Donor Pretreatment Research Articles

Donor pretreatment with gadolinium chloride improves early graft function and survival after porcine liver transplantation

Donor pretreatment with gadolinium chloride improves early graft function and survival after porcine liver transplantation

Protective effect of donor pretreatment with carbon monoxide on ischemia/reperfusion injury after rat liver transplantation; possible role of Hsp 70 expression

Introduction: Stress response genes, such as heme oxygenase-1 (HO-1) and heat shock protein 70 (HSP70), are known to be involved in ischemia/reperfusion (I/R) injury and have beneficial effects against oxidative stress. Carbon monoxide (CO), the byproduct of heme degradation by HO-1, has been shown to provide protection against I/R injury. This study was designed to examine whether donor pretreatment with CO has beneficial effects on hepatic cold I/R injury. Methods: Orthotopic syngenic LEW rat liver transplantation (OLT) was performed after 18 hrs preservation in cold UW. Donor rats were exposed to a non-toxic dose of CO (250 ppm) in a CO chamber for 24 hrs before harvesting. Controls donors were maintained in room air. Hepatic I/R injury was evaluated by serum AST levels and histopathology. Expression of HSP70 protein in donor liver was investigated before harvest. After OLT, expression of TNF-α and ICAM-1 mRNA, iNOS protein, and cleaved caspase-3 were measured. Results (Table):TABLE—ABSTRACT 107AST (IU/L) 24 hNeutrophil (/mm2) 48 hTNF-α (fold increase) 1 hiNOS (band denity) 3 hCaspase-3 (band denity) 3 hAir5547 ± 97987.1 ± 15.214.7 ± 5.326.3 ± 5.33.8 ± 0.3CO1981 ± 381∗23.6 ± 8.6∗4.8 ± 1.5∗7.5 ± 2.1∗2.8 ± 0.1∗Mean ± SD, n = 2–6 animals per group,∗P < 0.05. CO-inhalation for 24 hrs significantly increased HSP70 expression in the donor liver. After OLT, donor CO pretreatment markedly attenuated hepatic I/R injury assessed by low AST levels, reduced necrosis, and decreased neutrophil infiltration. Hepatic expression of TNF-α and ICAM-1 mRNA, and iNOS protein was significantly reduced in CO-treated group. Additionally, cleaved caspase-3 expressing apoptotic cells were significantly decreased with donor CO-inhalation. Conclusion: These results demonstrate that donor CO pretreatment leads to attenuation of hepatic cold I/R injury through the inhibition of iNOS induction, pro-inflammatory responses, and pro-apoptotic reactions. The protective effect of donor CO pretreatment is in part mediated by HSP70 induction in the donor liver. Thus, low-dose CO inhalation would be a novel therapeutic strategy to combat hepatic cold I/R injury.

Donor pretreatment with gadolinium chloride improves early graft function and survival after porcine liver transplantation

Kupffer cell depletion by gadolinium chloride (GdCl(3)) in rat livers has previously been proven to minimize hepatic ischemia/reperfusion injury after experimental liver transplantation (LTX). In the current study, we evaluated the effects of donor pretreatment with GdCl(3) on hepatic ischemia/reperfusion injury, macro- and microcirculation, and endotoxin clearance of the liver in a porcine model of experimental LTX. Two groups of 12 pigs were treated either with intravenous NaCl (0.9%; control) or GdCl(3) (20 mg/kg). Twenty-four hours after pretreatment, hepatic macrocirculation was quantified by Doppler flowmetry and liver parenchymous microcirculation by implanted thermodiffusion electrodes. The liver grafts were transplanted after 4-6 h of cold ischemia in University of Wisconsin (UW) solution. At 1 and 24 h after LTX, the perfusion values were re-evaluated and histology, biochemical (aspartate aminotransferase, AST) and functional parameters (partial thromboplastin time, prothrombin time, and bilirubin) were analyzed. Furthermore, endotoxin clearance of the liver was evaluated at all time points. In GdCl(3)-treated animals 80% of the Kupffer cells were destroyed, and 24 h after LTX ischemia/reperfusion injury in treated grafts was significantly lower in comparison to controls, as shown by histology, AST levels (741+/-490 U/l in controls vs 379+/-159 U/l in treated grafts, P<0.05), survival (67% vs 92%), and enhanced macro- (total transhepatic blood flow [THBF]=112+/-22 ml/min per 100 g in controls vs 157+/-45 ml/min per 100 g in treated grafts, P<or=0.05) and microcirculation (thermodiffusion [TD]=73+/-9 ml/min per 100 g in controls vs 90+/-16 ml/min per 100 g in treated grafts, P<or=0.05). Despite destruction of the macrophage system in the liver, the transhepatic endotoxin gradient of treated livers was enhanced before and 1 h after transplantation (58% in controls vs 85% in treated grafts, P<0.05). Destruction of Kupffer cells of donors by pretreatment with GdCl(3) in pigs is effective in preventing liver graft dys- and nonfunction after LTX. Pretreatment with GdCl(3) does not diminish but increase hepatic endotoxin clearance.

Graft immunogenicity and treatment of kidney donors

Abstract Allorecognition is induced when the host immune system detects alloantigens in the context of danger signals. As an implication of this theory, one should be able to prevent the alloimmune response by eliminating danger signals that could be achieved by modifying the graft before transplantation. Factors that are known to play a role in the induction of graft immunogenicity include brain death, cold and warm ischemia, and organ reperfusion. The molecules that are responsible for the danger signals have not yet been fully identified, but free oxygen radicals and interferon γ are important candidates. One of the most important protective mechanisms against oxidative stress is the heme oxygenase 1 system. Up-regulation of heme oxygenase 1 in grafts has been shown to prevent ischemia-reperfusion damage and improve long-term graft survival in various transplantation models. The end result of preventing the action of interferon γ in kidney transplants is less clear because the compound plays such a complex and pivotal role in the immune response. For example, when kidneys that do not produce interferon γ are transplanted into wild-type mice, an increased early graft necrosis is observed that can be treated with interferon γ. On the other hand, kidneys from CIIAKO mice, in which interferon γ is unable to induce major histocompatibility complex class II, are relatively protected against rejection. It has recently become clear that catecholamines are important graft-modifying agents. It has been shown in 2 independent clinical studies with a case-control design that treatment of kidney donors with catecholamines protects against rejection after kidney transplantation and is associated with a profound improvement of long-term graft survival. In vitro studies have elucidated that dopamine induces heme oxygenase 1 in endothelial and proximal tubular epithelial cells. Catecholamines can also modulate cytokine production and protect against cold-induced damage. Other substances such as proteoglycans and phosphatidylethanolamine-bound hyaluronic acid may interfere with the actions of interferon γ. Further studies of these compounds in experimental animal models and in prospective randomized clinical trials will help to establish their efficacy in donor pretreatment. It is important to underscore that donor pretreatment will have great advantages for the recipient because an improved long-term graft survival could thus be achieved cost-efficiently and without great effort or side effects.

Pretreatment of donors with interleukin-18 attenuates acute graft-versus-host disease via STAT6 and preserves graft-versus-leukemia effects

Interleukin-18 (IL-18) is a unique cytokine that modulates both T(H)1/T(H)2 responses, but its ability to modulate diseases through induction of T(H)2 cytokines is unclear. It has been shown to play an important role in allogeneic bone marrow transplantation (BMT). Because immune responses of allogeneic BM donors may affect acute graft-versus-host disease (GVHD), we investigated the effect of pretreating BM transplant donors with IL-18 on the severity of acute GVHD using a well-characterized experimental BMT model (BALB/c-->B6). Pretreatment of allogeneic BM transplant donors with IL-18 significantly improved survival (80% vs 0%; P <.001), and reduced clinical, biochemical, and pathologic indices of acute GVHD in BM transplant recipients. IL-18 pretreatment was associated with reduced interferon gamma (IFN-gamma) and greater IL-4 secretion by donor T cells after BMT. Acute GVHD mortality was reduced when IL-18 was administered to donors deficient in IFN-gamma and signal transducer and activator of transcription 4 (STAT4) but not STAT6 signaling molecules, suggesting a critical role for STAT6 signaling in IL-18's protective effect. IL-18 treatment did not alter donor CD8(+) cytotoxic T-lymphocyte (CTL) activity and preserved graft-versus-leukemia (GVL) effects after allogeneic BMT (70% vs 10%; P <.01). Together these data illustrate that pretreatment of donors with IL-18 prior to allogeneic BMT attenuates acute GVHD in a STAT6-dependent mechanism while preserving GVL effects.

Donor pretreatment with progenipoietin-1 is superior to granulocyte colony–stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation

The granulocyte colony-stimulating factor (G-CSF) and Flt-3 receptor agonist progenipoietin-1 (ProGP-1) has potent effects on dendritic cell (DC) expansion and may be an alternative to G-CSF for the mobilization of stem cells for allogeneic stem cell transplantation (SCT). We studied the ability of stem cell grafts mobilized with this agent to induce graft-versus-host disease (GVHD) to minor and major histocompatibility antigens in the well-described B6 --> B6D2F1 SCT model. ProGP-1, G-CSF, or control diluent was administered to donor B6 mice. ProGP-1 expanded all cell lineages in the spleen, and unseparated splenocytes from these animals produced large amounts of interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) whereas the expression of T-cell adhesion molecules was diminished. Transplantation survival was 0%, 50%, and 90% in recipients of control-, G-CSF-, and ProGP-1-treated allogeneic donor splenocytes, respectively (P <.0001). Donor pretreatment with ProGP-1 allowed a 4-fold escalation in T-cell dose over that possible with G-CSF. Donor CD4 T cells from allogeneic SCT recipients of ProGP-1 splenocytes demonstrated an anergic response to host antigen, and cytokine production (interferon gamma [IFNgamma], IL-4, and IL-10) was also reduced while CD8 T-cell cytotoxicity to host antigens remained intact. Neither CD11c(hi) DCs nor CD11c(dim)/B220(hi) DCs from ProGP-1-treated animals conferred protection from GVHD when added to control spleen. Conversely, when equal numbers of purified T cells from control-, G-CSF-, or ProGP-1-treated allogeneic donors were added to allogeneic T-cell-depleted control spleen, survival at day 60 was 0%, 15%, and 90%, respectively (P <.0001). The improved survival in recipients of ProGP-1 T cells was associated with reductions in systemic tumor necrosis factor alpha generation and GVHD of the gastrointestinal tract. We conclude that donor pretreatment with ProGP-1 is superior to G-CSF for the prevention of GVHD after allogeneic SCT, primarily due to incremental affects on T-cell phenotype and function.

16 Donor pretreatment with progenipoietin-1 prevents acute graft-versus-host disease in an IL-10 dependent fashion and augments the graft-versus-leukemia effect

16 Donor pretreatment with progenipoietin-1 prevents acute graft-versus-host disease in an IL-10 dependent fashion and augments the graft-versus-leukemia effect

88 Donor pretreatment with G-CSF prevents chronic graft-versus-host disease after allogeneic stem cell transplantation

88 Donor pretreatment with G-CSF prevents chronic graft-versus-host disease after allogeneic stem cell transplantation

Exogenous surfactant instillation in lung donors to reduce early graft dysfunction

Background: Application of exogenous surfactant has been proposed to ameliorate lung ischemia-reperfusion injury. Experimentally, donor pretreatment has been favored over posttransplant application. We now report on the first prospective randomized clinical study of donor surfactant instillation in lung transplantation.Methods: Lung donors were randomly assigned to the surfactant (Surf, n=14) or the control group (Co, n=15). Bovine surfactant (100 mg/kg BW) was instilled segmentally via bronchoscopy within 30 min before organ harvest. Diagnostic bronchoalveolar lavage (BAL) was performed before surfactant instillation and 8 h after reperfusion of the graft. Surface-tension lowering properties of surfactant and the ratio of small to large surfactant aggregates (SA/LA) were determined as indicators for preservation of surfactant function from BAL fluid. Clinical outcome of the surfactant group was compared to 187 consecutive cases with otherwise similar treatment.Results: Surface tension of BAL fluid after transplantation was slightly increased in the Surf group but markedly increased in the Co group, indicating loss of surfactant function (9.6±2.1 vs. 17.5±1.9 mN/m; Surf vs. Co, respectively; P<0.05). The SA/LA ratio, as a marker for metabolic conversion of the surfactant material, was high in both groups before explant (0.86±0.4 vs. 0.78±0.31). This ratio was improved after transplant in Surf lungs, but further deteriorated in controls (0.42±0.28 vs. 1.58±0.44). Early clinical outcome showed no death in both groups and was without significant differences between groups due to low sample size, but when compared to our overall recipient population, spirometry in the Surf group revealed higher FEV1 2 weeks after transplant (2.81±0.71 vs. 1.77±0.91; P<0.05).Conclusion: Exogenous surfactant is safe and markedly improves post transplant surfactant function. Its application before organ harvest is therefore encouraged, especially in borderline donors.

Donor pretreatment using the aerosolized prostacyclin analogue iloprost optimizes postischemic function of non heart beating donor lungs

Ischemia-reperfusion-injury accounts for one third of early deaths after lung transplantation. To expand the limited donor pool, lung retrieval from non-heart-beating donors (NHBD) has been introduced recently. However, due to potentially deleterious effects of warm ischemia on microvascular integrity, use of NHBD lungs is limited by short tolerable time periods prior to preservation. After intravenous prostanoids are routinely used to ameliorate reperfusion injury, latest evidence suggests similar efficacy of inhaled prostacyclin. Therefore, the impact of donor pretreatment using the prostacyclin-analogue Iloprost on postischemic NHBD lung function and preservation quality was evaluated.

Improvement of heart allograft acceptability by pretreatment of donors with granulocyte colony-stimulating factor

Improvement of heart allograft acceptability by pretreatment of donors with granulocyte colony-stimulating factor

Ischemic Preconditioning Increases the Tolerance of Fatty Liver to Hepatic Ischemia-Reperfusion Injury in the Rat

Hepatic steatosis is a major risk factor in ischemia-reperfusion. The present study evaluates whether preconditioning, demonstrated to be effective in normal livers, could also confer protection in the presence of steatosis and investigates the potential underlying protective mechanisms. Fatty rats had increased hepatic injury and decreased survival after 60 minutes of ischemia compared with lean rats. Fatty livers showed a degree of neutrophil accumulation and microcirculatory alterations similar to that of normal livers. However, in presence of steatosis, an increased lipid peroxidation that could be reduced with glutathione-ester pretreatment was observed after hepatic reperfusion. Ischemic preconditioning reduced hepatic injury and increased animal survival. Both in normal and fatty livers, this endogenous protective mechanism was found to control lipid peroxidation, hepatic microcirculation failure, and neutrophil accumulation, reducing the subsequent hepatic injury. These beneficial effects could be mediated by nitric oxide, because the inhibition of nitric oxide synthesis and nitric oxide donor pretreatment abolished and simulated, respectively, the benefits of preconditioning. Thus, ischemic preconditioning could be an effective surgical strategy to reduce the hepatic ischemia-reperfusion injury in normal and fatty livers under normothermic conditions, including hepatic resections, and liver transplantation.

New solutions in organ preservation

Over the last 20 years, a number of methods and solutions have been developed to preserve donor organs for transplantation. Collins et al first introduced the simple cold-storage technique to store and transport kidneys up to 30 hours. The development of the University of Wisconsin cold-storage solution (UW-CSS) in 1986 improved organ preservation and resulted in a better understanding in preservation related injury. Since its introduction, several UW-CSS-derived solutions have been tested, and important components for hypothermic preservation were identified. Pathophysiologic mechanisms in static hypothermic preserved organs are discussed, including hypothermia-induced cell swelling, compromised membrane function, adenosine triphosphate usage, cellular acidosis, and prevention of precursor formation of reactive oxygen species. Counteracting these pathophysiologic mechanisms can be divided into components acting by two protective principles. The first is to create a physical environment, minimizing cellular and interstitial edema formation. The second mechanism involves biochemically active components that ideally enter the cell and remain effective during reperfusion. Some preservation solutions currently in use are discussed on the basis of these two protective mechanisms. In respect of these principles in preservation, new solutions in preservation are discussed. Although cold-storage solutions improved organ viability, maintaining or even improving organ quality during preservation remains an important goal. Future advances in preservation might be found in donor pretreatment, cytoprotection, or the use of dynamic preservation systems.

Immunodulation of intestinal transplantation: antilymphocyte serum donor pretreatment vs. ex vivo graft irradiation

We have shown the beneficial effects of an immunomodulation strategy for small intestinal transplantation (SITx) by depleting mature lymphocytes from the graft and simultaneously infusing progenitor-rich donor bone marrow (BM) cells.1 In this study we evaluated the relative benefit of leukocyte elimination between ex vivo graft irradiation versus donor pretreatment with antilymphocyte serum (ALS) in a model of fully allogenic rat SITx. Special attention was paid to their impact on early immune responses after SITx by analyzing the levels and lineages of chimerism as well as the cytokine up-regulation in recipient spleen and allografts.

Donor pretreatment with FTY720 increases graft lymphocytes but does not affect graft survival following rat small bowel transplantation

Donor pretreatment with FTY720 increases graft lymphocytes but does not affect graft survival following rat small bowel transplantation

Reduction of immunogenicity by cyclophosphamide pretreatment and culture on xenograft survival of monkey pancreatic islets transplanted into rats.

Islet cell transplantation offers a new approach for the treatment of diabetes. Before transplantation, immunomodulation procedures are conducted to reduce the immunogenicity of the pancreatic islets. To study the effect of cyclophosphamide pretreatment and culture on the xenograft survival of monkey pancreatic islets. Islets were isolated from normal monkeys and transplanted under the renal capsule of the rats. The grafts were removed 7 days after transplantation and processed for histologic study to examine graft survival. Islets from normal monkeys without cyclosporine treatment were destroyed in 7 days. These islets required cyclosporine (30 mg/kg) to prevent rejection. In the second group, the donor monkeys were treated with cyclophosphamide (60 mg/kg body weight) -4 and -2 days before harvesting of the islets. On day 0, the islets were isolated and cultured in RPMI-1640 medium for 7 days in 95% air and 5% CO2. After culture, the islets were transplanted into the rats. The grafts were removed and processed for histologic study to examine graft survival. The pretreated and cultured islets required 15 mg/kg cyclosporine to prevent rejection. Half of the usual dose of cyclosporine is enough to prevent rejection if the donor monkeys were pretreated and the islets cultured. This study shows that donor pretreatment and culture reduces immunogenicity of the xenotransplanted primate islets into rats.

Pretreatment of Donors with Endothelin Receptor Antagonist TAK-044 Improves Cardiac Functional Recovery Following Preservation with University of Wisconsin Solution

Objective : Previous studies suggest that endothelin-1 (ET-1) plays a role in myocardial ischemia/reperfusion injury. Although administration of an endothelin receptor antagonist to the recipient has been shown to improve myocardial function after ischemia/reperfusion in a rat heart transplantation model, the effect of administering an endothelin receptor antagonist to the donor has not yet been examined. This study was designed to investigate the effects of pretreating donors with an ET A /ET B endothelin receptor antagonist (TAK-044) on myocardial function after cold preservation of a rat heart. Design : Male rats were pretreated with normal saline (control group, n = 8), TAK-044 (TAK group, n = 8, 1 mg/kg). Following cardiac arrest using cardioplegia, we washed out the coronary vascular beds with cold University of Wisconsin solution followed by 6-h preservation. After preservation, the hearts were mounted on a Langendorff apparatus to estimate aortic flow (AF), coronary flow (CF), cardiac output (CO), systolic pressure (SP), heart rate (HR), and rate-pressure product (RPP: HR 2 SP). The concentration of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK) within the coronary perfusate during reperfusion was measured. Results : AF, SP, and CO were significantly greater in the TAK group than in the control group ( p = 0.0045, 0.004, and 0.0295, respectively). Conclusion : Pretreatment of donors with a nonselective endothelin receptor antagonist (TAK-044) improved cardiac functional recovery following preservation and may be beneficial for prolonged myocardial preservation.

Gene therapy and graft modification

The application of gene therapy technology in the setting of solid organ transplantation has been greatly enhanced by the recent development of new gene delivery vehicles that are safer and more efficient. The transplant setting provides a unique advantage in that a graft can be efficiently transduced before transplantation by donor pretreatment or gene delivery during cold preservation. The expression of the transferred sequences within the graft may then allow control of local events, such as inhibition of the alloimmune response, prevention of cell death, or assistance in cellular recovery. It is the selection and design of the shuttle vector that determines the length and timing of expression of the protein. This review summarizes recent and novel advances in the application of gene therapy in organ and cellular transplantation.

PHARMACOLOGICAL PRECONDITIONING WITH LOW-DOSE CYCLOSPORINE OR FK506 REDUCES SUBSEQUENT ISCHEMIA/REPERFUSION INJURY IN RAT KIDNEY1,2

Ischemia/reperfusion (I/R) injury in the early posttransplant period is closely associated with delayed recovery of graft function, increased acute rejection, and late allograft dysfunction. Pharmacological preconditioning with low-dose cyclosporine (CsA) or FK506 was performed to induce ischemic tolerance in rat kidney with I/R injury. Low-dose CsA (3 mg/kg, administered i.v.) or FK506 (0.3 mg/kg i.v.) were used to induce ischemic tolerance in Sprague-Dawley rats, and the induction of heat shock protein (hsp) 70 by CsA or FK506 was evaluated overtime. Rats were pretreated with CsA or FK506 6 hr before I/R injury when hsp70 was maximally expressed, and were killed 24 hr later. The effect of pharmacological preconditioning on subsequent I/R injury was evaluated in terms of renal function, histopathology score, assays for apoptosis (DNA fragmentation analysis, TUNEL staining, expressions of pro-apoptotic genes, and caspase activity), and the expression of inflammatory cytokine genes (interleukin-1 and tumor necrosis factor-alpha). Preconditioning with low-dose CsA or FK506 significantly improved renal function and renal histology, compared to rats with I/R injury. Apoptotic cell death (typical DNA laddering and increased TUNEL-positive cells) in rat kidneys with I/R injury, was decreased by pretreatment with low-dose CsA or FK506. Increased expression of pro-apoptotic genes (Fas, Fas-ligand, caspase 1 and 3) and activated caspases in ischemic rat kidneys were decreased after CsA or FK506 pretreatment. Pretreatment with low-dose CsA or FK506 prevents subsequent I/R injury, and this effect may be related to the induction of hsp70. Pretreatment of renal donors with low-dose CsA or FK506 may result in an improvement in immediate posttransplant function.

Immunomodulation based on a two-way paradigm with deoxyspergualin alleviates graft-versus-host reaction in small-bowel transplantation in rats.

In this study, we investigated whether or not deoxyspergualin used as donor pretreatment, with and without pretreatment using bone-marrow-cell injection, could alleviate graft-versus-host reaction (GVHR) following small-bowel transplantation in an unidirectional GVHR model with Lewis (LEW)-to-F1 rats. In addition, we studied the effect of deoxyspergualin plus bone-marrow-cell donor pre-operative treatment in combination with recipient postoperative treatment using deoxyspergualin. When the donor was pretreated with bone-marrow cells from recipient rats, the recipient died at 7.2+/-1.4 days, showing significantly shorter survival compared with the control group. Deoxyspergualin, when employed either alone as recipient post-treatment or as donor pretreatment, both with and without additional pretreatment with F1 recipient bone-marrow-cell injection, did not result in significant prolongation of recipient survival. The combination of donor pretreatment with deoxyspergualin plus F1 bone-marrow-cell injection followed by post-operative deoxyspergualin administration, however, resulted in significant prolongation in recipient survival compared with control (26.1+/-1.7 days). In addition, no severe cutaneous lesions on GVHR were seen throughout the observation period. This suggests that donor pretreatment with deoxyspergualin and recipient bone-marrow-cell injection combined with postoperative deoxyspergualin administration can lead to resistance to GVHR after parent-to-F1 small-bowel transplantation.