Howard N. Sankary

PROTECTIVE EFFECT OF ISCHEMIC PRECONDITIONING ON LIVER PRESERVATION-REPERFUSION INJURY IN RATS

BACKGROUND Ischemic-preconditioning is a process whereby a brief ischemic episode confers a state of protection against subsequent long-term ischemia-reperfusion injury. Ischemic preconditioning has been studied in heart and liver ischemia-reperfusion injury; however, few studies have been performed in the model of preservation-reperfusion injury in liver transplantation. The current study was designed to evaluate the ability of ischemic preconditioning to protect liver grafts from long-term preservation-reperfusion injury. METHODS Male Sprague Dawley rats were used as donors and recipients of orthotopic liver transplantation. Ischemic preconditioning was done by interruption of the portal vein and hepatic artery for 5, 10, and 20 min (5-10, 10-10, and 20-10 groups). Reflow was initiated by removal of the clamp for another 10 min in all groups. The liver was removed and placed in a bath with Euro-Collins solution for different preservation times. Tolerance of the transplanted liver to cold ischemia was determined by survival time and liver function tests. Rat tumor necrosis factor was analyzed by a bioassay. Nomega-Nitro-L-arginine methyl ester, L-arginine, or adenosine was administered to block or stimulate the synthesis of nitric oxide (NO) in the rats that received long-term-preserved liver grafts. RESULTS Twenty percent of syngeneic rats (n=10) that received a liver graft with a 16-hr cold ischemia time in Euro-Collins solution survived for more than 1 day and 10% survived for more than 5 days. In contrast, 87.5% of rats (n=8) that received a liver graft with ischemic preconditioning (10-10 group) and 16 hr of cold ischemia survived for more than 1 day and 75% for more than 5 days. Recipients of liver grafts with ischemic preconditioning had significantly reduced levels of serum aspartate transaminase and tumor necrosis factor-alpha, as well as increased bile flow, compared with recipients of liver grafts without ischemic preconditioning. Blockage of the NO pathway using Nomega-nitro-L-arginine methyl ester, a stereospecific competitive inhibitor of NO formation, attenuated the protective effect of ischemic preconditioning. Administration of one of two precursors of NO synthesis, L-arginine or adenosine, prolonged the survival of rats that received 16-hr-preserved liver grafts. In addition, L-arginine synergized with short-term ischemic pre conditioning (5-10 group) to increase the survival of rats that received a liver graft with a 16-hr cold ischemia time, and the survival rate was 83% after 5 days. Finally, prolonged ischemic preconditioning (> or = 20 min; 20-10 group) resulted in liver damage and loss of function. CONCLUSION The current results show that ischemic preconditioning protects the liver graft from subsequent long-term cold preservation-reperfusion injury in a rat liver transplantation model. The protective role of ischemic preconditioning may be mediated by the endogenous production of NO.

Leflunomide in experimental transplantation. Control of rejection and alloantibody production, reversal of acute rejection, and interaction with cyclosporine.

Leflunomide is a compound recently shown to reduce T and B cell-mediated responses in a number of experimental rat, mouse, and human systems. To explore its potential as an immunosuppressant, we studied leflunomide in 128 Brown-Norway/Lewis cardiac transplants and in 48 unoperated Lewis rats. At doses ranging from 0.63 mg/kg to 10 mg/kg given for 7 days, leflunomide significantly prolonged graft survival compared with controls. When cyclosporine or leflunomide was given for 21 days at a dose of 5 mg/kg, indefinite graft survival occurred in 3/6 animals receiving leflunomide but in none of the 21-day cyclosporine-treated animals. When acute rejection was allowed to develop for four days in untreated rats, leflunomide but not cyclosporine reversed the rejection, returning histology to a normal appearance by seven days. Alloantibody responses measured in microcytoxicity assays as well as total allospecific IgG and IgM in the rejecting animals also were returned to baseline levels by leflunomide but not cyclosporine. When both drugs were used together, a synergistic effect was observed at low doses of both drugs. Pharmacokinetics studies showed that their combined use for up to 28 days did not affect the trough levels of cyclosporine or cyclosporine elimination, suggesting that the synergistic effect was not caused by reduced elimination. The toxicity of each drug was negligible in a group of 32 rats receiving the drugs alone or in combination as measured by serial observation of general appearance, testing of serum ALT, AST, bilirubin, creatinine, white blood cell counts, hemoglobin, and gross necropsy appearance. Weight gain was slightly reduced by both drugs but combined drug use did not alter the pattern. The results of these experiments show leflunomide to be a potent, well-tolerated immunosuppressant, synergistic in its activity with cyclosporine, and would seem to encourage a closer look at this drug for potential use in man.

Leflunomide controls rejection in hamster to rat cardiac xenografts.

Leflunomide is an isoxazole derivative that has the ability to prevent acute rejection of cardiac, renal, and skin transplants in strongly rejecting rat models. Furthermore, leflunomide is able to interact synergistically with CsA to inhibit allograft rejection and also reverse ongoing allograft rejection. In vitro studies suggest that the mechanism of action of leflunomide is via an interruption of cytokine signaling in T cells. This study defines the ability of leflunomide to prevent and reverse rejection of concordant xenografts. One hundred nine adult Lewis rats in 13 groups received abdominal heterotopic cardiac transplants from Golden Syrian hamsters. The xenograft survived 3.9±0.3 days without treatment. When leflunomide was given at 2.5, 5, 10, 15, or 20 mg/kg by gavage daily, the cardiac xenograft survivals were 5.0±0.6, 8.0±3.0, 52.0±20.2, 76.5±21.14, and 58.9±28.1 days, respectively. The survival rates were 4.0±0 and 27.7±28.7 days when CsA was given at 10 and 20 mg/kg i.m., respectively. The combination of CsA at 10 mg/kg with leflunomide at 10 mg/kg or 5 mg/kg prolonged cardiac xenograft survival to 106.0±50.2 days and > 90 days, respectively. There were no observable side effects in the latter combination. Histologic studies of untreated graft hearts 4 days after transplantation revealed infarction of myocardium and severe RBC extravasation. In contrast, the rejected hamster hearts from long-term survivors showed massive mononuclear cell infiltration and myocardium fibrosis in contrast to the early rejected picture. Therapy with leflunomide begun on day 2 reversed these rejection responses by day 6. In addition, the increase in allospecific IgM titers observed on day 2 was reversed, and the allospecific IgM to IgG isotype switch that occurred in untreated animals was prevented by leflunomide. These observations demonstrate that leflunomide, at nontoxic doses, effectively controlled acute rejection of concordant xenografts and synergistic immunosuppressive effect was achieved with leflunomide and CsA.

LEFLUNOMIDE, A NOVEL IMMUNOSUPPRESSIVE AGENT: THE MECHANISM OF INHIBITION OF T CELL PROLIFERATION

Leflunomide is a novel immunomodulating drug that has recently been demonstrated to prevent acute rejection and reverse ongoing rejection of kidney and cardiac allografts in rats. In vitro studies here demonstrate that leflunomide suppresses proliferation of human PBL stimulated with (1) allogeneic PBL in a one-way MLR (50% inhibition with 50–25 μM); (2) anti-CD3 mABs plus PMA (50% inhibition with 70 μM leflunomide); and (3) anti-CD28 mABs plus PMA (50% inhibition with 65 μM leflunomide). In contrast, CsA only inhibited T cell proliferation stimulated by anti-CD3 plus PMA. Leflunomide partially inhibited IL-2 production of T cells stimulated with anti-CD3 plus PMA or anti-CD28 plus PMA, whereas CsA completely inhibited IL-2 production by T cells stimulated by the CD3 pathway and only partially inhibited IL-2 production by T cells stimulated by the CD28 pathway. Because comparable levels of IL-2 were produced by CD28-stimulated T cells treated with either CsA or leflunomide, but no inhibition of proliferation was observed in the CsA-treated cultures, we hypothesized that the lowering of IL-2 levels was not the mechanism by which leflunomide inhibited T cell proliferation. This hypothesis was supported by the observations that exogenous IL-2 failed to restore the T cell proliferation in the presence of leflunomide. Loss of T cell responsiveness to IL-2 in the presence of leflunomide was not due loss of expression of IL-2 receptors. Collectively, our data suggest that inhibition of T cell proliferation by leflunomide occurs via inhibition of responsiveness to IL-2.

Blood and graft eosinophilia as predictors of rejection in human liver transplantation.

This study attempts to define the relationship of blood and graft eosinophilia to acute hepatic allograft rejection. Sixty liver transplant patients were studied for the first 30 days postoperatively, with daily serum bilirubin and liver enzyme levels, white blood cell counts and differential counts, and biweekly core liver biopsies. Graft eosinophilia was established if 7% or greater of the cells infiltrating the portal triads were eosinophils. Blood eosinophilia is an absolute eosinophil count greater than 500 cells/mm3 occurring on any of the 5 days preceding the day of rejection. Acute rejection was diagnosed when 2 days of hepatic allograft dysfunction occurred with histologic evidence of rejection. The 2nd day of dysfunction with appropriate histologic findings was arbitrarily chosen as the day of rejection. Graft eosinophilia predicted rejection with 92% sensitivity and 98% specificity. Blood eosinophilia occurred on the average on the day of rejection and on the 2 preceding days, while graft eosinophilia occurred on the day of rejection and on 1 preceding day. Blood eosinophilia followed by graft eosinophilia specifically occurred in cases of rejection. Blood eosinophilia not followed by graft eosinophilia was not associated with rejection. Following treatment of rejection with high-dose corticosteroids, blood and graft eosinophil counts decreased markedly. In summary: (1) graft eosinophilia is very sensitive and specific for acute hepatic allograft rejection; (2) blood eosinophilia closely precedes and parallels graft eosinophilia specifically during acute hepatic allograft rejection; and (3) elevated blood and graft eosinophil counts are markedly reduced following treatment of rejection with high-dose corticosteroids.

An evaluation of leflunomide in the canine renal transplantation model

Leflunomide is an isoxazole with newly discovered immunosuppressive properties. Its mechanism of action operates later in the cell cycle than cyclosporine and appears to interfere with lymphocyte IL-2 responsiveness. With the encouraging results from in vitro and small-animal studies, we subjected leflunomide to the rigorous canine renal transplantation model in a dose response protocol. Thirty-eight female mongrel dogs underwent renal transplantation and bilateral nephrectomy. Immunosuppression was stratified from controls with no immunosuppression to monotherapy with leflunomide at 2, 4, 8, and 16 mg/kg/day given orally and in a combination therapy with cyclosporine. To evaluate its toxicity while maintaining a low constant blood level, eight dogs were treated by continuous intravenous infusion at doses of 2, 4, 6, and 8 mg/kg/day. The mean survival time for nonimmuno suppressed controls (n=2) was 9 days, leflunomide 2 mg/kg/day (n=2) was 9 days, leflunomide 4 mg/kg/day (n=4) was 16 days, leflunomide 8 mg/kg/day (n=5) was 28 days, leflunomide 16 mg/kg/day (n=7) was 21 days. Cyclosporine alone at 10 mg/kg/day (n=4) resulted in a mean survival time of 13 days. The mean survival time with the combination of cyclosporine 10 mg/kg/day with leflunomide 4 mg/kg/day (n=6) was 68 days. The mean survival time for continuous intravenous leflunomide 2 mg/kg/day (n=2) was 10 days; for leflunomide 4 mg/kg/day, 20 days; for leflunomide 6 mg/kg/day, 14 days; and leflunomide 8 mg/kg/day, 21 days. The mean serum trough levels of leflunomide ranged from 10 μUg/ml at the 2 mg dose to 55 μUg/ml for the 16 mg dose, levels that have been well tolerated in man. Leflunomide at 16 mg/kg/day reliably prevented acute al-lograft rejection, but the dogs died of inanition with normal renal function. Leflunomide at a nontoxic dose of 4 mg/kg/day extended survival to 16 days, but all dogs died of rejection. A combination of inadequate doses of leflunomide (4 mg/kg/day) and cyclosporine (10 mg/kg/day) resulted in all animals having normal renal function and weight for 30 days. Even at a high dose of 16 mg/kg/day, no viral or bacterial infections were noted. These observations in a canine system add to the growing enthusiasm for the evaluation of leflunomide in human transplantation.

Pharmacologically induced regression of chronic transplant rejection.

Chronic rejection, characterized by a progressive obliterative arteriopathy, is a major cause of graft failure in long-surviving human transplants for which there is no effective treatment. Leflunomide, an isoxazol derivative, has been shown to be a novel immunomodulatory drug that profoundly suppresses the immune response. In this study, 58 Fisher-344 rats received cardiac transplantation from Lewis rats. All the recipients were given CsA at 2.5 mg/kg for 5 days postoperatively. Without further treatments, the arterial intima was progressively injured by mononuclear cell infiltration and Ab deposition. Smooth muscle cell and fibroblast proliferation in the intima became a predominant phenomenon by day 90. CsA was ineffective in controlling the progress of arterial intimal thickening when treatment began on day 30. Leflunomide at 5 mg/kg failed to control arterial intimal thickening by day 60 when therapy began on day 30. However, the progress of arterial intimal thickening was significantly inhibited by day 90 when the dosage of leflunomide had been increased to 10 mg/kg on day 60. Combined therapy with leflunomide and CsA at 5 mg/kg for 30 days dramatically reversed the arterial thickening by day 60. After increasing the dosages of both leflunomide and CsA to 10 mg/kg on day 60, the combination therapy steadily controlled the chronic rejection. Only the combination therapy significantly down-regulated circulating antidonor IgM and IgG titers. In rat smooth muscle cell culture, this same drug combination had a synergistic inhibitory effect on proliferation. Therefore, the combination therapy of leflunomide and CsA could reverse and control the progress of chronic rejection, while leflunomide, at higher dosage as a monotherapy, could stabilize chronic rejection in this model. The mechanism of the regression of chronic rejection by leflunomide and cyclosporine may be related to their in vitro abilities to control not only lymphocyte but smooth muscle cell proliferation, as well. The synergistic effect of these two drugs on vascular smooth muscle cell proliferation in vitro may be an important part of this novel activity. This unique feature holds intriguing possibilities for treating established chronic rejection.

Delayed xenograft rejection in the concordant hamster heart into Lewis rat model

The inability to provide an adequate supply of human organs for clinical transplantation has created a strong interest in the use of nonhuman, especially nonprimate, organs. The first biological obstacle confronting such discordant transplantations is a series of violent reactions that result in hyperacute rejection of the xenograft. Significant advances in controlling hyperacute rejection have been achieved recently through the generation of transgenic pig donors bearing human complement regulatory proteins. However, when hyperacute rejection is averted, the xenografts are rejected in 2-70 days in spite of high-dose immunosuppression, by a process collectively termed delayed xenograft rejection. Delayed xenograft rejection is characterized by a refractoriness to conventional immunosuppression, extensive xenoreactive antibody deposition, and cellular infiltration that is dominated by macrophages. We have examined the features of extended host and graft response in the concordant hamster-to-rat xenotransplant model, where such features have historically been obscured by early graft destruction. Hamster hearts transplanted into rats do not encounter hyperacute rejection but are rejected within 3-4 days when xenoreactive antibody titers rise exponentially to levels that elicit a classical antibody- and complement-mediated acute xenograft rejection. We have successfully blocked acute xenograft rejection by a combination of immunosuppressive agents, leflunomide, and cyclosporine. Stopping the immunosuppression resulted in graft rejection that is histologically characterized by extensive xenoreactive antibody deposition and cellular infiltration that is predominantly composed of macrophages. We have noted the similarities between the histopathology of rejection of long-surviving concordant xenografts and that described for discordant xenografts and refer to the process of rejection of concordant grafts that have escaped acute xenograft rejection, delayed xenograft rejection.

Biliary Strictures in Hepatic Transplantation

Between August 1985 and December 1990, 198 liver transplantations were performed. Among 18 patients, 20 biliary strictures were identified, which were categorized as anastomotic (n = 6), nonanastomotic central hilar (n = 8), and nonanastomotic peripheral (n = 6). Pretransplant disease, hepatic artery patency, presence of acute or chronic rejection, and donor cold ischemia times were tabulated for each case. Among the six patients with peripheral strictures, three had sclerosing cholangitis prior to transplantation. Three patients with nonanastomotic strictures experienced chronic rejection. The mean cold ischemia time for patients with nonanastomotic strictures was 9.75 hours versus 8.1 hours for nonstrictured transplants (P = .025). Balloon dilation was performed in 13 patients; follow-up longer than 6 months was available for nine patients. Dilation was successful in four cases. Among the five failures, only one patient has needed surgery. An association was noted between nonanastomotic biliary strictures and prolonged donor cold ischemia time, between peripheral nonanastomotic strictures and pretransplant sclerosing cholangitis, and between nonanastomotic strictures and chronic rejection. Percutaneous balloon dilation was found useful in the treatment of the strictured transplant.

The use of granulocyte colony-stimulating factor after liver transplantation

Granulocyte colony-stimulating factor (G-CSF) increases the number of circulating granulocytes and decreases TNF production while improving survival in sepsis models. To study the effects of G-CSF administration on sepsis and rejection, 37 primary liver allograft recipients received intravenous recombinant human G-CSF (rhG-CSF; 5–10 μg/kg/day) for the first 7–10 days following transplantation, targeting a blood absolute granulocyte count of between 10,000 and 20,000 cells/mm3. These recipients were monitored prospectively for sepsis and rejection, as were the previous 49 primary liver allograft recipients who did not receive G-CSF. Both groups utilized identical protocol immunosuppression and standardized diagnosis and treatment of sepsis and rejection. Univariate and logistic regression analysis of risk factors for sepsis and rejection revealed no difference between the two patient groups. G-CSF-treated patients developed an increased absolute granulocyte count over time (P<0.0001, repeated-measures analysis of variance). G-CSF-treated patients had a decreased number of sepsis episodes per patient (0.92±.5 vs. 2.18±2.8, P<0.02, t test), and a lower percentage of sepsis-related deaths (8% vs. 22%, P<0.04, chi-square test). The incidence of acute rejection was decreased in the G-CSF-treated group (22% vs. 51%, P<0.01, chi-square test). These pilot data support further investigation into G-CSFs favorable effects on sepsis and rejection.