Lewis Liver Research Articles

The Role of Tacrolimus Nanomicelles in Acute Rejection After Liver Transplantation in Rats.

Although there is some progress in immunosuppressive therapy of acute rejection, there is still a lack of standardized diagnosis and treatment. For the acute rejection after liver transplantation, there is still a lack of an exact treatment at this stage. Tacrolimus (TAC) side effects will also affect the survival rate and quality of life of recipients after transplantation to a large extent. Rat orthotopic liver transplantation model was established and divided into three groups. In the tolerance group, Brown Norway (BN) to Lewis liver transplantation was used; in the rejection group, Lewis to BN liver transplantation was used; in the TAC group, TAC was injected after operation on the basis of establishing rejection model. The expression of GITRL in Kupffer cells and the change of cytokines were detected 7 days after operation. In this study, the animal model of acute rejection of rat liver transplantation was established to simulate the clinical allogeneic liver transplantation, and the important role of TAC in the acute rejection of rat liver transplantation was evaluated.

Chronic Antibody-Mediated Responses May Mediate Chronic Rejection in Rat Orthotopic Liver Transplantation

The category of chronic antibody-mediated rejection (AMR) is not included in Banff schema for liver allograft rejection. In the present study, we examined the pathology of chronic rejection using rat liver transplantation. Orthotopic liver transplantation from Lewis to BN rats was performed without immunosuppression, and with or without HA reconstruction. We studied grafts at day 120 for arterialized and day 39 for nonarterialized transplants focusing on the immunoglobulin G (IgG) deposition and the pathologic characteristics of rejection. About 20% of arterialized grafts survived more than 120 days. Between day 7 and day 120, T-cell infiltration to arterialized grafts was accompanied by IgG deposition in portal veins, hepatic arteries, and bile ducts in portal areas, sinusoids and hepatocytes. At day 120, arterialized grafts were morphologically characterized by late chronic rejection with IgG deposition, intraluminal portal veins fibrosis, intimal fibrous thickening of hepatic arteries, diffuse sinusoidal fibrosis, as well as injury and loss of bile ducts due to fibrosis. The severities of T cell-mediated rejection and AMR were higher in nonarterialized than arterialized grafts. Nonarterialized Lewis liver grafts in BN rats were rejected by day 39, as characterized by late chronic rejection with IgG deposition and cellular infiltration. In conclusion, chronic AMR may be involved in chronic rejection of liver transplantations. When chronic AMR was involved in chronic liver graft rejection, typical late morphological changes emerged within a short period.

Chronic Antibody-Mediated Rejection in Rat Orthotopic Liver Transplantation

Introduction: In chronic rejection in Banff schema for liver allograft rejection, the early and late chronic rejection has been defined. However, the pathological characteristics of chronic antibody-mediated rejection in liver allogarfts have not been fully investigated. In the present study, we examined the pathology of chronic antibody-mediated rejection using rat liver transplantation. Method: Orthotopic liver transplantation was performed from Lewis (RT1l) to BN (RT1n) rats without immunosspression. In this allogenic combination, the liver grfts transplantated with hepatic artery reconstruction could survive more than 100 days, although the grafts transplanted without hepatic artery reconstruction were rejected by day 35 with extensive T cell-mediated and antibody-mediated rejection. We studied Lewis liver grafts that were transplnated into BN rats with hepatic artery reconstruction, focusing on the pathological characteristics of chronic rejection accompanied with chronic antibody-mediated rejection in liver grafts. Results: The grafts transplanted with hepatic artery reconstruction could survived more than 100 days with mild graft dysfunction (T-Bil 0.4 ± 0.1 mg/dl, AST 198.1 ± 19.9 mg/dl), after the eraly severe T cell- and antibody-mediated rejection and graft dysfunction (T-Bil 4.9 ± 1.4 mg/dl, AST 420.8 ± 134.4 mg/dl). Between day 7 and day 28, in addition to the development of T cell-mediated rejection, the deposition of IgM and IgG was detected on endothelial cells in portal veins and hepatic arteries, epithelial cells in bile ducts, and hepatic cells in lobules. After day 28, T cell-mediated rejection gradually subsided but continued more than day 100. The deposition of IgM and IgG was also contnued mainly on endothelial cells in portal veins and hepatic arteries, suggesting the development of chronic antibody-mediated rejection. In light and electron microscopic studies at more than day 100, most prominent features were persistent endothelial cell injuries in portal veins and hepatic arteries with obliterative vasculopathy, diffuse sinusoidal fibrosis, and obstructive cholangiopathy and ductopenia. Conclusion: In chronic liver allograft rejection, chronic antibodymediated rejection could develop with graft dysfunction. The pathological characteristics of chronic rejection accompanied with chronic antibodymediated rejection were similar features as late chronic rejection, but they progressed within short periods with typical obliterative vasculopathy and ductopenia. This animal model is useful for the evaluation of the mechanism and pathological characteristics of chronic antibodymediated liver allograft rejection.

Identification of the rat NKG2D ligands, RAE1L and RRLT, and their role in allograft rejection

NKG2D is a receptor expressed by NK cells and subsets of T lymphocytes. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We cloned and expressed two rat NKG2D ligands, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that these ligands can induce IFN-gamma secretion and cytotoxicity by rat NK cells. To examine changes in expression of NKG2D and the NKG2D ligands RAE1L and RRLT after transplantation, we used a Dark Agouti (DA)-->Lewis rat model of liver transplantation. NKG2D expression was significantly increased in allogeneic liver grafts by day 7 post-transplant. Ligands of NKG2D, absent in normal liver, were readily detected in both syngeneic and allogeneic liver grafts by day 1 post-transplant. By day 7 post-transplant, hepatocyte RAE1L and RRLT expression was significantly and specifically increased in liver allografts. In contrast to acute rejection that develops in the DA-->Lewis model, transplantation of Lewis livers into DA recipients (Lewis-->DA) results in spontaneous tolerance. Interestingly, expression of RAE1L and RRLT is low in Lewis-->DA liver allografts, but significantly increased in DA-->Lewis liver allografts undergoing rejection. In conclusion, our results suggest that expression of NKG2D ligands may be important in allograft rejection.

Establishment of an Acute Graft-Versus-Host Disease Model Following Liver Transplantation in Donor-Dominant One-Way Major Histocompatibility Complex Matching Rats

Acute graft-versus-host disease is an uncommon but devastating complication following liver transplantation (LTx-aGVHD). We investigated whether a rat model of LTx-aGVHD could be established using Lewis rat donors and (LewisXBN)F1 rats as recipients, which provides favorable conditions for studies of graft-versus-host reaction or disease. Replacement of (LewisXBN)F1 livers by Lewis livers alone was not sufficient to induce aGVHD; all recipients grew in a normal pattern as the syngeneic liver transplantation (LT) from Lewis to Lewis rat. However, when various numbers of donor splenocytes (1 × 10 8, 2 × 10 8, 3 × 10 8, 4 × 10 8) were transferred simultaneously with the LT, the morbidities of lethal aGVHD were 16.7%, 50%, 83.3%, and 100%, respectively. The clinical courses as well as histologic analyses of skin and colon showed typical aGVHD characteristics. However, unlike transfusion-associated aGVHD, the liver graft was not involved. These clinical and histologic characteristics of aGVHD were consistent with those in humans who develop aGVHD after LT. Thus, a reproducible rat model of LTx-aGVHD was developed by performing LT from Lewis to (LewisXBN)F1 rats in combination with donor splenocyte transfusion. This model may be useful for further studies on the mechanisms and effective treatment modalities for LTx-aGVHD.

Insights into the Mechanism Responsible for Spontaneous Acceptance of Liver Allografts (141.41)

Abstract Transplantation of fully histoincompatable livers between strains of rodents often results in spontaneous acceptance of the graft. Lewis rats reject DA livers in 14 days while DA recipients of Lewis livers have long-term graft survival. We sought to determine the mechanism of this spontaneous graft survival. NK cells and subsets of T cells express NKG2D. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We examined NKG2D receptor and ligand expression post-transplant. Groups of Lewis rats received livers from DA (rejection combination) or Lewis donors (syngeneic) and DA rats received livers from Lewis (non-rejection combination) or DA donors (syngeneic). NKG2D expression was low in syngeneic grafts but increased 7-fold in Lewis→DA grafts and 17-fold in DA→Lewis grafts by day 7 post-transplant. Ligands of NKG2D were not expressed in normal liver but were up regulated in all liver grafts by day 1 post-transplant, suggesting that ischemia/reperfusion injury and trauma induce expression of these ligands. RAE1L and RRLT were significantly up regulated specifically in DA→Lewis liver grafts by day 7 post-transplant. In marked contrast, the expression of RAE1L and RRLT in Lewis→ DA liver grafts was similar to the levels detected in syngeneic liver grafts suggesting that signals that lead to an increase in NKG2D ligand expression are absent in this strain combination. In conclusion, the absence of NKG2D ligand expression in the liver graft may play a role in preventing rejection, and decreasing the expression of these proteins may be a useful strategy to promote graft non-responsiveness. Supported by AI044095

Regulatory T cells contribute to the immunoregulatory effect on graft versus host reaction after liver transplantation in donor-dominant one-way MHC matching rats

Acute graft-versus-host disease following liver transplantation (LTx-aGVHD) poses a major diagnostic and therapeutic challenge, and the mortality rate is as high as 85%. Even the liver contains large numbers of lymphoid cells in the parenchyma and surgical damage of liver transplantation would induce an immunosuppressive, the incidence of LTx-aGVHD is just approximately 1–2%. CD4 +CD25 +regulatory T (Treg) cells have recently been shown to suppress proliferative responses of CD4 +CD25 −T cells to alloantigenic stimulation in vitro and are required for ex vivo tolerization of donor T cells, which results in their reduced potential to induce aGVHD after bone marrow transplantation. To investigate the role of Treg in LTx-aGVHD, we compared the proportional frequency of Treg in syngeneic liver transplantation recipients (sLT group), semiallogeneic liver transplantation recipients (Semi-LT group), LTx-aGVHD induced recipients (LTx-aGVHD group) and healthy controls. Here we show that replacement of (LewisXBN)F1 liver by Lewis liver alone in Semi-LT group was not sufficient to induce aGVHD, and all recipients grew in a normal pattern as the syngeneic LT from Lewis to Lewis rat. However, when 4 × 10 8 of donor splenocytes were transferred simultaneously with LT, the morbidity of lethal aGVHD were 100%. A relative stable percentage of Treg, defined as CD4, CD25 and Foxp3, was detected in peripheral blood mononuclear cells (PBMCs) of sLT group compared with healthy controls. In early time after transplantation, no significantly change of Treg population was observed in these recipients after semiallogeneic liver transplantation in comparison with healthy controls ( P > 0.05). However, Treg levels showed a relative increase 4 days after transplantation. Especially on 12th and 16th day after transplantation, there was significantly increased proportion of Treg cells compared with healthy controls ( P < 0.05). The present of Treg decreased progressively in LTx-aGVHD group, which was significantly lower than group 1 and group 2 on the 12th and 16th day after liver transplantation ( p < 0.05). In conclusion, recipients in semi-LT group harbor an increased percentage of Treg in peripheral blood compared with controls. Treg have an immunoregulatory effect on graft versus host reaction after liver transplantation. Additional donor splenocyte transplanted with the liver provoke the development of aGVHD after liver transplantation, and reversed the change of Treg in PBMCs as in Semi-LT group, which destroy the balance between Treg and conventional effector T cells which determine the outcome of aGVHD after liver transplantation.

Characteristics of acute rejection model of DA to LEW orthotopic liver transplantation in inbred rats

Objective To study the characteristics of liver transplantation models in inbred rats DA to LEW and judge the grades of acute rejection. Methods Surgical skills were trained by using closed colony SD and Wistar rats, followed by DA to LEW orthotopic liver transplantation with Kama-da's two-cuff technique. The grades of acute rejection were judged by their clinical manifestations and Banff schema. Results Fifteen models of DA to LEW liver transplantation were established. The suc-cessful rate was 86.7 %. The causes of death were hemorrhage and thrombosis of suprahepatic and in-frahepatic vena cava and obstruction of biliary tract. Grade I rejection occurred on the 3rd d and reached the peak after the 7th d after liver transplantation. Remaining models died of grade Ⅲ rejec-tion besides postoperative complication within 12 d after the transplantation. Conclusion The acute rejection of DA to LEW orthotopic liver transplantation models is stable and intensive. It can be effec-tively used to study allograft rejection and tolerance. But it is difficult to establish this kind of ortho-topic liver transplantation models in inbred rats because of their own tissue characteristics. Key words: Liver transplantation; Inbred rat; Model; Rejection

Relative contribution of direct and indirect allorecognition in developing tolerance after liver transplantation.

The interaction of donor passenger leukocytes and host leukocytes in recipient secondary lymphoid tissues during the early posttransplantation period is crucial in directing host immune reactions toward allograft rejection or acceptance. Responsible T cell clones could be activated through the direct and indirect pathways of allorecognition. We examined the role of the indirect pathway in liver transplantation (LT) tolerance by depleting host antigen-presenting cells (APC) with phagocytic activity [e.g., cluster domain (CD)68+/CD163+ macrophages, CD11c+ dendritic cells (DC)] using liposome-encapsulating clodronate (LP-CL). After Lewis rat cell or liver graft transplantation, Brown Norway (BN) rat recipients pretreated with LP-CL showed a significantly reduced type 1 helper T cell cytokine up-regulation than control-LP-treated recipients. In the LT model, LP-CL treatment and host APC depletion abrogated hepatic tolerance; Lewis liver grafts in LP-CL-treated-BN recipients developed mild allograft rejection, failed to maintain donor major histocompatibility complex (MHC) class II+ leukocytes, and developed chronic rejection in challenged donor heart allografts, while control-LP-treated BN recipients maintained tolerance status and donor MHC class II+ hepatic leukocytes. Furthermore, in the BN to Lewis LT model, LP-CL recipient treatment abrogated spontaneous hepatic allograft acceptance, and graft survival rate was reduced to 43% from 100% in the control-LP group. In conclusion, the study suggests that host cells with phagocytic activity could play significant roles in developing LT tolerance.

The Difficulty of Eliminating Donor Leukocyte Microchimerism in Rat Recipients Bearing Established Organ Allografts

Unequivocal eradication of donor leukocyte microchimerism from recipients of long-surviving organ transplants has never been reported. Here we describe a drastic attempt to accomplish this objective. In control experiments, a rank order of microchimerism and of associated donor specific nonreactivity was produced in Brown-Norway (BN) rats by transplantation of Lewis (LEW) liver, bone marrow cell (BMC) and heart allografts under a brief course of tacrolimus. The degree of microchimerism at 60 and 110 days was estimated with semiquanitative immunocytochemical and PCR techniques. Tolerance at 110 days was assessed in the different control groups by challenge transplantation of naïve LEW hearts. In parallel experimental groups, an attempt was made to eliminate microchimerism from the BN recipients. The animals were submitted at 60 days to 9.5-Gy total body irradiation (TBI), reconstituted immediately with naïve BN BMC, and tested for donor specific nonreactivity by LEW heart transplantation at 110 days. After the TBI-reconstitution at 60 days, microchimerism was undetectable in BMC recipients at 110 days, significantly reduced in heart recipients, and least affected in liver recipients. Except in liver recipients, abrogation of LEW-specific nonreactivity was demonstrated by rejection of the priming grafts, or by rejection of the challenge heart grafts, and by in vitro immune assay. It is difficult to eliminate microchimerism in organ recipients once the donor cells have settled into tissue niches.

Gene array analysis of a rat model of liver transplant tolerance identifies increased complement C3 and the STAT-1/IRF-1 pathway during tolerance induction

This study aimed to define the molecular mechanism during induction of spontaneous liver transplant tolerance using microarrays and to focus on molecular pathways associated with tolerance by meta-analysis with published studies. The differences in the early immune response between PVG to DA liver transplant recipients that are spontaneously tolerant (TOL) and PVG to Lewis liver transplants that reject (REJ) were examined. Spleens from TOL and REJ on days 1 and 3 were compared by 2 color microarray. Forty-six of 199 genes differentially expressed between TOL and REJ had an immunological function. More immune genes were increased in TOL vs. REJ on day 1, including STAT-1, IRF-1 and complement C3. Differential expression of selected genes was confirmed by quantitative RT-PCR. The results were compared to two published high-throughput studies of rat liver transplant tolerance and showed that C3 was increased in all three models, while STAT-1 and IRF-1 were increased in two models. The early increases in immune genes in TOL confirmed previous reports of an active early immune response in TOL. In conclusion, the increase in STAT-1, IRF-1 and complement component C3 in several models of liver transplant tolerance suggests that the STAT-1/IRF-1 apoptotic pathway and C3 may be involved in the tolerogenic mechanism.

The persistence of regulatory cells developing after rat spontaneous liver acceptance

We have previously reported that the spontaneous acceptance of Lewis (LEW, RT1(l)) to Dark Agouti (DA, RT1(a)) rat orthotopic liver transplant (OLT) is eliminated by donor gamma-irradiation. The acceptance of the irradiated LEW liver is also reestablished in a naïve rat after the adoptive transfer of T regulatory (T-reg) cells from a LEW to a DA liver-tolerant long-term (>60 days) survivor (LTS) into a naïve DA rat. However, little is known about the growth conditions required to maintain T-reg function. In this study, we examined the need for continued donor-specific alloantigen stimulation for the maintenance and function of T-reg cells. Splenocytes (SCs; 1.5 x 10(8) cells) from a LEW liver allograft-tolerant LTS DA recipient were adoptively transferred fresh or after in vitro stimulation into another naive DA rat on day 1, 4, or 7 before an irradiated (1000R) LEW liver transplant. For in vitro alloantigen stimulation, SCs from LEW to DA LTS were co-cultured with mitomycin-C (MMC)-treated naïve LEW (donor alloantigen-specific) or Brown Norway (BN) (RT1(n); third party) SCs for 72 hours. Graft rejection, as defined by death of the recipient, was confirmed histologically. All LEW liver grafts were accepted spontaneously by DA recipients for more than 60 days (n=32), while all irradiated LEW livers were acutely rejected (n=9; mean survival time [MST]=12.8 +/- 4.0 days). When LTS DA SCs were adoptively transferred into a naive DA rat 1 day before OLT, all irradiated LEW grafts were accepted greater than 60 days (n=9). However, when fresh LTS DA SCs were transferred to a new naïve DA rat on 4 or 7 days before OLT, all irradiated LEW liver grafts were acutely rejected (MST=10.2 +/- 0.5 days [n=4] and MST=13.5 +/- 5.0 days [n=4], respectively). When LTS DA SCs were stimulated in vitro before adoptive transfer, irradiated LEW liver grafts after 4 days (n=5) were then accepted. In vitro culture of LTS DA SCs with MMC-treated BN SCs (third-party) for 72 hours before adoptive transfer resulted in 3 of 5 irradiated LEW livers at day 4 being accepted (n=5). The maintenance of T-reg function requires continuous LEW donor-specific alloantigen stimulation.

Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on postischemic reperfusion injury, apoptotic cell death graft regeneration and survival*

Inhibition or destruction of Kupffer cells (KC) may protect against ischemia-reperfusion (IR) induced primary graft nonfunction (PNF) in liver transplantation. Besides KC activation, PNF is characterized by microvascular perfusion failure, intrahepatic leukocyte accumulation, cell death and hepatocellular dysfunction. KCs can be inactivated by different agents including gadolinium chloride (GdCl3), methyl palmitate (MP) and glycine. The effects of three KC inactivators on IR-injury after rat liver transplantation were compared in the present study. Lewis liver donors were treated with GdCl3, MP, glycine or saline (control). Liver grafts were transplanted following 24 h storage (UW solution). KC populations and IR damage were assessed by histologic analysis, quantitative real-time polymerase chain reaction (RT-PCR) and intravital microscopy. The number of hepatic ED-1 positive macrophages was diminished after GdCl3 (114.8+/-4.4/mm2 liver tissue) and MP treatment (176.0+/-5.0), versus the glycine (263.9+/-5.5) and control (272.1+/-5.6) groups. All three treatment modalities downregulated phagocytic activity for latex particles, paralleled by reduced microvascular injury (acinar perfusion index, GdCl3: 0.75+/-0.03; MP: 0.83+/-.03; glycine: 0.84+/-0.03; 0.63+/-0.03). Quantitative RT-PCR revealed elevated myeloperoxidase mRNA after glycine versus GdCl3 and MP pretreatment (3.2- and 3.4-fold, P=0.011, respectively), without difference to controls (2.9-fold of glycine). TNFalpha-mRNA was reduced after glycine- (5.2-fold), GdCl3- (19.7-fold), MP-treatment (39.5-fold) compared with controls. However, profound prevention of intrahepatic cell death and liver graft failure was solely achieved with glycine preconditioning. Different than GdCl3 and MP, glycine modulates rather than destroys KCs. Glycine appears to preserve cell viability and to TNFalpha/leukocyte dependent organ regeneration capacity, which is related to increase graft survival following liver transplantation.

Posttransplant Interleukin-4 Treatment Converts Rat Liver Allograft Tolerance to Rejection

Previous studies showed that liver transplant rejection in the Piebald Virol Glaxo (PVG)-to-Lewis combination was associated with more intragraft interleukin (IL)-4 mRNA expression than in spontaneously tolerant grafts in the PVG-to-Dark Agouti (DA) combination. There was also immunoglobulin (Ig) G1 antibody deposition, suggesting an IL-4-induced IgG class switch in rejection. The aim of this study was to investigate whether IL-4 treatment converts PVG-->DA liver transplant tolerance to rejection. DA (RT1a) rats were recipients of orthotopic PVG (RT1c) liver transplants and DA liver transplants were syngeneic controls. Supernatant from IL-4-transfected Chinese hamster ovary cells (0.5 mL, 30,000 U) or from untransfected cells was injected intraperitoneally on days 3 through 7. Samples were taken for immunohistochemical staining of frozen tissue sections to analyze cellular infiltrate and antibody deposition. IL-4 treatment significantly reduced survival of liver allografts from greater than 100 days in untreated animals to 9 days (P=0.004). Pathologic analysis of IL-4-treated animals showed that death was caused by liver transplant rejection, with a heavy infiltrate of mononuclear cells, disruption of portal tract areas, and infarction. Immunohistochemistry revealed an extensive infiltrate of T cells, CD25-expressing cells, and B cells that was similar to the level in PVG--> Lewis liver allograft recipients that reject the liver. There was also a more extensive monocyte-macrophage infiltrate and more major histocompatibility complex class II expression in IL-4-treated animals compared with untreated animals. There was moderate increase of IgM, little IgG1, and no IgE or IgG2a antibody deposition. IL-4, a T-helper type 2 cytokine that has previously been shown to inhibit delayed-type hypersensitivity responses such as rejection, was found to promote rejection of liver allografts. There was only slight evidence of a graft-specific antibody response, showing that IL-4 induces liver allograft rejection in association with some, but not all, of the changes accompanying rejection in the PVG-->Lewis strain combination.

FK778 controls acute rejection after rat liver allotransplantation showing positive interaction with FK506

This study including prevention and rescue experiments was performed to examine the efficacy of FK778 and its interactions with FK506. In the prevention experiment, Brown-Norway rats transplanted with a 7 Lewis livers received day-course of FK778 or a combination of FK778 and FK506 treatment. For the rescue experiment, the recipients were additionally treated with FK778 from days 7 to 13. Blood chemistry and histopathological findings were used to examine the host and the graft condition. Donor-specific IgM was measured using enzyme-linked immunosorbent assays. The serum trough level of FK778 was examined by high-performance liquid chromatography. FK778 suppressed acute rejection in a dose-dependent manner. The optimal FK778 dosage was 20 mg/kg body weight (BW) d. FK778 treatment from days 7 to 13 rescued liver grafts from ongoing rejection. The combination of FK506 (0.125 mg/kg BW/d) and FK778 (20 mg/kg BW/d) maintained better graft condition than FK778 (20 mg/kg BW/d) monotherapy. In conclusion, FK778 prevents acute rejection in and rescues transplant recipients from ongoing rejection after rat liver transplantation. The optimal monotherapy dosage of FK778 was 20 mg/kg BW/d. Combination therapy with FK506 was more beneficial than FK778 monotherapy.

Regulatory cells develop after the spontaneous acceptance of rat liver allografts

After donor-specific transfusion, tolerance to heart transplants is serially passed to naive rats by the adoptive transfer of long-term survivor (LTS)-tolerant splenocytes (SC). We examined whether regulatory cells similarly develop after the spontaneously accepted Lewis (LEW) to Dark Agouti (DA) liver transplants. SC from a LTS DA rat with a LEW liver were adoptively transferred to a naive DA 1 day before transplantation of an irradiated (1000 rad) LEW liver. Untreated LEW to DA liver allografts were uniformly accepted; whereas all irradiated LEW liver grafts were rejected. In contrast, when 1.5 x 10 8 DA LTS SC were transferred to a naive DA recipient, all irradiated LEW liver grafts were accepted. When decreased to 1.0 x 10 8 LTS DA SC, only 1 of 4 irradiated LEW grafts was accepted. However, if 1.5 x 10 8 DA SC harvested only 30 days after liver transplantation were transferred, only 2 of 5 irradiated LEW liver grafts were accepted. The serial second and third adoptive transfers of 1.5 x 10 8 DA LTS SC also resulted in the uniform acceptance of irradiated LEW livers. Regulatory cells that develop after the spontaneous acceptance of a LEW to DA liver transplant can serially transfer tolerance to new naive LEW liver allograft DA recipients. This "infectious tolerance" is dependent on the time of cell harvest after transplantation and on the cell dose given.

FK778 CONTROL ACUTE REJECTION AFTER RAT LIVER ALLOTRANSPLANTATION INTERACTING WITH FK506 POSITIVELY

P1128 Aims: FK778 is one of synthetic malononitrilamide derives from A771726, which is an active metabolite of leflunomide. This agent is used for kidney and heart transplantation and acute or chronic GVHD proved to prevent immunoreactions. The efficacy of FK778 for preventing rejection after liver transplantation was studied. In addition, the synergistic effect of FK778 with FK506 for inhibition of immunoreaction after rat liver transplantation was examined. Methods: Lewis livers were orthotopically transplanted into Brown-Norway under a 7 day-course of 0 (U), 10 (M1), 20 (M2) or 30 (M3) of FK778 (mg/kg BW) oral treatments. In addition, recipients were treated with 0 (UL) and 20 (M2L) from day 7 to 13 for rescue experiment. To examine the synergistic effect of FK778 with FK506, following three groups were administrated FK778 (mg/kg BW) / FK506 (mg/kg BW) under 7 day-course; 0/ 0.125 (T), 10/ 0.125 (TM1) and 20/ 0.125 (TM2). For exam of the graft state, serum AST and ALT were measured and rejection activity index (RAI) based on Banff’s schema was scored. Lymphocyte proliferations in grafts and recipient spleens were analyzed by BrdU uptake. Donor hepatocyte specific IgM were measured using ELISA. Serum trough level of FK778 was examined by HPLC. Results: RAI score was suppressed by FK778 treatment in dose dependent manners (U; 8.2±0.8, M1; 5.6±1.1, M2; 1.4±0.9, M3; 0.8±0.9). Infiltrates in the graft and proliferating splenocytes were decreased remarkably in M1 rats and diminished in M2 and M3 rats. AST and ALT value were both jumped up at U and M1. AST and ALT values of M2 and M3 maintained as normal level. Serum total protein levels of U, M1 and M2 rats were almost nearly equal to that of normal. Only the serum protein level of M3 were remarkable low (P < 0.01 vs.M2). Four of M3 rats suffered from intestinal bleeding with high trough levels (over 4.0 mmol/L) of FK778. Donor specific IgM production was suppressed in dose dependent manners. In rescue experiment, RAI scores were decreased by the M2L treatment (P<0.05 vs. UL). Number of BrdU+ cells in the M2L graft and spleen were almost equal to that of M2 graft and spleen. AST and ALT values were improved by the ML treatment. The RAI scores of the combination therapy (TM1; 3.8±1.5, TM2; 0.8±0.5) were more improved than that of T (4.8±1.0). By FK778 treatment with/ without FK506, number of BrdU+ cells in graft and spleen significantly decreased. Number of BrdU+ cells of TM1 and TM2 in the graft and spleen expressed significantly smaller population than that of T. Especially RAI score and number of BrdU+ cells in graft and spleen of TM2 were suppressed as M3 level without adverse effects. TM2 treatment maintained serum AST and ALT values. IgM value was not be influenced by additional FK506 treatment and was determined by administrated FK778 dose. Conclusions: FK778 was effective for prevention from acute rejection and rescue from ongoing rejection after rat liver transplantation. Appropriate dose was 20 mg/kg BW. FK778 suppressed IgM production by itself. Combination therapy was more useful than FK778 monotherapy.

Cytotoxic T-cell elimination during anti-CD4-induced rat liver acceptance and rapid replacement of functional graft antigen-presenting cells.

In previous studies, we showed that primed T cells were eliminated in long-term survival Wistar Furth (WF) recipient rats with spontaneously accepted Lewis (LEW) liver graft and that the grafted liver lost the ability to elicit rejection reaction early after liver transplantation. We hypothesized that the same phenomenon may be observed in tolerant animals after immunosuppression in a rejector rat strain combination (WF-->LEW). Furthermore, we proposed the repopulation of liver allograft with host antigen-presenting cells rapidly after transplantation. Recipient LEW rats that underwent anti-CD4 therapy accepted the WF liver allografts after a transient rejection reaction. In tolerant animals, alloreactive CD8 T cell precursors were present, but primed T cells were absent. Intraperitoneal challenge with grafted WF liver homogenates obtained from recipient LEW rats on day 4 after transplantation did not induce transient rejection responses in long-term survival recipient LEW rats, a finding that differed from the results of experiments using normal WF liver homogenates. However, challenge with grafted WF liver homogenates, similar to those of normal LEW liver homogenates, induced rejection responses in long-term survival recipient WF rats with LEW liver allograft. Flow cytometric analysis confirmed that most of nonparenchymal cells in the grafted WF liver were recipient (LEW) genotype. These observations showed that the deletional mechanism of effector T cells also is observed in this setting, and professional donor antigen-presenting cells are replaced by those of recipient genotype within the graft during the early phase of 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.

SPLENIC-INJECTION OF AUTOLOGOUS HEPATOCYTES GENETICALLY MODIFIED TO EXPRESS SOLUBLE DONOR-MHC CLASS I ANTIGEN PROLONGS HEART ALLOGRAFT SURVIVAL

881 Previous studies suggest that liver allografts can provide protection for other transplanted organs. In this respect heart allograft rejection can be prevented, and even reversed, with a liver transplant from the same donor. One possible explanation for this phenomenon is that hepatocytes produce relatively large amounts of potentially immunosuppressive soluble MHC class I antigen (Ag). In support of this theory we have recently shown that hepatocyte-secreted, soluble, allogeneic-MHC class I Ag downregulates both cytotoxic and helper T lymphocyte responses in vivo. In the present study we examined the potential immunosuppressive effects of hepatocyte-secreted donor-MHC Ag in a rat heart allograft model. Hepatocytes were obtained by collagenase digestion of a Lewis (RT11) liver. Cells were cultured and subsequently transfected by lipofection to express the soluble donor-MHC class I Ag, RT1.Aa (plasmid: pcRQ.B3). One day prior to ACI (RT1a) heterotopic heart allografting, Lewis recipients were injected with 1 × 107 pcRQ.B3-transfected hepatocytes via the spleen. Controls did not receive hepatocytes or were injected with hepatocytes transfected with a gene encoding firefly-luciferase (pCMVLux). All animals received a short-course of cyclosporin A (10 mg/kg/day, i.p.) on days 0 to +7, relative to heart transplantation. Graft survival results showed that recipients injected with hepatocytes expressing soluble donor-MHC class I Ag experienced a prolongation of ACI heart allograft survival, when compared to control. (Table)TableThese results suggest that the immunosuppressive effects of a single mismatched soluble donor-MHC class I Ag are sufficiently potent to promote heart allograft survival in a fully MHC-disparate, high-responder, rat-strain combination. This is one of the first steps in the development of a gene transfer-based system to deliver soluble donor-MHC class I molecules to organ transplant recipients. We are currently refining this gene transfer system to allow for more prolonged soluble donor-MHC expression in vivo, which we predict is a critical factor to provide for longer immunologic protection of heart allografts. Also, it is likely that delivery of a single type of mismatched donor-MHC Ag will be more effective between strain combinations where the MHC is not completely different. In summary, these results provide incentive to determine the full potential of soluble donor-MHC class I gene therapy in organ transplantation.