoding a thermolabile mutant simian virus 40 T antigen (SV40ts). The cloned, immortalized hepatocytes proliferate at 33 degrees C. At the nonpermissive temperatures (37-39 degrees C), they stop growing and exhibit characteristics of differentiated hepatocytes. These cells did not produce tumors when transplanted in mice with severe combined immunodeficiency disease or in syngeneic rats. To induce acute liver failure, Lewis rats were subjected to 90% hepatectomy (Hpx) and given 5% oral dextrose. All rats that did not undergo hepatocyte transplantation died within 96 hr. Fifty percent of rats that received intrasplenic injection of 10 x 10(6) primary Lewis rat hepatocytes (G2, n=6) or 10 x 10(6) SV40ts-conditionally immortalized (SV40ts-ci) hepatocytes (G3, n=8) 1 day before 90% hepatectomy survived, whereas 80% of rats that received an intraperitoneal injection of 200 x 10(6) primary Lewis rat hepatocytes (G4, n=10) or 200 x 10(6) SV40ts-ci hepatocytes (G5, n=10) on the day of hepatectomy survived. Survival after intraperitoneal injection of a cellular homogenate of 200 x 10(6) primary Lewis rat (G7, n=9) or SV40ts-ci hepatocytes (G8, n=10) on the day of Hpx was 33% and 40%, respectively, whereas survival after intraperitoneal injection of 200 x 10(6) Lewis rat bone marrow cells (G6, n=7) was 29%. Thus, transplanted, conditionally immortalized hepatocytes can be as effective as primary hepatocytes in supporting life during acute liver insufficiency. This work represents the first step in developing an hepatocyte cell line that would partially alleviate the organ-donor shortage and could be of potential clinical value.

Viral vectors and protein carriers utilizing asialoglycoprotein receptor (ASGR)-mediated endocytosis are being developed to transfer genes for the correction of bilirubin-UDP-glucuronosyltransferase (bilirubin-UGT) deficiency. Ex vivo evaluation of these gene transfer vectors would be facilitated by a cell system that lacks bilirubin-UGT, but expresses differentiated liver functions, including ASGR. We immortalized primary Gunn rat hepatocytes by transduction with a recombinant Moloney murine leukemia virus expressing a thermolabile mutant SV40 large T antigen (tsA58). At 33 degrees C, the immortalized hepatocyte clones expressed SV40 large T antigen, synthesized DNA, and doubled in number every 2 to 3 days. At this temperature, differentiated hepatocyte markers, e.g., albumin, ASGR, and androsterone-UGT, were expressed at 5% to 10% of the levels found in primary hepatocytes maintained in culture for 24 hours. Glutathione-S-transferase Yp (GST-Yp), an oncofetal protein, was expressed in these cells at 33 degrees C, but was undetectable in primary hepatocytes. In contrast, when the cells were cultured at 39 degrees C or 37 degrees C, the large T antigen was degraded, DNA synthesis and cell growth stopped, and morphologic characteristics of differentiated hepatocytes were observed. The expression of albumin, ASGR, and androsterone-UGT, and their corresponding mRNAs, increased to 25% to 40% of the level in primary hepatocytes, whereas GST-Yp expression decreased. Functionality of ASGR was demonstrated by internalization of Texas red-labeled asialoorosomucoid, and binding and degradation of 125I-asialoorosomucoid. After liposome-mediated transfer of a plasmid containing the coding region of human bilirubin-UGT1, driven by the SV40 large T promoter, active human bilirubin-UGT1 was expressed in these cells. The immortalized cells were not tumorigenic after transplantation into severe combined immunodeficiency mice. These conditionally immortalized cells will be useful for ex vivo evaluation of bilirubin-UGT gene transfer vectors.

The copper content and ceruloplasmin activity were determined in mice bearing benzo(a)pyrene induced fibrosarcoma. The copper level and ceruloplasmin activity in different organs of fibrosarcomatous mice varied when compared to their controls. Significant changes in copper and ceruloplasmin concentration were observed in the liver and tumor tissue of host mice bearing fibrosarcoma compared to controls. Disturbed copper metabolism at the hepatic level may account for the hypercupremia observed during malignancy.

Hepatocytes from donor transgenic mice that produce an easily assayable circulating marker have been used to develop a novel hepatocyte transplantation system. Isolated G7 HBV transgenic donor hepatocytes secreting HBsAg were transplanted into congeneic or allogeneic mouse recipients. Serum HBsAg was present three days after hepatocyte transplantation in congeneic animals and persisted indefinitely when hepatocytes were transplanted into the spleen. Transplanted hepatocytes within the splenic pulp were identified by morphologic and histochemical analysis. Migration of hepatocytes injected into the spleen to the liver was demonstrated by in situ hybridization using an RNA probe for HBsAg. Transplantation into nonimmunosuppressed allogeneic recipients resulted in disappearance of detectable hepatocytes in the spleen within two weeks. This novel transplantation system should facilitate studies of hepatocyte engraftment and survival, modulation of allograft rejection, and development of hepatocyte-directed gene therapy.

Effect of tumor growth on the survival of circulating erythrocytes was studied in mice bearing a wide spectrum of experimental tumors. RBC half-life (t1/2), measured by 51Cr-labeling technique, decreased significantly (p less than 0.05) in all the tumor types studied, particularly in transplantable Sarcoma-180 and benzo(a)pyrene-induced primary fibrosarcoma. Changes in erythrocyte morphology like anisopoikilocytosis were also observed in the tumor hosts. Cross-transfusion of 51Cr-labeled RBCs between normal and tumor-bearing animals revealed that both intrinsic and extrinsic factors are responsible for shortened RBC survival. As far as the cellular abnormalities are concerned, the decrease in RBC t1/2 was not attributable to increased osmotic fragility as the cells were observed to be osmotically more resistant. Similarly, membrane sialic acid content was markedly elevated in the tumor hosts, thus the shortened erythrocyte life-span cannot be attributed to decrease in sialic acid content of the erythrocyte membrane.

The erythropoietic response of Strain A mice with benzo(a)pyrene-induced fibrosarcoma has been studied. The rate of erythropoiesis, expressed in terms of 59Fe incorporation into circulating erythrocytes, was slightly increased in the fibrosarcomatous mice compared to the matched controls. The femoral marrow of the tumor hosts became hypocellular with reduced number of erythroblasts. The spleen, on the other hand, was hypercellular with an increased number of erythroid progenitor cells. Quantitative assessment of erythropoietic activity in the hematopoietic organs by measuring the 6-hr organ uptake of 59Fe revealed erythropoietic suppression in the marrow but enhancement in the spleen following tumor development. While the number of CFU-s in the femoral marrow of the tumor-bearing animals was slightly increased, marked increase in the concentration as well as absolute number of CFU-s was found in the spleen. Bioassay for erythropoietin revealed appreciable increase in the level of plasma erythropoietin in the tumor-bearing animals.

A mammary tumor-associated antigen (MTAA) of the murine mammary tumor virus (MuMTV)-induced spontaneous mammary tumors of C3H/J mice was purified and partially characterized. The crude extract of the mammary tumor, when subjected to DEAE-cellulose chromatography and eluted with a discontinuous NaCl gradient, provided three major protein peaks, of which only the first (F1) possessed the MTAA activity. The antigen was further purified by subjecting F1 to polyacrylamide gel electrophoresis. The MTAA was a glycoprotein with a molecular weight of approximately 83,000. The antigen was localized in the plasma membrane and was different from the MuMTV structural antigens. Circulating antibodies against the MTAA were observed in the sera of tumor-bearing mice but not in that of tumor-free mice.

Immunological relatedness of a non-virion, murine mammary tumor-associated antigen (MTAA) and human breast cancer was demonstrated in this study. The MTAA was isolated from spontaneous mammary tumors (MMT) of C3H/J mice. An anti-serum specific for the MTAA was prepared by exhaustively absorbing the rabbit anti-serum to the MMT extract with tissue pellets of the pooled organs of tumor-free female mice. The observed cross-reaction of human malignant breast tumor (MBT) tissues with the absorbed antiserum further absorbed with normal human breast tissues suggested the presence of the MTAA or a related antigen in the MBT. No MTAA reactivity was found in human benign breast tumor. Circulating antibodies against the MTAA were recorded in all of 14 preoperative patients with MBT. Up to 18 months following surgical removal of the MBT, antibodies could be detected in the sera, though the reactivity was significantly lower than that of the sera of preoperative patients, and eventually disappeared. The antibodies were absent in patients with benign breast tumor and other neoplastic diseases, except in one patient with cancer of the cervix, and in healthy women.

Tumor-induced changes in the pattern of erythropoiesis have been studied in A/RB mice bearing a transplantable ascites tumor, DBA lymphoma. The tumor growth was accompanied by diminished erythropoiesis, and the 51Cr-labeled red cell half-life was also decreased. Plasma iron turnover rate of the tumor bearing hosts, however, was increased and the level of erythropoietic stimulating factor (erythropoietin) in the plasma was not reduced. The existence of an inhibitor(s) to erythropoietic activity was unlikely. Comparative study of the erythropoietic activity revealed suppression in the marrow, but an increment in the spleen. The distribution of pluripotent hematopoietic stem cells (CFUs) also paralleled this alteration. However, the degree of ineffective erythropoiesis, determined by in vitro technique, was significantly elevated in the spleen. Possibly, this has reduced the effectiveness of splenic compensatory erythropoiesis.

To understand the nature of copper distribution during malignancy, 27 strains A/RB mice with fibrosarcoma were used. Tracer methodology with radioactive isotope of copper (64Cu) was applied. In the study made at different time intervals after isotope administration whole blood plasma and different tissues like liver, heart, spleen kidney and brain were investigated. Liver nuclear fractions were also examined. Distribution of 64Cu in different tissues reflected interesting results. While the incorporation pattern in the liver showed gradual fall from 8 h of isotope administration, the tumor tissue exhibited gradual elevation, compared to the controls. The effect of this finding is discussed.

Red cell survival studies in mice with benzo(a) pyrene [B(a)p]-induced fibrosarcoma indicate marked reduction in RBC 51Cr t1/2. In contrast, the t1/2 of erythrocytes from B(a)p injected but tumor-free mice was not declined. Increasing tumor burden resulted in increment in the severity of this disorder, and preincubation of normal red cells with tumor supernatant caused significant reduction (p less than 0.05) in 51Cr t1/2. It is suggested that the observed alteration in RBC life span may be due to the effect of tumor. Apparently, both intrinsic and extrinsic cellular defects could be implicated for this abnormality. However, the shortened survival cannot be attributed to altered osmotic fragility, since the erythrocytes of the fibrosarcomatous mice were more resistant to hypotonic hemolysis in vitro.