Although immunosuppressive regimens are effective, rejection occurs in up to 50% of patients after orthotopic liver transplantation (OLT), and there is concern about side effects from long-term therapy. Knowledge of clinical and immunogenetic variables may allow tailoring of immunosuppressive therapy to patients according to their potential risks. We studied the association between transforming growth factor-beta, interleukin-10, and tumor necrosis factor alpha (TNF-alpha) gene polymorphisms and graft rejection and renal impairment in 121 white liver transplant recipients. Clinical variables were collected retrospectively, and creatinine clearance was estimated using the formula of Cockcroft and Gault. Biallelic polymorphisms were detected using polymerase chain reaction-based methods. Thirty-seven of 121 patients (30.6%) developed at least 1 episode of rejection. Multivariate analysis showed that Child-Pugh score (P =.001), immune-mediated liver disease (P =.018), normal pre-OLT creatinine clearance (P =.037), and fewer HLA class 1 mismatches (P =.038) were independently associated with rejection, Renal impairment occurred in 80% of patients and was moderate or severe in 39%, Clinical variables independently associated with renal impairment were female sex (P =.001), pre-OLT renal dysfunction (P =.0001), and a diagnosis of viral hepatitis (P =.0008), There was a significant difference in the frequency of TNF-alpha -308 alleles among the primary liver diseases. After adjustment for potential confounders and a Bonferroni correction, the association between the TNF-alpha -308 polymorphism and graft rejection approached significance (P =.06). Recipient cytokine genotypes do not have a major independent role in graft rejection or renal impairment after OLT, Additional studies of immunogenetic factors require analysis of large numbers of patients with appropriate phenotypic information to avoid population stratification, which may lead to inappropriate conclusions.

BACKGROUND/AIMS:Liver sinusoids contain a large population of spontaneously cytotoxic cells (NK cells), CD8+ T cells and macrophages. The physiological role of these leucocytes remains unclear. They may participate in immune surveillance and peripheral tolerance by deleting tumour cells, virus-infected cells and activated T cells as they traffic through the liver. In order to gain further information about the function of these leucocytes within the hepatic sinusoids, we examined their production of immunomodulatory cytokines and apoptosis-related molecules. METHODS:Semi-quantitative polymerase chain reaction and immunohistochemistry were used to determine the spontaneous production of cytokines and apoptosis-related molecules by sinusoidal leucocytes isolated from donor liver preservation solution. RESULTS:In comparison with matched peripheral blood mononuclear cells, sinusoidal leucocytes produced more mRNA for IL-10, IL-15, TNF-alpha, IL-18, IFN-gamma, FasL, perforin and granzyme. IL-4 and IL-12 were not detected and IL-2 was only faintly detected in the liver-derived CD4+ population. Less bcl-2 was expressed in liver-derived CD4+ and CD8+ cells in comparison with matched peripheral blood cell populations. CONCLUSIONS:The cytokines produced spontaneously by sinusoidal leucocytes are consistent with their high level of activation and spontaneous cytotoxicity. Their strong expression of apoptosis-mediating molecules (FasL, perforin, granzyme and TNF-alpha) support a role for these cells in immune surveillance and peripheral tolerance induction.

The fascinating aspect of the liver is the capacity to regenerate after injury or resection. A variety of genes, cytokines, growth factors, and cells are involved in liver regeneration. The exact mechanism of regeneration and the interaction between cells and cytokines are not fully understood. There seems to exist a sequence of stages that result in liver regeneration, while at the same time inhibitors control the size of the regenerated liver. It has been proven that hepatocyte growth factor, transforming growth factor, epidermal growth factor, tumor necrosis factor-alpha, interleukins -1 and -6 are the main growth and promoter factors secreted after hepatic injury, partial hepatectomy and after a sequence of different and complex reactions to activate transcription factors, mainly nuclear factor kappaB and signal transduction and activator of transcription-3, affects specific genes to promote liver regeneration. Unraveling the complex processes of liver regeneration may provide novel strategies in the management of patients with end-stage liver disease. In particular, inducing liver regeneration should reduce morbidity for the donor and increase faster recovery for the liver transplantation recipient.

Background, The quantitation of donor leukocyte chimerism may aid in establishing the etiology of neutropenia after liver transplantation.Methods. The incidence and clinical and laboratory characteristics of severe neutropenia were studied in adults who have undergone liver transplantation at our institution over the last 4 years.Results. Severe neutropenia developed in 5 of 156 patients (3%). The clinical and pathological features were nonspecific, In two patients with a delayed diagnosis of graft-versus-host disease (GVHD), donor leukocytes comprised greater than or equal to 50% of the circulating peripheral blood mononuclear cells. In a third patient, an earlier diagnosis of GVBD was suspected on the basis of a donor leukocyte count of 3-10% in the peripheral blood. In contrast, donor leukocyte chimerism was less than or equal to 0.01% in two patients with probable drug-induced neutropeniaConclusions. The determination of donor leukocyte chimerism has an important role in the investigation of neutropenia after liver transplantation, allowing early diagnosis and treatment of GVHD.

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the nonrejector combination of DA donors into PVG recipients. In the present study, the purification of proteins from rat OLT serum taken 60 days after transplantation was performed by affinity chromatography using the anti-LSF-1 polyclonal antibody (pAb). The assessment of column eluates using anti-LSF-1 and OLT serum was studied using rat heart and liver transplantation models. Rejection was not suppressed by the administration of OLT serum in heart or liver allografts. However, heart allografts treated with peak eluates (450 micrograms single shot im, dissolved in Intralipos) taken from the affinity OLT serum survived significantly longer than untreated rats (median = 36.5 days; n = 7 vs 6.5 days; n = 5, respectively, P = 0.011). The same treatment with anti-LSF-1 column eluates also prolonged liver allografts significantly (>200 days) than those in either the untreated group (median = 11 days; n = 7) or those which received only Intralipos (median = 10.5 days; n = 5, P = 0.019). Subsequent analysis of the N-terminal sequences of some of the proteins which were eluted from the affinity column revealed that the homology of a 30-kDa protein was identical to hemoglobin alpha-chain, a 59-kDa protein to granulocyte inhibitory factor, a 70-kDa and a 90-kDa to albumin and its precursor, respectively. Although the specific immunosuppressive component has not been isolated, our results suggested that the anti-LSF-1 column can extract immunosuppressive moiety of LSF-1 from OLT serum.

The number involved in and the rate of migration of donor leucocytes into the following recipient organs (spleen, thymus, bone marrow, lung and mesenteric lymph nodes) were measured in two rat models of orthotopic liver transplantation (OLT) using donor-specific MHC class I antibodies. The first OLT model is one that does not require immunosuppression in order to achieve tolerance and involved the transplantation of DA (MHC haplotype, RT1(a)) livers into PVG (RT1(c)) recipients. The second model was one that required a 7-day (10 mg/kg) treatment with cyclosporin A (CsA) to achieve tolerance and used DA donors into Lewis (RT1(1)) recipients. Recipient organs were biopsied on days 3, 20 and 87 following OLT and donor leucocyte migration was quantified by immunohistochemistry and computer densitometry of immunoblots of detergent-solublized tissues in order to resolve both membrane-bound and soluble donor MHC class I antigen. In a separate experiment, spleen biopsies were taken following OLT on days 3 and 15 from the naturally tolerizing OLT model (DA into PVG), treated with and without CsA for 7 days and compared with the (DA into Lewis) model. The initial rate of leucocyte migration between days 3 and 21 following OLT was found to be the most rapid into the spleen, followed by the bone marrow and mesenteric lymph nodes in the naturally tolerant (DA into PVG) model when compared with the (DA into Lewis) model. The number of donor leucocytes in the spleen and mesenteric lymph nodes in both models was, however, approximately the same by 87 days. No real difference in the rate of leucocyte migration was seen in the thymus or the lung for both transplant models over the time course assayed. CsA was found to lower the rate of donor leucocyte migration only over the period it was administered. The rate of donor leucocyte migration into the spleen was still much lower 15 days after OLT in the (DA into Lewis) model compared with the (DA into PVG) model treated with and without CsA. Thus the differences in the rate of donor leucocyte migration into the spleen, bone marrow and mesenteric lymph nodes immediately following OLT may offer an explanation as to why the (DA into PVG) combination is able to accept a transplanted liver without immunosuppressive therapy.

The effect of serum from orthotopic liver retransplanted rats (re-OLT serum) on graft-versus-host disease (GVHD) was studied in rats. In the re-transplantation model of rat liver, orthotopic liver transplantation (OLT) was carried out in the DA (RT1(a)) into PVG (RT1(c)) combination; two days later the DA liver was removed and a new PVG liver implanted into the same recipient (re-OLT). In the in vivo GVHD model, male PVG rats were sublethally irradiated and injected intravenously with 3 x 10(8) DA or BN (RT1(n)) spleen cells through the penial vein. Within 1 h of the inoculation, rats of the experimental group were injected with 1 mi of re-OLT serum taken at postoperative day (POD) 7. Rats in the control group received 1 mi of normal PVG serum or syngeneic re-OLT serum (PVG-PVG, PVG-PVG). All PVG rats in the control groups died of GVHD within 21 days after the inoculation of DA or BN spleen lymphocytes. However, when the animals were treated with re-OLT serum, 100% (6/6) of the rats survived more than 60 days, following inoculation with DA lymphocytes but not with BN lymphocytes. The POD 7 re-OLT serum showed a strong inhibition against DA anti-PVG mixed lymphocyte reaction (MLR), although re-OLT serum did not contain soluble DA class I antigens, anti-DA class I or II antibody. The potential GVHD inhibitory factors in re-OLT serum may be two unique immunosuppressive proteins, which have been detected by SDS PAGE and reported previously.We conclude that re-OLT serum has immunosuppressive factors, which, at least in part, prevented the induction of GVHD in rats.

The tolerance induced by orthotopic liver transplantation [DA (RT1a) rats to PVG (RT1c) rats] can be prevented by total body irradiation of the donor rat. Reconstitution of the irradiated donor with DA splenic leukocytes reintroduces this tolerance. To investigate the major histocompatibility complex (MHC) specificity of passenger leukocytes, irradiated DA donors were reconstituted by third-party BN (RT1n) splenic leukocytes. The reconstitution with BN splenocytes re-established DA-specific tolerance in PVG recipients, as confirmed by subsequent DA cardiac allografting, while BN hearts were rejected with second-set tempo. To determine which cell components play an important role in re-establishing liver graft tolerance, DA splenic leukocytes were further purified into three types: T, B, and adherent cells. Only "T-cell-enriched" preparations restored liver graft tolerance in three out of five PVG recipients. These results suggest that passenger leukocytes of differing MHC types can help to induce liver-specific tolerance and that T cells in the liver graft may be essential to regulate tolerance induction.

We investigated the effect of a prostacyclin analogue (OP 2507) on PVG (RT1c) recipients subjected to an extended anhepatic phase (AH) and transplanted orthotopically with PVG livers. All of the animals that underwent orthotopic liver transplantation (OLT) with a 20-min AH survived for 1 week with or without OP 2507, (OP) treatment (10/10, 100%). When the AH was lengthened to 45 min, the 1-week survival rate of recipients was poor in the OP-untreated groups (1/10, 10%). Treatment of the recipient with OP 2507, 0.15 micrograms/kg per minute for 30 min, prior to the 45-min AH substantially improved 1-week survival (5/6, 83.3%, P < 0.05). The serum TNF-alpha level at day 1 in OP-treated animals that underwent OLT with a 45-min AH was significantly lower than that in animals with 45-min AH OLT without OP treatment. We conclude that OP 2507 treatment has potential usefulness as a perioperative treatment when the AH is extended during OLT.

In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart grafts in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination. DA (RT1a) donor into PVG (RT1c) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosupressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-1) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 μg of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RT1n) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-1), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In 56 cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the induction of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in vivo results, suggests a role for LSF-1 in the induction of tolerance.

The tolerance induced by orthotopic liver transplantation (OLT) in certain combinations of rat strains can be prevented by total body irradiation (TBI) of the donor. We demonstrate here that the intravenous inoculation of splenic leukocytes into irradiated donors before OLT could re-establish tolerance in association with a state of microchimerism detected in the recipients. When donor DA (RT1a) strain rats were irradiated with 1000 rad 24 h before liver harvesting and subsequent liver implantation into PVG recipients, five out of six rats died from rejection in this normally tolerogenic OLT (DA-PVG) combination. Injection of 1.5 x 10(8) splenic leukocytes from naive DA rats into the irradiated DA donor rats 24 h before OLT restored the tolerogenic potential of the liver allografts. Immunofluorescence assay revealed an increased number of donor (DA) type cells in the PVG recipient bearing a repopulated DA liver, compared to the PVG recipient of an irradiated liver. These results suggest that passenger leukocytes reconstituted by splenic leukocytes have the capacity to protect liver allografts.

Liver grafts between certain rat strain combinations, such as DA (RT1a)-into-PVG (RT1c), are accepted without the use of immunosuppressive agents. To explore the nature and role of serum proteins in liver-induced immunosuppression, we have developed a retransplantation model of rat liver grafting. In this procedure, orthotopic liver transplantation (OLT) is carried out in the DA-into-PVG combination; two days later the DA liver is removed and a new PVG liver implanted into the same recipient (re-OLT). Serum from re-OLT rats was immunosuppressive when tested in mixed lymphocyte reactions (MLR). Three novel proteins were detected in re-OLT serum by SDS-PAGE, with sizes of 180 kD, 87 kD, and 10 kD. The N-terminal sequences of these were distinct and did not match protein sequences in the computer databases, although there was some homology between the 10 kD sequence and the beta-chain of rat hemoglobin. Purified 87 kD and 10 kD proteins were immunosuppressive in MLR; in both cases suppression was dose-dependent and nonstimulator-specific. Production of the 180 kD and 87 kD molecules required the presence of the recipient spleen. We conclude that re-OLT serum contains novel immunosuppressive proteins, which may be products of immune recognition and associated with the immediate termination of graft rejection.

A surgical experience with a method of rat orthotopic liver retransplantation (OLRT), and a preliminary study of immunological responses after OLRT are reported. OLRT was performed on the same recipient after the first orthotopic liver transplantation (1st‐OLT) according to our original (Kamada's) cuff method. Replacement of the portal vein (PV) and infra‐hepatic vena cava (IHVC) cuffs was not technically difficult. However, there were no survivors from the first 6 retransplanted rats, mainly due to complications from defective supra‐hepatic vena cava (SHVC) anastomoses. Unlike the human intra‐abdominal SHVC, the posterior wall of the intraabdominal SHVC in rats is too short and fragile to perform an end‐to‐end anastomosis twice between donor and recipient SHVC. For a second group of seven retransplants, a modification of the SHVC anastomosis was made between donor and recipient SHVC in conjunction with the recipient's cuff diaphragm. This enabled reanastomosis to be secure, resulting in the improved 1‐week survival after isogeneic OLRT (85.7%). This OLRT model has been applied to the fully allogeneic combination for several immunological studies and led to novel findings. Thus, an experimental model of a rat orthotopic liver retransplant model has the potential to allow more valuable insights into the immunological study of chronic rejection, sensitization and chimerism following liver retransplantation. © 1995 Wiley‐Liss, Inc.