Liver Isografts Research Articles

Cholangiocyte Epithelial to Mesenchymal Transition (EMT) is a potential molecular mechanism driving ischemic cholangiopathy in liver transplantation.

Donation after circulatory death (DCD) has expanded the donor pool for liver transplantation. However, ischemic cholangiopathy (IC) after DCD liver transplantation causes inferior outcomes. The molecular mechanisms of IC are currently unknown but may depend on ischemia-induced genetic reprograming of the biliary epithelium to mesenchymal-like cells. The main objective of this study was to determine if cholangiocytes undergo epithelial to mesenchymal transition (EMT) after exposure to DCD conditions and if this causally contributes to the phenotype of IC. Human cholangiocyte cultures were exposed to periods of warm and cold ischemia to mimic DCD liver donation. EMT was tested by assays of cell migration, cell morphology, and differential gene expression. Transplantation of syngeneic rat livers recovered under DCD conditions were evaluated for EMT changes by immunohistochemistry. Human cholangiocytes exposed to DCD conditions displayed migratory behavior and gene expression patterns consistent with EMT. E-cadherin and CK-7 expressions fell while N-cadherin, vimentin, TGFβ, and SNAIL rose, starting 24 hours and peaking 1–3 weeks after exposure. Cholangiocyte morphology changed from cuboidal (epithelial) before to spindle shaped (mesenchymal) a week after ischemia. These changes were blocked by pretreating cells with the Transforming Growth Factor beta (TGFβ) receptor antagonist Galunisertib (1 μM). Finally, rats with liver isografts cold stored for 20 hours in UW solution and exposed to warm ischemia (30 minutes) at recovery had elevated plasma bilirubin 1 week after transplantation and the liver tissue showed immunohistochemical evidence of early cholangiocyte EMT. Our findings show EMT occurs after exposure of human cholangiocytes to DCD conditions, which may be initiated by upstream signaling from autocrine derived TGFβ to cause mesenchymal specific morphological and migratory changes.

Gene expression profiling of acute cellular rejection in rat liver transplantation using DNA microarrays

Acute cellular rejection (ACR) is still a major problem in organ transplantation, and its genetic and molecular mechanisms remain poorly understood. We used DNA microarrays to investigate the gene expression profiles in ACR. We hypothesized that changes of gene expression in grafts could also be detected in peripheral blood leukocytes. We first compared the gene expression profiles in liver isografts (Lewis to Lewis) and allografts (Dark Agouti to Lewis) harvested from rats at days 1, 3, 5, and 7 after transplantation. Hierarchical clustering analysis indicated that gene expression started to change on day 3, and 89 differentially expressed genes were extracted from allografts in comparison with isografts at day 3. Most of the up-regulated genes were associated with graft-infiltrating leukocytes. We then confirmed the similarity of gene expression changes in peripheral leukocytes by quantitative real-time polymerase chain reaction. We also investigated the gene expression changes in other inflammatory and liver dysfunction models. Two interferon-gamma inducible genes, interferon regulatory factor 1 and guanylate nucleotide binding protein 2, were overexpressed in both the peripheral leukocytes and liver graft during ACR. Although further studies are necessary, these 2 genes in peripheral leukocytes could be potentially useful markers for rejection or immunosuppression.

Hepatic Regenerative Response in Small-Sized Liver Isografts in the Rat

To investigate hepatic regenerative response and associated mechanisms in different-size liver grafts in the rat. Rat models of different-size-graft liver transplantation (whole, 50%-size, or 30%-size) were established, with a sham operation group serving as a control. Portal pressure, graft injury, interleukin 6 (IL-6), signal transducer and activator of transcription (Stat3), mitogen-activated protein kinase (MAPK), cyclin D1, and proliferating cell nuclear antigen (PCNA) were all assessed. The portal pressure was significantly higher and hepatic injury more severe in the smaller sized groups than in the whole graft group, especially in the 30%-size grafts. Hepatic IL-6 and tumor necrosis factor-alpha (TNF-alpha) levels in the two smaller sized groups were significantly higher than in the whole graft group, while IL-6 levels appeared to be negatively associated with graft sizes. Downstream markers of IL-6, Stat3 and MAPK phosphorylation, cyclin D1, and PCNA expression were also markedly increased in the small-sized grafts compared with the whole grafts, and appeared to positively correlate with early measurements of portal pressure and subsequent hepatic injury. Vigorous hepatic regeneration in small-for-size liver grafts may be associated with highly activated IL-6/Stat3 and MAPK signaling, which may in turn correlate with graft size, portal pressure, and hepatic injury.

Overexpression of indoleamine dioxygenase in rat liver allografts using a high-efficiency adeno-associated virus vector does not prevent acute rejection

The aim of this study was to evaluate the ability of local overexpression of indoleamine dioxygenase (IDO) to abrogate rat liver transplant rejection by the use of an adeno-associated virus vector [recombinant adeno-associated virus 2/8 (rAAV2/8)] to deliver the transgene to the allograft prior to transplantation. A green fluorescent protein (GFP)-expressing vector [recombinant adeno-associated virus 2/8-liver-specific promoter 1-enhanced green fluorescent protein (rAAV2/8-LSP1-eGFP)] was used to examine the kinetics of expression and optimal dosing for transduction of Piebald Virol Glaxo (PVG) rat livers. A vector encoding the rat IDO gene (rAAV2/8-LSP1-rIDO) was constructed and tested by its ability to induce tryptophan catabolism and kynurenine production in vitro and in vivo. PVG donor rats were injected, via the portal vein, with rAAV2/8-LSP1-rIDO 2 weeks before transplantation into PVG strain isograft or Lewis (LEW) strain allograft recipients. With the enhanced GFP vector, 29.5% and 47.4% of hepatocytes were found to express GFP at 3 and 6 weeks after injection, respectively. In untransplanted PVG animals, the rAAV2/8-LSP1-rIDO vector induced, 3 weeks after administration, a 1.8-fold increase (P = 0.0161) in liver IDO activity, which was associated with a fall in serum tryptophan to 0.5 times the baseline level (P < 0.001). PVG recipients of PVG liver isografts pretreated with the IDO-expressing vector had a 45% lower level of serum tryptophan than recipients of isografts pretreated with the GFP-expressing vector (P = 0.03). LEW recipients of PVG liver allografts pretreated with the rat IDO vector had a median survival time of 12 days, whereas recipients of allografts pretreated with rAAV2/8-LSP1-eGFP had a median survival time of 13 days (P = 0.38). Both groups displayed similar histological features of acute cellular rejection. In conclusion, rAAV2/8 vectors produce highly efficient, though delayed, hepatocyte transduction in vivo and provide a useful gene delivery tool for transplantation models. However, gene delivery using IDO was unsuccessful in prolonging rat liver allograft survival.

Downregulation of Cytokine Release by Heat Preconditioning of Livers Used for Transplantation in Rats

We evaluated the changes in the levels of released cytokines following heat preconditioning of the livers used in rat liver transplantation. The donor rats in the heat preconditioning (HP) group were subjected to heat preconditioning 48 h before graft harvesting. The liver isografts were preserved in Euro-Collins solution for 8 h, and then transplanted orthotopically. The one-week survival rate of the HP group was significantly better than that of the control (C) group. The serum levels of interleukin-6 and interleukin-10 were significantly lower in the HP group than in the C group. Histological staining revealed that the stagnation of red blood cells and infiltration of neutrocytes were reduced in the HP group. The expression of intercellular adhesion molecule-1 was decreased around the central vein in the HP group, as revealed by immunohistochemistry. These results indicate that heat preconditioning downregulates cytokine release and reduces the frequency of microcirculation disorders.

Activation pattern of mitogen-activated protein kinases in early phase of different size liver isografts in rats

Mitogen-activated protein kinases (MAPK) play a pivotal role in ischemia reperfusion injuries of heart and liver, but the activation pattern of MAPKs in the early phase of different size liver isografts remains unclear. The experiment is designed to investigate the activation pattern and role of MAPKs in isografts of the rat with different size liver transplantation. The animal models of different size graft liver transplantation (whole graft, 50% size, or 30% size, respectively) were established and the sham operation group served as a control. The recipients were sacrificed at 0.5-, 2-, 6-, and 24-hour time points after transplantation to harvest the graft specimens and blood samples. The serum aspartate amino transferase (AST), alanine amino transferase (ALT) and tumor necrosis factor-alpha (TNF-alpha) levels, and histological findings were evaluated. The expressions of the total and phosphorylated p46/p54 JNKs, p38 MAPK, and p42/p44 ERKs were detected by Western blot. The serum ALT and AST levels increased significantly at the 0.5-hour time point and maintained high with the peak levels at the 6-hour time point after liver transplantation. The different sizes of liver isografts did not change the expressions of total p46/p54JNKs, p38MAPK, and p42/p44 ERKs. While the expressions of phosphorylated p46/p54JNKs, p38 MAPK, and p42/p44 ERKs were either negative or mildly up-regulated in the sham operation group, they were significantly activated in the transplanted liver at the 0.5-hour time point, especially in the 30% size liver transplantation group. In conclusion, the activation of three MAPKs in liver isografts correlates with graft size and the JNK and p38 MAPK are responsible for the graft injury while the ERK signal pathway maybe participate in the regulation of cell growth and differentiation after small-for-size liver transplantation.

Novel technique for suprahepatic vena cava reconstruction in rat orthotopic liver transplantation

In previous studies, the suture technique and the cuff method were applied to anastomoses of the suprahepatic vena cava (SHVC) in rat orthotopic liver transplantation. However, the anastomosis of SHVC is difficult during transplantation because of the short length of the SHVC. Here, we developed a novel method for anastomoses of SHVC, using a veno-lined stent technique. The veno-lined stent for SHVC anastomosis was prepared after the donor operation. The special veno-lined stent was a 4.0-mm-long polythene tube in which a venous segment from the donor was lined. During the recipient operation, the donor SHVC was anastomosed end-to-end to the recipient SHVC, using the veno-lined stent. Anastomoses of the portal vein and infrahepatic vena cava were performed using a cuff technique. Continuity of the bile duct was established using a stent. The hepatic artery was ligated, and the graft was not arterialized. As controls, the unlined stent, the suture technique, and the cuff method were also used for SHVC anastomoses, respectively, as three control groups with the identical procedures above. In total, 30 orthotopic liver isografts were performed using the veno-lined stent technique. The survival rate was 90% (27/30) after 1 week and 70% (21/30) after 2 months, with normal hepatocellular function. The SHVC anastomosis using a veno-lined stent took 10 +/- 2 (mean +/- SD) min. The anhepatic phase, recipient operative time, and complete operation time were about 14 +/- 2 min, 40 +/- 5 min, and 120 +/- 10 min, respectively. However, in the nonlined stent control group, a total of 20 orthotopic liver isografts used the nonlined stent for SHVC anastomoses, and all failed because of venous thrombosis in SHVC; none survived over 1 week. In the suture technique control group, 40 orthotopic liver isografts were performed using a suture technique for SHVC anastomoses. The results showed no significant difference with those of the veno-lined stent method. But in the cuff method control group, of all 20 orthotopic liver isografts performed using the cuff method for SHVC anastomoses, 10 failed because of failed ligation on the anastomostic site. The survival rates at 1 week and 2 months postoperatively were significantly different from those of the veno-lined stent method. The veno-lined stent technique provides a novel, simple, and reliable method for SHVC anastomoses. It avoids bleeding during suture and the ligation difficulties found with the cuff method. The operation's success rate is satisfactory. This model is successful, and could be applied in various experimental studies.

Therapeutic potential of interleukin-6 in preventing obesity- and alcohol-associated fatty liver transplant failure

Donor organ shortage significantly hinders orthotopic liver transplantation therapy, the only effective treatment for chronic end-stage liver disease and acute liver failure. Further complicating this matter is the prevalence of steatosis in 13% to 50% of donor livers obtained from obese and alcoholic individuals. When transplanted, these livers are associated with primary nonfunction and an elevated risk of dysfunction. New therapeutic approaches to render marginal fatty livers worthy for clinical transplantation are actively being sought. Study findings obtained from my group show that in vitro treatment with interleukin-6 (IL-6) dramatically reduces mortality, liver injury, and necrapoptosis in steatotic Zucker rat liver isografts. Findings of additional studies indicate that IL-6 induces hepatoprotection of steatotic liver isografts by preventing sinusoidal endothelial cell damage and, consequently, the amelioration of hepatic microcirculation, and by protecting against hepatocyte death, which is likely mediated through activation of signal transducer and activator of transcription 3/Bcl-x L. Finally, in vitro IL-6 treatment also prevents mortality associated with alcoholic fatty liver transplants. Relative to the protective effect of IL-6 on steatotic Zucker rat liver, IL-6 is less effective in alcoholic fatty livers, which may be due to the inhibitory effects of ethanol on IL-6 activation of signal transducer and activator of transcription 3 in hepatocytes and sinusoidal endothelial cells. Collectively, these results support the assertion that in vitro IL-6 treatment of steatotic livers may render allografts usable for clinical transplantation, thereby decreasing the gap between the short supply of cadaver liver allografts and high demands for replacement livers. Higher concentrations of IL-6 may be required to protect against alcoholic fatty liver isograft injury because alcohol inhibits IL-6 signaling in the liver.

Protective effect of matrine on sinusoidal endothelial cells of rat liver isograft

Protective effect of matrine on sinusoidal endothelial cells of rat liver isograft

In vitro interleukin-6 treatment prevents mortality associated with fatty liver transplants in rats

Background & aims: Orthotopic liver transplantation is currently the only curative therapy for chronic end-stage liver disease and acute liver failure. However, a scarcity of cadaveric donors has led to a critical shortage of organs available for transplant. This is further complicated by the prevalence of steatosis in about 13%–50% of donor livers, which is associated with a high risk of dysfunction and primary nonfunction. Methods: Steatotic Zucker rat livers and livers from alcohol-fed rats were transplanted into lean control rats. Liver injury, activation of survival signals, and hepatic microcirculation were compared in nontreated and interleukin-6 (IL-6)-treated steatotic isografts. Results: IL-6 pretreatment of steatotic Zucker rat liver isografts dramatically reduces mortality and liver injury following transplantation. Reperfusion after transplantation induces significant sinusoidal endothelial cell necrapoptosis in steatotic Zucker rat liver isografts, which is prevented by in vitro IL-6 pretreatment. IL-6 treatment activates cell survival signal transducer and activator of transcription factor 3 (STAT3) in hepatocytes and sinusoidal endothelial cells. Laser Doppler imaging and microsphere analyses demonstrate that IL-6 treatment markedly improves hepatic microcirculation, which is impaired in steatotic Zucker rat liver transplants. Finally, in vitro IL-6 treatment of donor livers also markedly reduces mortality associated with fatty liver transplants from alcohol-fed rats. Conclusions: IL-6 induces hepatoprotection of steatotic liver isografts via preventing sinusoidal endothelial cell necrapoptosis and consequent amelioration of hepatic microcirculation, and protecting against hepatocyte death. IL-6 pretreatment of steatotic livers may render such allografts useable for clinical transplantation.

Single vs Dual Vessel Porcine Extracorporeal Liver Perfusion

Background. The use of porcine extracorporeal liver perfusion (PECLP) to provide temporary hepatic support for patients in fulminant hepatic failure has been limited by the fact that individual perfusions can be sustained for only a few hours. Inadequate liver function and/or hemodynamic instability are the major contributing factors for early interruption of PECLP. Recent reports suggest that the choice of single (portal vein only) vs dual (portal vein and hepatic artery) vessel perfusion may influence the duration of perfusion. We hypothesize that PECLP with single vessel perfusion (SVP) is associated with worse liver function and greater hemodynamic instability than PECLP with dual vessel perfusion (DVP).Materials and methods. To eliminate the potentially confounding influences of liver failure and xenograft rejection, liver isografts procured from White-Landrace pig donors were perfused by either SVP or DVP via an extracorporeal circuit established with normal White-Landrace pig recipients. The function of perfused livers was evaluated by measuring production of bile and Factors V and VIII, clearance of ammonia and lactate, and extraction of O2 at baseline and at 0, 1, 3, 6, 12, and 24 h after initiation of PECLP. The impact of PECLP on recipient hemodynamic status was assessed by monitoring BP, heart rate, urine output, O2 saturation, etc. Among other parameters evaluated were serum albumin and total protein and hepatic release of IL-1β and nitric oxide to assess their possible contributions to hemodynamic instability.Results. DVP and SVP livers cleared ammonia and lactate similarly. Both approaches were associated with progressive hypoalbuminemia and hypoproteinemia. DVP livers produced more bile and Factor V and were associated with less recipient hypotension and IL-1β and NO release than SVP livers.Conclusions. Livers with DVP function better than livers with SVP. The duration of PECLP can be limited by recipient hypotension, although this complication is less severe with DVP than with SVP.

New approaches to inducing the death of alloreactive lymphocytes

New approaches to inducing the death of alloreactive lymphocytes

Protective effect of heat preconditioning of rat liver graft resulting in improved transplant survival.

The protective effect of heat preconditioning of the liver against ischemia-reperfusion injury has been reported mostly in models of transient ischemia in relation to heat shock protein 70 (HSP70). We estimated the effect of heat preconditioning of liver grafts on the transplant survival rate and on apoptosis of sinusoidal endothelial cells (SEC) as well as hepatocytes in a rat model of liver transplantation. Donor rats of the heat shock (HS) group were subjected to heat preconditioning 48 hr before graft harvest, and HSP70 levels were estimated by. Western blot analysis and immunohistochemistry. The liver isografts from the HS group and control (C) group were preserved in Euro-Collins solution for 6 or 8 hr and transplanted orthotopically. Serum hyaluronic acid and alanine aminotransferase were measured, and apoptosis of the SEC and hepatocytes was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling staining and electron microscopy. HSP70 expression was detected not only in hepatocytes but also in SEC. In the 8-hr preservation model, the 1-week survival rate was 60% in the HS group and 0% in the C group. Serum hyaluronic acid and alanine aminotransferase levels in the HS group were significantly lower than those in the C group at 3 hr after reperfusion, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling-positive SEC in the C group (35.2%) was markedly increased compared with the HS group (10.1%). Electron microscopic examination confirmed the features of apoptosis of SEC. Heat preconditioning of the graft improved the survival rate of the liver transplants. Induction of HSP70 in SEC as well as in hepatocytes might attenuate preservation-reperfusion injury by inhibiting apoptosis of SEC.

LIVER PRESERVATION: A COMPARISON OF CELSIOR TO COLLOID-FREE UNIVERSITY OF WISCONSIN SOLUTION1

Celsior (CEL) was formulated specifically for heart preservation. Recently some preliminary reports have suggested that CEL is also effective for liver preservation. In this study liver preservation with CEL was compared to colloid-free University of Wisconsin solution (MUW). Arterialized rat liver isografts were flushed and stored for 24 hr at 0 degrees C in CEL or MUW before orthotopic transplantation. Plasma albumin, bilirubin, glucose, aspartate aminotransferase, and alkaline phosphatase were measured 1, 2, 3, 7, 14, 21, and 28 days after surgery. All recipients of MUW-preserved livers survived, none of the recipients of CEL-preserved grafts lived beyond 3 days. On day 1, AST was raised in all rats but rats receiving CEL-preserved liver grafts were also markedly hypoglycemic, hypoalbuminemic and had elevated alkaline phosphatase. Celsior is not an effective solution for long-term liver preservation in its present composition.

Adenoviral-mediated gene delivery to liver isografts: improved model of ex vivo gene transfer

Adenoviral-mediated gene delivery to liver isografts: improved model of ex vivo gene transfer

Reduction of hepatic ischemia/reperfusion injury by a soluble P-selectin glycoprotein ligand-1.

The authors' goal was to determine the effects of specific binding and blockade of P- and E-selectins by a soluble P-selectin glycoprotein ligand-1 (PSGL-1) in rat models of hepatic in vivo warm ischemia and ex vivo cold ischemia. The authors also sought to determine the effect of selectin blockade on isograft survival in a syngeneic rat orthotopic liver transplant model. Ischemia/reperfusion (I/R) injury is a major factor in poor graft function after liver transplantation, which may profoundly influence early graft function and late changes. It is hypothesized that I/R injury leads to the upregulation of P-selectin, which is then rapidly translocated to endothelial cell surfaces within 5 minutes of reperfusion of the liver, initiating steps leading to tethering of polymorphonuclear neutrophil leukocytes to the vascular intima. Local production by leukocytes of interleukin-1, tumor necrosis factor-alpha, or both induces P-selectin expression on the endothelium and continues the cascade of events, which increases cell adherence and infiltration of the organ. To examine directly the effects of selectins in a warm hepatic I/R injury model, 100 microg of PSGL-1 or saline was given through the portal vein at the time of total hepatic inflow occlusion. The effects of PSGL-1 in cold ischemia were assessed using an isolated perfused rat liver after 6 hours of 4 degrees C storage in University of Wisconsin (UW) solution, with or without the instillation of PSGL-1 before the storage. To evaluate the effect of selectin blockade on liver transplant survival, syngeneic orthotopic liver transplants were performed between inbred male Sprague-Dawley rats after 24 hours of cold ischemic storage in UW solution. A separate group of animals received two doses of 100 microg of PSGL-1 through the portal vein before storage and before reperfusion of the transplanted liver. Recipient survival was assessed at 7 days, and the Kaplan-Meier product limit estimate method was used for univariate calculations of time-dependent recipient survival events. In an in vivo warm rat liver ischemia model, perfusion with PSGL-1 afforded considerable protection from I/R injury, as demonstrated by decreased transaminase release, reduced histologic hepatocyte damage, and suppressed neutrophil infiltration, versus controls (p < 0.05). When cold stored livers were reperfused, PSGL-1 reduced the degree of hepatocyte transaminase release, reduced neutrophil infiltration, and decreased histologic hepatocyte damage (p < 0.05 vs. UW-only controls). On reperfusion, livers treated with PSGL-1 demonstrated increased portal vein blood flow and bile production (p < 0.05 vs. UW-only controls). In addition, 90% of the rats receiving liver isografts stored in UW solution supplemented with PSGL-1 survived 7 days versus 50% of those whose transplanted syngeneic livers had been stored in UW alone (p < 0.05). Selectins play an important role in I/R injury of the liver. Early modulation of the interaction between P-selectin and its ligand decreases hepatocyte injury, neutrophil adhesion, and subsequent migration in both warm and cold rat liver ischemia models. In addition, the use of PSGL-1 before ischemic storage and before transplantation prevents hepatic injury, as documented by a significant increase in liver isograft survival. These findings have important clinical ramifications: early inhibition of alloantigen-independent mechanisms during the I/R damage may influence both short- and long-term survival of liver allografts.

Impairment of Hepatic Mitochondrial Respiratory Function Following Storage and Orthotopic Transplantation of Rat Livers

Prolonged storage of organs for transplant results in tissue damage which may be compounded on reperfusion of the graft tissue. The effect of storage times was examined on hepatic mitochondrial oxygen consumption and activities of complexes I, II–III, IV, and V in mitochondria isolated from rat liver isografts stored for 25 min and 24 h pre- and posttransplantation. While Complex I activity was significantly (P< 0.05) inhibited under all the conditions studied, Complex II–III activity was only significantly (P< 0.05) reduced following transplantation of 24-h stored tissue. Complex IV activity remained unchanged under all the conditions studied. Although Complex V activity was significantly damaged within the first 25 min of ischemia, activity values were partially recovered to control levels following 3 h of reperfusion after transplantation. Prolonged (24 h) storage induced decreases in Complex V activity which were irrecoverable. Mitochondria subjected to 25 min ischemia alone also showed a significant (P< 0.01) decrease in NAD+-linked respiratory control indices due to a stimulated state 4 rate. The 24-h storage and transplantation brought about a significantly (P< 0.001) greater inhibition of respiratory control and state 3 respiration. FAD-linked respiration parameters were significantly (P< 0.05) affected in livers subjected to prolonged (24 h) storage or transplantation. These data suggest that a loss of membrane integrity coupled with an inhibition of Complexes I and V and an involvement of Complex II–III in 24-h stored hepatic transplants accounts for mitochondrial respiratory dysfunction in hepatic transplantation injury. No indication of Complex IV damage was found in this study. This study shows that damage to specific mitochondrial complexes occurs as a consequence of hypothermic ischemic injury.

Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction.

MHC-mismatched liver grafts are accepted spontaneously between many mouse strains. The underlying mechanism(s) is unclear. In the B10 (H2(b)) to C3H (H2(k)) strain combination used in this study, donor T cells within the liver were rapidly replaced within 2 to 4 days of transplantation with those of the recipient. Freshly isolated liver graft-infiltrating cells harvested on days 4 and 7 exhibited strong CTL responses against donor alloantigens. CTL activity was reduced substantially, however, by day 14, although levels of CTL precursors in the spleen and liver remained high. Examination of the liver allografts by in situ terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick end labeling on days 4, 7, and 14 after transplantation revealed prominent apoptotic cells dispersed throughout the nonparenchymal cell population. When acute liver allograft rejection was induced by administration of IL-2 from days 0 to 4 post-transplant (median survival time, 5 days), apoptotic activity (day 4) was reduced substantially, whereas CTL activity was enhanced. Nonparenchymal cells isolated from allografts of unmodified recipients 4, 7, and 14 days after transplantation exhibited significantly higher DNA fragmentation after 18-h culture than cells from liver isografts. Moreover, the level was 4 to 5 times higher than that of cells from IL-2-treated mice (on day 4). These observations suggest that T cell deletion, not regulation, may be responsible for spontaneous liver allograft acceptance. The molecular recognition events that cause apoptosis of infiltrating T cells and why this occurs within liver grafts, but not heart or skin grafts, remain to be elucidated.

Paracrine enhancement of porcine to murine islet xenograft survival by simultaneous fetal pancreas and fetal liver isografts.

Paracrine enhancement of porcine to murine islet xenograft survival by simultaneous fetal pancreas and fetal liver isografts.

Rejection of spontaneously accepted rat liver allografts with recipientinterleukin-2 treatment or donor irradiation.

While MHC incompatible DA (RTl(a)) to Lewis (RT1(1), LEW) rat liver allografts are acutely rejected, the reciprocal LEW to DA liver grafts are spontaneously accepted. The mechanism of this acceptance remains unclear. We evaluated the effect of donor treatment with total body irradiation (TBI) or gadolinium chloride (GdCl3), and recipient treatment with exogenous IL-2 after transplantation on the survival of the spontaneously accepted liver grafts. Male LEW and DA rats were used as donors and recipients for orthotopic liver allo- or iso-graft transplants. The LEW liver donor was treated by TBI (10 gray) 7 days before transplantation, or LEW donor Kupffer cell phagocytosis was blocked with GdCl3 (7 mg/kg) on days -2 and -1 pretransplant. In an attempt to reverse LEW liver graft acceptance, 180,000 units human IL-2 (hIL-2) were administered daily IP to the DA liver recipients from days 1 to 7 after liver grafting. While untreated LEW recipients rejected DA liver grafts within 13 days, DA recipients accepted LEW livers indefinitely (>302 days). In contrast, irradiation of the LEW liver donor prevented the spontaneous acceptance by DA recipients, and resulted in acute rejection of the liver grafts in 9-20 days. However, spontaneous graft tolerance was restored by parking the irradiated LEW donor liver in naive LEW rats for 48 hr before retransplantation to DA recipients (>50 days). When LEW donors were treated with GdCl3, which is known to block Kupffer cell phagocytosis and antigen processing, the spontaneous acceptance of the LEW liver grafts by DA recipients was unaffected. However, when exogenous rhIL-2 was given daily, LEW liver allografts were rejected by the DA recipients. The resulting liver failure correlated with a progressive increase in serum bilirubin and the development of a predominantly lymphocytic portal tract infiltration, bile duct epithelial damage, and portal vein endothelitis, which is consistent with acute allograft rejection. LEW and DA recipients of liver isografts developed no toxicity and survived indefinitely (>100 days) when treated with the same dose of IL-2. These results indicate that spontaneous rat liver allograft acceptance is associated with the presence of radiosensitive cells in the donor liver that may interact with recipient T cells to inhibit (Th1) production of IL-2.