Anergic lymphocytes generated by blocking CD28 and ICOS pathways in vitro prolong rat cardiac graft survival.

Regulatory cells may play a pivotal role in inducing and maintaining transplantation tolerance. We investigated the mechanism of anergic lymphocytes with regulatory cell potential generated in vitro by ICOS and CD 28 co-stimulatory blockades as a source of cellular therapy for treating allograft rejection. Anergic lymphocytes were generated by a mixed lymphocyte reaction consisting of DA splenocytes as the stimulator and Lewis splenocytes as the responder in the presence of anti-ICOS mAb and rCTLA-4I g. Immunoregulatory effects of these lymphocytes were evaluated by secondary MLR and using other various stimulations. DA heart was transplanted into 7.5 Gy-irradiated Lewis rat after intravenous administration of these cells and/or Lewis spleen lymphocytes. We observed that these lymphocytes were not only anergic to alloantigen and polyclonal stimulations but also exhibited regulative activity to inhibit the alloreactive T-cell response. Our adoptive transfer studies revealed that irradiated recipients that received both anergic lymphocytes and naIve Lewis lymphocytes had significantly prolonged DA cardiac graft survival (mean 17.5 days) compared with a group that received Lewis lymphocytes alone (mean 10.8 days). Furthermore, some of the recipients accepted the graft indefinitely after receiving anergic lymphocytes alone (>100 days). These results demonstrated that anergic lymphocytes with regulatory activities can be generated through blocking co-stimulatory signals, CD 28 and ICOS, simultaneously in vitro, and may advance a new immunomodulatory strategy for preventing allorejection in organ transplantation.

Genetic heterogeneity and efficiency of two different methods of adenovirus-mediated gene transfer in a rat liver transplantation model.

PURPOSE: We used recombinant adenoviral vectors for gene therapy in liver transplantation, and investigated the efficacy of gene transfer and expression on the grafts and genetic heterogeneity, with two exogenous gene transfer methods in three different syngeneic rat strains. METHODS: We transferred adenoviral vector encoding Escherichia coli beta-galactosidase via a donor tail vein 3 days before transplantation; via a recipient tail vein immediately after grafting; and ex vivo by perfusion and clamping during transplantation. RESULTS: The high efficacy of beta-galactosidase gene transfer and expression was seen in both delivery systems, with 70% positivity for hepatocytes on day 3, which persisted for at least 3 weeks after transplantation. The efficacy of gene transfer and expression was similar in the three strains (DA, Lewis, and PVG). CONCLUSIONS: These data suggest that adenovirus-mediated gene transfer delivers effective gene therapy by tail vein injection of a donor or a recipient, or by ex vivo graft perfusion in rat liver transplantation. It is not necessary to consider the differences in the strains. Furthermore, ex vivo graft perfusion is probably more suitable not only for rat liver transplantation but also possibly for future clinical application.

Characterization and gene transfer in mesenchymal stem cells derived from human umbilical-cord blood.

It has been shown that the stromal-cell population found in bone marrow can be expanded and differentiated into cells with the phenotypes of bone, cartilage, muscle, neural, and fat cells. However, whether mesenchymal stem cells (MSCs) are present in human umbilical-cord blood (UCB) has been the subject of ongoing debate. In this study, we report on a population of fibroblastlike cells derived from the mononuclear fraction of human UCB with osteogenic and adipogenic potential, as well as the presence of a subset of cells that have been maintained in continuous culture for more than 6 months. These cells were found to express CD29, CD44, CD90, CD95, CD105, CD166, and MHC class, but not CD14, CD34, CD40, CD45, CD80, CD86, CD117, CD152, or MHC class II. We also compared gene expression after gene transfer using lenti- and adenoviral vectors carrying the green fluorescence protein to the MSCs derived from UCB because a reliable gene-delivery system is required to transfer target genes into MSCs, which have attracted attention as potential platforms for the systemic delivery of therapeutic genes. The lentiviral vectors can transduce these cells more efficiently than can adenoviral vectors, and we maintained transgene expression for at least 5 weeks. This is the first report showing that UCB-derived MSCs can express exogenous genes by way of a lentivirus vector. These results demonstrate that human UCB is a source of mesenchymal progenitors and may be used in cell transplantation and a wide range of gene-therapy treatments.

Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model.

Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34(+/-) cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34(+), or CD34(-) cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of alpha-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34(+) and CD34(-) cells were not observed in human hepatocyte-specific expression.

Inhibition of allogeneic T-cell responses by dendritic cells expressing transduced indoleamine 2,3-dioxygenase.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an enzyme involved in the catabolism of tryptophan and has been shown to prevent rejection of the fetus during pregnancy by inhibiting alloreactive T cells. METHODS: In this study we investigated dendritic cells (DCs) that are transfected with IDO cDNA in the inhibition of T-cell proliferation after antigen-specific interaction. XS106 DCs, derived from A/J mice (H-2k), were transduced with IDO with a gene-delivery system using a recombinant adenoviral vector. RESULTS: Western blotting and immune staining revealed IDO expression in XS106 DCs transduced with IDO (XS106-IDO DCs), and its catabolic effect was confirmed by an increase in kynurenine concentration. Fluorescence-activated cell sorting revealed that XS106-IDO DCs were not changeable for Ia, CD80, and CD86 expression. After XS106-IDO DCs were co-cultured with C57BL/6 allogeneic splenic T cells, the proliferation of the T cell was significantly inhibited. The co-cultured T cells with XS106-IDO DCs exhibited cell-cycle arrest. Furthermore, injection of XS160-IDO DCs into the footpads of C57BL/6 (H-2b) mice demonstrated a reduced T-cell response against allo-antigen. CONCLUSIONS: These results suggest that overexpression of IDO in the DCs effectively inhibited T-cell proliferation, and may expand a new immunomodulatory strategy for the prevention of allo-rejection of organ transplantation.

Significant enhancement by anti-ICOS antibody of suboptimal tacrolimus immunosuppression in rat liver transplantation.

A member of the costimulatory molecule family, inducible costimulator (ICOS), is expressed on activated T cells and plays a critical role in their primary activation and cytokine production. ICOS is involved in different immune phenomena, such as Th1-mediated autoimmune disease and graft rejection. Although blockade of ICOS costimulation theoretically may protect grafts from rejection, a single dose of anti-ICOS antibody did not result in the prolongation of rat liver allograft survival. However, in this article, we report that anti-rat ICOS antibody markedly enhanced the immunosuppressive activity of a suboptimal dose of tacrolimus (FK506). After fully allogenic DA to LEW liver transplantation, recipients received a single injection of tacrolimus (1 mg/kg, intramuscularly) with or without anti-ICOS antibody (1 mg/kg, intravenously). Recipient survival was significantly prolonged in rats treated with both the antibody and suboptimal tacrolimus (median survival time 44 days vs. 28 days with tacrolimus alone, P <.01). The extent of cell infiltration into the graft was closely associated with prolongation of recipient survival. Our findings thus demonstrate that anti-ICOS antibody immunotherapy combined with suboptimal tacrolimus has a synergistic effect in preventing hepatic allograft rejection and that it may induce long-term graft acceptance intimately associated with a marked reduction of intragraft T lymphocyte infiltration.

Biodegradable microsphere-loaded tacrolimus enhanced the effect on mice islet allograft and reduced the adverse effect on insulin secretion.

The adverse effects of tacrolimus have limited the use of this potent immunosuppressive drug in clinical transplantation. To improve the therapeutic effects, we developed a new type of tacrolimus with biodegradable microsphere technology and examined the immunosuppressive effects on allogeneic islet transplantation and the side-effects on insulin secretion in vivo. With a single subcutaneous injection, mouse blood concentrations of tacrolimus (M-FK) carried in biodegradable microspheres remained flat for 2 weeks (only 10 h for conventional tacrolimus). A single subcutaneous administration of 20 mg/kg of M-FK significantly prolonged the survival of islet allografts (MST = 28 days) compared with the control group (MST = 10 days). Series administration of 10 mg/kg of M-FK at 7-day intervals markedly prolonged the survival of islet grafts, and resulted in 60% allograft acceptance. In mice with syngeneic islet transplantations, a single administration of 30 mg/kg of tacrolimus inhibited insulin secretion, whereas a single administration of an equal dosage of M-FK did not. The results suggested that M-FK enhanced the immunosuppressive effects on islet allograft rejection more effectively with reduced side-effects on insulin secretion.

Differences in lymphocyte gene expression between tolerant and syngeneic liver grafted rats.

Induction of tolerance to allogeneic donor grafts is a clinically desirable goal in bone marrow and solid organ transplantation. We have taken the advantage of DNA microarray technology to investigate gene expression mechanism in regulatory cells. In the present study, using a tacrolimus (FK506) induced tolerance of the fully mismatched liver allograft rat model, we demonstrated that, in contrast with peripheral blood lymphocytes (PBLs) from syngeneic recipients, PBLs taken from tolerant recipients 100 days after transplantation were able to suppress the in vitro proliferation of allogeneic PBLs and to prolong the survival of second syngeneic recipients. We also compared messenger RNA profiles in PBLs from tolerant recipients with those from syngeneic recipients using a DNA microarray with probe sets corresponding to more than 8000 rat genes. There were 96 up-regulated and 103 down-regulated genes in the tolerant recipients. In the up-regulated group, there were 76 known genes and 20 expressed sequence tags (ESTs). In the down-regulated groups, there were 87 known genes and 16 ESTs. Our data indicated that FK506 treatment induced tolerance and expansion of regulatory cells and the DNA microarray technology was useful for this application and provided many informative insights into the mechanism of lymphocyte regulation.

AdCTLA-4Ig combined with donor splenocytes, bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model.

It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti-ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25+ CD4+ regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.

Inducible liver injury in the transgenic rat by expressing liver-specific suicide gene.

Suicide gene expression in specific tissue of transgenic animals has been used for cell-specific ablation. To examine the influence of hepatocyte removal, we produced the herpes simplex virus thymidine kinase (HSVtk) transgenic rat, whose gene was regulated by an albumin enhancer promoter. The liver presence of HSVtk was demonstrated in one line of the transgenic rats. We injected ganciclovir (GCV, 50mg/kg) into the rat on alternate days. After 28 days of GCV administration, liver tissues, and blood of the rats were collected. The histological investigation revealed infiltration of T cells, macrophages, granulocytes/neutrophils, and hepatocyte cell death. The biochemistry analysis demonstrated elevated levels of AST, ALT, and total bilirubin in transgenic rat. In conclusion, the transgenic rat with expressed albumin-specific HSVtk developed experimental hepatitis with administration of GCV, and will be a useful model to facilitate the evaluation of drug effects for clinical control of liver disease.

Simultaneous blockade of co-stimulatory signals, CD28 and ICOS, induced a stable tolerance in rat heart transplantation.

An inducible co-stimulator (ICOS), a recently identified co-stimulatory receptor with a close structural homology of CD28 and CTLA4, is expressed on activated T cells. Anti-ICOS antibody was demonstrated to be effective on prolongation of graft survival after liver transplantation in rats. In this study, we investigated the potency of tolerance induction using the antibody combined with a recombinant adenovirus vector containing CTLA-4Ig cDNA (AdCTLA-4Ig) in rat heart transplantation model. Using a DA-to-Lewis rat heart transplantation model, an anti-rat ICOS antibody and AdCTLA-4Ig were simultaneously administered i.v. into recipients. The tissue specimens from the grafts were removed on various days after transplantation for histological evaluation. Donor-strain skin and heart grafts, and third-party heart allografts were challenged in the recipients with a long-term surviving graft. Splenocytes from the tolerance-induced recipients were used for adoptive transfer study. Anti-ICOS antibody alone did not prolong the survival of heart allograft. AdCTLA-4Ig monotherapy significantly prolonged the survival of heart allograft (Group 4). With a combination of Anti-ICOS antibody and AdCTLA-4Ig, all recipients were resulted in a long-term allograft acceptance for more than 200 days (Group 8). When challenged donor-strain skin grafts in the tolerant rats of Group 4, the skin was rejected, which also lead to a rejection of primary heart allografts. The recipients in Group 8 also rejected donor-strain skin grafts with no rejection of the primary heart grafts. These recipients accepted secondary heart grafts from donor-strain but not third-party. In Group 8 long-term survival recipients showed a high population of CD4+CD25+ regulatory T cell in peripheral blood, and in adoptive transfer study subtraction of these CD4+CD25+ T cells accelerate the rejection of heart graft in secondary irradiated recipients. The present results demonstrated that anti-ICOS antibody combined with AdCTLA-4Ig potently induces a stable immune tolerance after heart allografting in rat, which is mediated by the induction of CD4+CD25+ regulatory T cells. This strategy may be attractive for clinical employment to induce transplantation tolerance.

Prolonged survival of rat liver allograft with adenoviral gene transfection of human immunodeficiency virus type 1 nef.

HIV-1 nef is believed to allow immune evasion by modifying cell surface molecules because of certain mechanisms such as downregulation of the major histocompatibility complex (MHC) class I molecule complex as well as upregulation of FasL. In the present study, we successfully generated a recombinant adenovirus vector containing HIV-1 nef. We detected the expression of nef in liver infected with AxCANef by immune staining and Western blotting, and confirmed its expression as persistent for more than 4 weeks. Furthermore, the surface expression of MHC class I was downregulated in AxCANef-infected hepatic cells. In addition, we also observed nef-induced FasL upregulation of gene-transfected hepatic cells. Using a DA-to-Lewis orthotopic liver transplantation model, we transfected AxCANef to a liver graft to determine whether nef expression could have an effect on recipient survival. AxCANef significantly prolonged recipient survival time (14.5 days) compared with the uninfected group (11 days) (P <.001) and the AxCALacZ-infected group (11 days) (P <.001). Histologic analysis showed reduction in the number of accumulated inflammatory cells and an increase in apoptotic cells in grafts expressing nef. In conclusion, we showed that the nef gene could prolong survival of rat liver allografts, and this result suggested the potential clinical use of its transfection.

Prolongation of liver xenograft survival by adenovirus vector-mediated CTLA-4Ig gene transfer.

Cytotoxic T lymphocyte-associated antigen-4/immunoglobulin fusion products (CTLA-4Ig), a structural homologue of CD28, has been shown to inhibit cellular and humoral immune responses. In this study, we investigated the efficacy of an adenovirus vector containing the CTLA-4Ig gene (AdCTLA-4Ig) on recipient survival after hamster-to-rat liver xenografting. AdCTLA-4Ig was administrated intravenously immediately after grafting. Gene expression was achieved a maximum of 7 days after vector injection and continued for more than 4 weeks. The proportion of CD25(+) T-cells in recipient lymph nodes was significantly reduced 7 days after administration of AdCTLA-4Ig, compared to a group given an adenoviral vector containing LacZ gene (AdLacZ) or to an untreated control group. AdCTLA-4Ig markedly reduced CD2(+) T-cell infiltration into the graft and significantly prolonged recipient survival time (9.2+/-4.12 days), compared to the untreated group (5.4+/-0.78 days) (P<0.001) and the AdLacZ-treated group (5.2+/-0.28 days) (P<0.001). These results indicate that a blockade of T-cell co-stimulation by AdCTLA-4Ig inhibited T-cell activation and attenuated CD2(+) T-cell infiltration into the xenograft, resulting in significant prolongation of recipient survival time. Thus, AdCTLA-4Ig therapy may provide a novel approach to immune regulation.

Gene expression profile in the regenerating rat liver after partial hepatectomy.

BACKGROUND/AIMS: When a loss of hepatic mass occurs, the expression of a large number of genes is either induced or altered, accompanying hepatocyte proliferation. In the present study, we made an in-house cDNA microarray containing 4608 elements (Liver chip), and analyzed extensively gene expression profiles of the regenerating liver after 70% partial hepatectomy (PHx) in rats. METHODS: RNAs were prepared from three rat livers at each time point (taken at 0, 6, 12, 18, 24, 48, 72 h, and 1 week after PHx). Using the liver chip, we performed large-scale analysis of gene expression during liver regeneration. Elements either up- or down-regulated more than twofold at one or more time points were selected. RESULTS: Among the 4608, 382 were identified. Using cluster analysis, we found great similarity between gene-expression profiles at 12 and 18 h after PHx as well as between 48 and 72 h after PHx. We also found that there are at least six distinct temporal patterns of gene expression in the regenerating rat liver after PHx. CONCLUSIONS: These results indicated that microarray analysis is a powerful approach for monitoring molecular events in the regenerating liver.

Amelioration of experimental autoimmune encephalomyelitis in Lewis rats by FTY720 treatment.

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-dependent autoimmune disease that reproduces the inflammatory demyelinating pathology of multiple sclerosis (MS). We investigated the efficacy and mechanism of immunosuppression against EAE by administering 2-amino-[2-(4-octylphenyl) ethyl]-1,3-propanediol hydrochloride (FTY720) in Lewis rats immunized with myelin basic protein together with complete Freund's adjuvant. FTY720 treatment almost completely protected the rats against disease. The protection by FTY720 was associated with a dramatic reduction in the number of lymphocytes staining for T-cell receptors in the spinal cord as examined by immunohistochemistry. The mRNA expression of Th1 cytokines interleukin (IL)-2, IL-6, and interferon-gamma in the spinal cord was also reduced dramatically as assessed by reverse-transcription polymerase chain reaction. Furthermore, lymphocytes isolated from the spleen of FTY720-treated rats were transferred into naive recipient rats against EAE manifestation by reducing both disease incidence and clinical score. These results suggested that the protective anti-inflammatory effect of treatment with FTY720 was, to a large extent, due to the inhibition of encephalitogenic T-cell responses and/or their migration into the central nervous system and may be a potential candidate for use in treating patients with MS.

Prolonged survival in rat liver transplantation with mouse monoclonal antibody against an inducible costimulator (ICOS).

BACKGROUND: An inducible costimulator (ICOS), a recently identified costimulatory receptor with a close structural homology to CD28 and CTLA4, is expressed on activated T cells. Interaction with its ligand on antigen-presenting cells stimulates T-cell proliferation to produce a different spectrum of cytokine. The inhibition of ICOS-mediated signal transduction by an anti-ICOS antibody is considered to be capable of protecting against graft rejection in organ transplantation. METHODS: An anti-rat ICOS antibody was intravenously administered into recipients of dark Agouti-to-Lewis liver transplantations. The recipient lymphocytes from mesenteric lymph nodes were harvested on day 7 after transplantation for fluorescence-activated cell sorting analysis, and tissue specimens from the grafts were removed for histologic evaluation. Antigen-specific T-cell proliferation responses were assessed in vitro with anti-ICOS antibody. RESULTS: Monotherapy with the antibody significantly prolonged the graft survival time by inhibiting T-cell activation and its proliferation response. The graft-infiltrating cells, both CD4 and CD8 T cells, were not completely reduced even when rats were administered the antibody, whereas the expression of ICOS almost completely disappeared in these cells. CONCLUSIONS: T-cell activation through the ICOS costimulatory pathway plays an important role in graft rejection, and manipulating its pathway is an effective method for modulating transplantation immunity.

Distinct pathways of apoptosis triggered by FTY720, etoposide, and anti-Fas antibody in human T-lymphoma cell line (Jurkat cells).

2-amino-2-[2-(4-octylphenyl)ethyl] propane-1,3-diol hydrochloride (FTY720), a synthetic product derived from a metabolite of Isaria sinclairii, has been demonstrated to have a potent immunosuppressive activity that induces apoptotic cell death in T cells and several other cell lines. In this study, using the human T-lymphoma cell line, Jurkat cells, we investigated the apoptotic signal transduction mediated by FTY720, in particular comparing its role on the cleavage of caspases, with that mediated by etoposide or anti-Fas antibody. All of these agents cleaved caspases, inducing their active form in the affected cells. Pretreatment with a broad caspase inhibitor [benzyloxycarbonyl-Val-Ala-Asp-(Ome) fluoromethyl ketone] markedly decreased the incidence of apoptotic cells induced by FTY720, etoposide, and anti-Fas antibody, through the abrogation of cleavage of Bid, poly(ADP-ribose) polymerase, and caspases 3, 8, and 9. The overexpression of Bcl-2 gene prevented FTY720- and etoposide-mediated apoptosis, but not Fas-mediated apoptosis. In addition, mitochondria were demonstrated to play a critical role in FTY720-triggered cell death, suggesting that this drug has a potent anticancer activity.

Fas-mediated apoptosis is involved in the elimination of gene-transduced hepatocytes with E1/E3-deleted adenoviral vectors.

Gene-transduced hepatocytes with E1/E3-deleted adenoviral vectors are eliminated immediately and the expression of transduced genes disappears rapidly following the vector administration. In this report, we analysed the involvement of apoptotic cell death in the elimination of hepatocytes infected with adenoviral vectors. An E1/E3-deleted adenoviral vector expressing Escherichia coli ß-galactosidase (LacZ) was injected via the portal vein into congenitally Fas-deficient mice (lpr), Fas ligand-deficient mice (gld) and their control mice, MRL and C3H. 5-Bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) staining of the liver specimens showed that 80-100% of hepatocytes were LacZ positive at 7 days after virus administration, suggesting that most of the hepatocytes received the injected adenoviral vectors. In normal mice, the number of LacZ-positive cells decreased dramatically at 14 and 21 days after transduction and few positive cells were observed at day 28. ß-Galactosidase activity, quantified by the O-nitrophenyl-beta-D-galactopyranoside assay, gave comparable results to X-gal staining. At days 14 or 21, many apoptotic hepatocytes and apoptotic infiltrating cells were detected with the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) in situ apoptosis detection method. This observation suggested that the apoptotic process was associated with the elimination of adenovirus-infected hepatocytes. To test the involvement of the Fas-Fas ligand interaction in this apoptotic process, the period of transgene expression was measured in 1pr and gld mice, which had received the same amount of AxCALacZ. X-Gal histochemical analysis detected many LacZ-positive cells in 1pr or gld mice liver even at 21 or 28 days after AxCALacZ injection. There were significant differences in the reduction rates of ß-galactosidase activity of liver homogenates between lpr and MRL, or gld and C3H mice. Based on these observations, we conclude that the Fas-mediated apoptotic process is involved in the elimination of hepatocytes infected with E1/E3-deleted adenoviral vectors.