Donor Pretreatment Research Articles

Intravenous glycine improves survival in rat liver transplantation.

In situ manipulation by touching, retracting, and moving liver lobes gently during harvest dramatically reduces survival after transplantation (P. Schemmer, R. Schoonhoven, J. A. Swenberg, H. Bunzendahl, and R. G. Thurman. Transplantation 65: 1015-1020, 1998). The development of harvest-dependent graft injury upon reperfusion can be prevented with GdCl3, a rare earth metal and Kupffer cell toxicant, but it cannot be used in clinical liver transplantation because of its potential toxicity. Thus the effect of glycine, which prevents activation of Kupffer cells, was assessed here. Minimal dissection of the liver for 12 min plus 13 min without manipulation had no effect on survival (100%). However, gentle manipulation decreased survival to 46% in the control group. Furthermore, serum transaminases and liver necrosis were elevated 4- to 12-fold 8 h after transplantation. After organ harvest, the rate of entry and exit of fluorescein dextran, a dye confined to the vascular space, was decreased about twofold, indicating disturbances in the hepatic microcirculation. Pimonidazole binding, which detects hypoxia, increased about twofold after organ manipulation, and Kupffer cells isolated from manipulated livers produced threefold more tumor necrosis factor-alpha after lipopolysaccharide than controls. Glycine given intravenously to the donor increased the serum glycine concentration about sevenfold and largely prevented the effect of gentle organ manipulation on all parameters studied. These data indicate for the first time that pretreatment of donors with intravenous glycine minimizes reperfusion injury due to organ manipulation during harvest and after liver transplantation.

ADENOSINE DEAMINASE INHIBITION INCREASES INTERSTITIAL ADENOSINE CONCENTRTIONS, POST-REPERFUSION BLOOD FLOW AND SURVIVAL IN RAT LIVERS

335 Adenosine was added to the UW solution to improve postreperfusion ATP synthesis. We recently demonstrated that 1. only <1% of protectants like adenosine added to preservation solutions are absorbed by the graft and 2. that pretreatment with protectants extented graft survival more than a simple addition of protectants to the solution. We here investigated the effect of donor pretreatment with an adenosine deaminase inhibitor (EHNA) on interstitial adenosine concentrations, postreperfusion microcirculation and graft survival after rat liver preservation. Methods: Rat livers were preserved in UW solution (4°C) for 44 h. Donors were pretreated with various EHNA concentrations (0μM, 0.1μM, 1μM) injected intraportally before flush out with UW. Interstitial adenosine concentrations were measured by the microdialysis technique and blood flow by Laser Doppler flowmetry (given in perfusion units) after donor laparotomy (T0), after EHNA injection (T1), and after reperfusion (T2 and T3), in an orthotop liver transplant model. Also graft survival was monitored in rat livers after preservation in UW (for 44 hrs) with and without EHNA pretreatment. Results: Interstitial nucleoside concentrations (in ng/ml - upper line for 1μM EHNA pretreatment) (Figure)FigureBlood flow in donor livers (T0 and T1) and recipients (T3, T4) (Table)TableDiscussion: Donor pretreatment with EHNA increases interstitial adenosine concentrations in a dose dependent manner. Pretreatment with 1μM EHNA improves hepatic blood flow before flush out and after reperfusion and increases graft survival after critical preservation times. Adenosine deaminase inhibition appears to be a valuable adjunct to improve low flow during reperfusion and might improve clinical liver preservation.

Donor and recipient treatment with the Lazaroid U-74006F do not improve post-transplant lung function in swine.

U-74006F is the only Lazaroid which is currently in clinical use. A number of experimental studies demonstrate that Lazaroids reduce ischemia/reperfusion injury in various organ systems. We evaluated the effect of U-74006F on reperfusion injury in a large animal model of lung allo-transplantation. Two different treatment modalities were evaluated and compared with corresponding control groups. Unilateral left lung transplantation was performed in 21 weight-matched pigs (24-31 kg). Donor lungs were flushed with 1.51 cold (1 degrees C) LPD solution and preserved for 20 h. In group I (n = 5), donor animals were pretreated with U-74006F (10 mg/ kg i.v.) 20 min before harvest. In addition U-74006F was added to the flush solution (10 mg/l). In group III (n = 6), the Lazaroid was given to the donor before flush and to the recipient before reperfusion (3 mg/kg i.v.). Group II and IV (n = 5) served as control. One hour after reperfusion, the recipient contralateral right pulmonary artery and bronchus were ligated to assess graft function only. Extravascular lung water index (EVLWI), mean pulmonary artery pressure, cardiac output, and gas exchange were assessed during a 5 h observation period. Lipid peroxidation (TBARS) and neutrophil migration (MPO activity) were measured at the end of the assessment in lung allograft tissue. A significant change of TBARS concentration was shown in group III (group III 78.7+/-4.6 pmol/g vs. group IV 120.8+/-7.2 pmol/g (P = 0.0065) normal lung tissue 41.3+/-4.2 pmol/g). MPO activity was reduced in group III 3.74+/-0.25 deltaOD/mg per min vs. group IV 4.97+/-0.26 deltaOD/mg per min (P = 0.027), normal lung tissue 1.04+/-0.27 deltaOD/mg per min). Pulmonary hemodynamics and gas exchange after reperfusion did not differ between groups. In group I and III, a tendency towards a reduced EVLWI was noted. We conclude that combined treatment of donor and recipient with U-74006F reduces free radical mediated injury in the allograft. However, this intervention did not result in a significant reduction of post-transplant lung edema or improvement of pulmonary hemodynamics. Donor pretreatment alone did not improve lung allograft reperfusion injury. These results indicate that the benefit of U-74006F is too small to consider clinical application in lung transplantation.

Trafficking of APC from liver allografts of Flt3L-treated donors: augmentation of potent allostimulatory cells in recipient lymphoid tissue is associated with a switch from tolerance to rejection

Livers transplanted across major histocompatibility complex (MHC) barriers in mice are normally accepted without recipient immune suppression, and induce a state of functional tolerance. However, markedly increasing functional dendritic cells (DC) in the ‘passenger leucocyte’ population by donor pretreatment with the hematopoietic growth factor Flt3-ligand (Flt3L;10 μg/day for 10 days) results in acute allograft rejection. In this study, molecular, immunohistochemical and flow cytometric analysis of donor cell traffick into recipient lymphoid tissue 24 h after liver transplantation (C57BL/10 [H2 b]→C3H [H2 k]) was performed. In addition, the capacity of donor-derived cells in these tissues to stimulate host T cell proliferation was examined. Reverse transcriptase polymerase chain reaction analysis revealed increases in donor genomic DNA in both thymi and spleens of mice given livers from Flt3L-treated donors compared to controls. Donor MHC class II + (IA b+) cells in spleens were strikingly elevated (10-fold) in the former group. Two-colour flow cytometry revealed a similar increase in donor-derived H2-K b+/I-A b+ cells, and in the incidence of donor leucocytes expressing CD40, CD80, and CD86. CD11c + DC comprised ∼40% of the I-A b+ cells in spleens of mice given livers from Flt3L-treated donors. These changes were associated with the presence, in spleens, of potent allostimulatory activity for naive recipient strain T cells, that was not observed in normal liver recipients. Elicitation of allograft rejection, associated with enhanced trafficking of stimulatory donor antigen-presenting cells (APC), in particular DC, suggests that normal liver graft survival and tolerance induction may be linked to failure/counter-regulation of APC-driven stimulation of effective anti-donor T cell responses.

Pretreatment of small bowel isograft donors with cobalt-protoporphyrin decreases preservation injury

Pretreatment of small bowel isograft donors with cobalt-protoporphyrin decreases preservation injury

Twenty-four-hour preservation in gamma-hydroxybutyrate improves lung function in a canine single-lung allotransplantation.

We investigated the effect of gamma-hydroxybutyrate (GHB) when added to the low-potassium University of Wisconsin (LPUW) solution used for the preservation of canine lung for 24 hr. We also examined the effect of pretreatment of donor and recipient dogs with GHB on lung function after transplantation. Two groups were investigated. In the LPUW group, donor lungs were flushed with LPUW solution without GHB. In the GHB group, donor and recipient dogs were pretreated with GHB, and donor lungs were flushed with LPUW containing GHB. Posttransplant graft function was best in the GHB group. At 1 hr after reperfusion, PaO2 in the GHB group (475.7+/-96.2 mmHg) was significantly higher than in the LPUW group (188.3+/-102.7 mmHg, P<0.05). Furthermore, the use of GHB resulted in a significant increase in lung compliance (28.3+/-6.5 ml/cm H2O) compared with LPUW group (21.5+/-2.8 ml/cm H2O). Our results suggest that GHB is potentially useful for functional improvement of hypothermically preserved canine lung allografts after reperfusion.

Donor pre-treatment with inhaled nitric oxide attenuates acute lung injury following single lung transplantation

Donor pre-treatment with inhaled nitric oxide attenuates acute lung injury following single lung transplantation

Orthotopic transplantation of pig hearts harvested after 30 min of normothermic ischemia: controlled reperfusion with blood cardioplegia containing the Na+-H+-exchange inhibitor HOE 642.

The aim of our study was to develop a surgical technique for a successful transplantation of hearts harvested after 30 min of normothermic ischemia without donor pretreatment. Successful transplantation of ischemic compromised hearts could help to expand the severely limited donor pool. We used the pig model because this species is very susceptible to myocardial ischemia. Na+-H+-exchange (NHE) inhibitors have shown excellent protective properties in several in vitro and in vivo models of myocardial ischemia and reperfusion. In group I (n=12) hearts were harvested after 30 min of normothermic ischemia following cardiac arrest induced by exsanguination. Hearts were perfused with warm blood cardioplegia and transplanted orthotopically. In group II (n=9) controlled reperfusion with cold leucocyte-depleted blood cardioplegia was performed after 30 min of normothermic ischemia. In group III (n=8) the same procedure was performed as in group II but blood cardioplegia contained 1 mmol/l HOE 642. In group I massive myocardial oedema was observed and none of the animals could be weaned from cardiopulmonary bypass (CPB). In contrast, all animals in groups II and III could be weaned from CPB with low dose inotropic support. In groups II and III the contractility of the hearts, expressed as maximal left and right ventricular stroke work index was significantly impaired after transplantation as compared with the preoperative value. Supplementation of blood cardioplegia with HOE 642 resulted in a significantly better recovery of the LVSWImax (Group II vs. III). Successful transplantation of pig hearts is possible after 30 min of normothermic ischemia without donor pretreatment if a controlled reperfusion with cold leucocyte-depleted blood cardioplegia is performed. HOE 642 given during reperfusion only improves posttransplant left ventricular function.

Donor pretreatment with bone marrow cells for prevention of graft-versus-host reaction after small bowel transplantation

Donor pretreatment with bone marrow cells for prevention of graft-versus-host reaction after small bowel transplantation

Lazaroid donor pretreatment does not improve lung allograft reperfusion injury in swine.

Lazaroid donor pretreatment does not improve lung allograft reperfusion injury in swine.

Protection against allograft rejection with intercellular adhesion molecule-1 antisense oligodeoxynucleotides.

We designed an antisense phosphorothioate oligodeoxynucleotide (oligo) to specifically inhibit the expression of rat intercellular adhesion molecule-1 (ICAM-1) mRNA (IP-9125). IP-9125 oligo was delivered intravenously by osmotic pump alone or in combination with cyclosporine (CsA) to recipients in order to prevent the rejection of kidney or heart allografts. In additional experiments, kidney allografts were perfused with IP-9125 before grafting. IP-9125 inhibited ICAM-1 mRNA and ICAM-1 protein expression in rat aortic endothelial cells; scrambled controls IP-12140 and IP-13944 were ineffective. Untreated ACI (RT1a) recipients rejected Lewis (RT1l) kidney allografts at a mean survival time of 8.5+/-1.1 days. A 14-day intravenous administration of 2.5 mg/kg/day IP-9125 prolonged the survival of kidney allografts to 39.2+/-16.4 days; 5.0 mg/kg/day, to 43.0+/-17.5 days; and 10.0 mg/kg/day, to 50.4+/-21.6 days. In contrast, a scrambled control IP-12140 was not effective. A combination of 10 mg/kg/day IP-9125 and 1.0 mg/kg/day CsA delivered for 14 days synergistically extended kidney allograft survival times 88.5+/-7.5 days. In contrast, the combination of 10.0 mg/kg/day control IP-12140 with CsA was ineffective (20.7+/-3.2 days) when compared with CsA alone (20.2+/-4.0 days). Similar results were obtained for heart transplants in recipients treated with IP-9125 alone or in combination with CsA. Furthermore, in situ immunostaining showed that IP-9125 significantly reduced the expression of ICAM-1 protein in kidney allografts. Finally, perfusion of kidney grafts alone with 20.0 mg per 2 ml of IP-9125 protected kidney allografts from rejection (37.5+/-7.5 days; P < 0.001), whereas perfusion with 20 mg per 2 ml of control IP-12140 was ineffective (12.6+/-5.0 days). Rat ICAM-1 IP-9125 oligo inhibits ICAM-1 protein expression in vitro and in vivo as well as blocks allograft rejection when used for pretreatment of donors, graft perfusion, or postoperative treatment of recipients.

Donor pretreatment with ambroxol or dexamethasone fails to ameliorate reperfusion injury in experimental lung transplantation.

Based on the known properties of ambroxol and dexamethasone to inhibit inflammation and increase endogenous surfactant levels, the potential advantage of donor pretreatment with either drug was investigated in an acute rat double-lung transplant model. Donor animals were randomly assigned to one of three treatment groups: an ambroxol group (AMB; 0.4 mg/kg), a dexamethasone group (DX; 2 mg/kg); or an untreated control group (CN). Drugs were given intraperitoneally 6 h prior to harvest. Following standard preservation and 16 h of cold ischemia, the donor double lung block was implanted into syngeneic recipients using custom-designed stents for the vascular anastomosis. During reperfusion, serial measurements of graft pulmonary vascular resistance and alveolar-arterial oxygen difference were obtained. Separate graft ventilation allowed determination of graft dynamic lung compliance. Final assessment included weight gain and histology. For phospholipid analysis, lung lavages were performed in the three study groups at the end of reperfusion and compared to levels before graft harvest. Donor pretreatment did not significantly affect preharvest phospholipid levels. Survival following graft ischemia and reperfusion was shortest after AMB (92 +/- 5 min) and longest after DX (110 +/- 5 min; DX vs AMB P < 0.03) and CN (116 +/- 4 min; CN vs AMB P < 0.02). DX pretreatment provided better compliance (P < 0.02) and lower vascular resistance (P < 0.0001) than AMB treatment. Airway resistance was lower in the AMB and DX groups than in controls (P < 0.04 and P < 0.02, respectively). The alveolar-arterial oxygen difference was markedly similar in all groups. Graft weight gain amounted to 114% +/- 10% in AMB, 88% +/- 12% in DX, and 98% +/- 13% in CN (P = NS). Thus, in this rat lung transplantation model, donor pretreatment with dexamethasone did not improve graft function compared to untreated controls and donor pretreatment with ambroxol was found to be potentially detrimental to graft function during reperfusion.

Donor pretreatment with ambroxol or dexamethasone fails to ameliorate reperfusion injury in experimental lung transplantation

Abstract Based on the known properties of ambroxol and dexamethasone to inhibit inflammation and increase endogenous surfactant levels, the potential advantage of donor pretreatment with either drug was investigated in an acute rat double-lung transplant model. Donor animals were randomly assigned to one of three treatment groups: an ambroxol group (AMB; 0.4 mg/kg), a dexamethasone group (DX; 2 mg/kg); or an untreated control group (CN). Drugs were given intraperitoneally 6 h prior to harvest. Following standard preservation and 16 h of cold ischemia, the donor double lung block was implanted into syngeneic recipients using custom-designed stents for the vascular anastomosis. During reperfusion, serial measurements of graft pulmonary vascular resistance and alveolar-arterial oxygen difference were obtained. Separate graft ventilation allowed determination of graft dynamic lung compliance. Final assessment included weight gain and histology. For phospholipid analysis, lung lavages were performed in the three study groups at the end of reperfusion and compared to levels before graft harvest. Donor pretreatment did not significantly affect preharvest phospholipid levels. Survival following graft ischemia and reperfusion was shortest after AMB (92 ± 5 min) and longest after DX (110 ± 5 min; DX vs AMB P < 0.03) and CN (116 ± 4 min; CN vs AMB P < 0.02). DX pretreatment provided better compliance (P < 0.02) and lower vascular resistance (P < 0.0001) than AMB treatment. Airway resistance was lower in the AMB and DX groups than in controls (P < 0.04 and P < 0.02, respectively). The alveolar-arterial oxygen difference was markedly similar in all groups. Graft weight gain amounted to 114%± 10% in AMB, 88%± 12 % in DX, and 98 %± 13 % in CN (P= NS). Thus, in this rat lung transplantation model, donor pretreatment with dexamethasone did not improve graft function compared to untreated controls and donor pretreatment with ambroxol was found to be potentially detrimental to graft function during reperfusion.

Donor pretreatment with IL-10 decreases TNF, IFN-gamma release and liver injury after cold ischemia-reperfusion in mice

Donor pretreatment with IL-10 decreases TNF, IFN-gamma release and liver injury after cold ischemia-reperfusion in mice

The impact of donor organ quality on postoperative liver function after orthotopic rat liver transplantation.

The impact of donor factors for posttransplant liver function was evaluated in the model of orthotopic rearterialized liver transplantation in the rat. The effect of donor fasting, parenteral hyperalimentation, hypotension, warm ischemia and endotoxins on histology, clinical chemistry and MEGX test was analyzed in syngeneic and allogeneic recipients of livers stored for 4 hrs on ice. In syngeneic animals, 20 min of warm ischemia led to significantly elevated serum transaminase levels and degree 2 histological damage on POD 2. Endotoxins produced a grade 1 histological damage. All groups had a lower MEGX formation rate compared to controls. In allogeneic animals, warm ischemia was the single most detrimental parameter. The strength of the rejection response on POD 8 did not depend on the type of donor pretreatment. The major finding of this non-survival study is the deleterious effect of warm ischemia and endotoxin on the functional and structural integrity of liver grafts after 4 hrs of cold ischemia.

Beneficial Impact of FK 409 As Donor Pretreatment on Warm or Cold Ischemically Injured Hearts in the Rat

Beneficial Impact of FK 409 As Donor Pretreatment on Warm or Cold Ischemically Injured Hearts in the Rat

Augmentation of dendritic cells in murine organ donors by Flt3 ligand alters the balance between transplant tolerance and immunity.

Treatment of mice with the recently cloned hemopoietic growth factor Flt3 ligand (FL; 10 microg/day for 10 days) resulted in a large increase in myeloid lineage cells within the liver. While the number of nonparenchymal cells (NPC) harvested from liver increased about 9-fold, a 90-fold increase was observed in the proportion of CD11c+ dendritic cells (DC) recovered from NPC following overnight (18-h) culture in granulocyte-macrophage CSF. In contrast, only a 50% increase was seen in CD11c+ cells within heart single cell suspensions and in the number of DC obtained from hearts after 18-h culture. Liver NPC and heart cell suspensions freshly isolated from 10-day FL-treated animals exhibited increased T cell allostimulatory capacity compared with controls. Overnight cultured DC from livers of FL-treated animals expressed both higher levels of costimulatory molecules (CD80 and CD86) and allostimulatory activity than those from controls. Heart-derived DC also displayed enhanced stimulatory capacity. Pretreatment of organ donors with FL for either 5 or 10 days before transplant of organs to normal recipients abrogated the spontaneous liver allograft acceptance normally observed and resulted in delayed or acute graft rejection (median survival times, 40 and 12 days, respectively). Heart rejection was significantly accelerated by pretreatment of donors with FL for 5 or 10 days (median survival times, 8 and 7 days, respectively, vs 12 days in controls). These novel findings reveal the potent immunologic adjuvant properties of FL in vivo. They also show that substantial augmentation of the number of potential allostimulatory cells in donor organs before transplantation favors rejection rather than tolerance induction.

Efficiency of Pentoxifylline in Donor Pretreatment in Rat Liver Transplantation

Donor pretreatment is a new concept in organ preservation. Pentoxifylline (PTX) has been reported to suppress the activation of Kupffer cells and to decrease injury to the hepatic graft after rat liver transplantation. We evaluated the efficiency of PTX pretreatment on the donor against hepatic injury following cold ischemia (CI) or warm ischemia (WI) using the rat liver transplantation model. Dose dependency: every rat was injected intraperitoneally with PTX (30, 50, or 80 mg/kg) or saline. One hour later, the portal vein (PV) and the hepatic artery (HA) were clamped for 30 min. Transplantation: the donor rat was injected intraperitoneally with 50 mg/kg PTX or saline, 1 hr before laparotomy. Animals were divided into two groups. In the CI group, grafts were preserved for 12 hr in University of Wisconsin solution at 4°C and transplanted. In the WI group, the PV and the HA in the donor were clamped for 30 min before donor surgery, and the grafts were transplanted. Serum levels of tumor necrosis factor-α (TNF-α), glutathioneS-transferase-α (GST-α), and aspartate transaminase (AST) were measured at 30 min, 3 hr, and 24 hr after reperfusion of the PV. Compared with those of a control group, the serum levels of TNF-α, GST-α, and AST in the PTX-pretreated groups were significantly lower after both CI and WI at 30 min and further suppressed in the WI group at 24 hr. These results indicate that PTX pretreatment on the donor is effective for suppression of hepatic injury after both CI and WI.

Donor pretreatment with leflunomide has an adverse effect on rat cardiac allograft survival

The effect of leflunomide (Lef) donor pretreatment (DPT) on rat cardiac allograft survival was investigated. In untreated Lewis recipients of untreated DA hearts, median graft survival was 5 days. DPT (Lef 5 or 10 mg/kg per day for 30 days) reduced median grafts survival to 4.0 and 4.5 days, respectively. In immunosuppressed (Lef 5 mg/kg for 10 or 30 days) Lewis recipients of untreated DA hearts, median graft survival was 21 and 33 days, respectively. DPT with Lef 5 mg/kg per day for 5 days or 30 days reduced median graft survival to 12 and 23.5 days, respectively ( p < 0.05). DPT with Lef resulted in earlier graft rejection but the histological appearance at the time of rejection was the same as in untreated controls. DPT with lef resulted in a 40% reduction in MHC class II-psotive cells in the heart. Histological examination of rejecting hearts showed no obvious difference in the nature of the rejection process between DPT and untreated control hearts. The paradox between class II reduction but earlier rejection indicates that DPT is exerting a deleterious effect through some unrecognized property of the graft.

Effect in supralethally irradiated rats of granulocyte colony-stimulating factor and lisofylline on hematopoietic reconstitution by syngeneic bone marrow or whole organ passenger leukocytes.

We have previously shown the existence of migratory hematopoietic stem cells in adult solid organs. This study demonstrates that granulocyte colony-stimulating factor (G-CSF) and lisofylline, a phosphatidic acid inhibitor that suppresses hematopoiesis-inhibiting cytokines, can enhance the engraftment of organ-based hematopoietic stem cells. When syngeneic heart grafts or liver nonparenchymal cells were transplanted into lethally irradiated (9.5 Gy) Lewis rats, complete hematopoietic reconstitution and animal survival were significantly improved by treating the recipient with G-CSF or, to a lesser extent, with lisofylline. Pretreatment of hepatic nonparenchymal cell donors with G-CSF, but not lisofylline, also resulted in striking improvement of recipient survival which was associated with an augmented subpopulation of donor stem cells. The results suggest that these drugs can be used to enhance the chimerism that we postulate to be the basis of organ allograft acceptance.