Introduction: Cold ischemia is associated with delayed graft function and influences significantly long-term graft survival after transplantation. Recently we have demonstrated the protective effects of Simvastatin, a 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (HMG-CoARI) in a cold ischemia model of renal transplantation. Methods: Simvastatin (10 mg/kg/day) was applied for 3 days prior to transplantation to F344 donors. After a prolonged cold ischemic period of 24 h the kidneys were transplanted to Lewis recipients. The organs were harvested after 24 h (n=5), 14 d (n=5) and 6 mths. (n=5) and analyzed by RT-PCR and FACS-analysis. Results: The numbers of CD3+T-cells, CD4+CD3+T-cells and CD3-CD4+ monocytes in the recipients’ spleen were reduced after 24h and 14 days. Additionally the expression of the chemokine receptor CCR7 and its ligands CCL19 and CCL21 was markedly reduced in recipients’ spleens. Interestingly, by analyzing renal allografts after 6 months this observation was even more amplified. Spleens from recipients receiving an allograft from treated donors displayed a significant reduction of MHC class II (p<0.006), CCL19 (p<0.012), and CCL21 (p<0.0061), as well as decreased mRNA expression of immunproteasome subunits including PSMB8 (p<0.0061), PSMP9 (p<0.0061) and PSMB10 (p<0.042). Conclusion: Our data suggest that donor treatment with Simvastatin following prolonged cold ischemia reduces graft immunogenicity by modulating potential antigen presenting cells and their homing to lymphoid organs. Donor treatment with Simvastatin represents an attractive tool to preserve renal function after ischemia/reperfusion injury.

OBJECTIVE:To investigate potential beneficial effects of donor treatment with methylprednisolone on organ function and outcome after liver transplantation. SUMMARY BACKGROUND DATA:It is proven experimentally and clinically that the brain death of the donor leads to increased levels of inflammatory cytokines and is followed by an intensified ischemia/reperfusion injury after organ transplantation. In experiments, donor treatment with steroids successfully diminished these effects and led to better organ function after transplantation. METHODS:To investigate whether methylprednisolone treatment of the deceased donor is applicable to attenuate brain death-associated damage in clinical liver transplantation we conducted a prospective randomized treatment-versus-control study in 100 deceased donors. Donor treatment (n = 50) consisted of 250 mg methylprednisolone at the time of consent for organ donation and a subsequent infusion of 100 mg/h until recovery of organs. A liver biopsy was taken immediately after laparotomy and blood samples were obtained after brain death diagnosis and before organ recovery. Cytokines were assessed by real-time reverse transcriptase-polymerase chain reaction. Soluble serum cytokines were measured by cytometric bead array system. RESULTS:After methylprednisolone treatment, steroid plasma levels were significantly higher (P < 0.05), and a significant decrease in soluble interleukins, monocyte chemotactic protein-1, interleukin-2, interleukin-6, tumor necrosis factor-alpha, and inducible protein-10 was observed. Methylprednisolone treatment resulted in a significant downregulation of intercellular adhesion molecule-1, tumor necrosis factor-alpha, major histocompatibility complex class II, Fas-ligand, inducible protein-10, and CD68 intragraft mRNA expression. Significantly ameliorated ischemia/reperfusion injury in the posttransplant course was accompanied by a decreased incidence of acute rejection. CONCLUSIONS:Our present study verifies the protective effect of methylprednisolone treatment in deceased donor liver transplantation, suggesting it as a potential therapeutical approach.

Several strategies to induce graft tolerance have been developed in animal models. To understand the underlying mechanisms is essential for a clinical adaptation. Here we compared mechanisms of tolerance induction modifying signal 1 (non-depleting anti-CD4 mAb RIB 5/2) with those blocking co-stimulatory signal 2 (CTLA-4Ig). Kidneys from DA donors were grafted into bilaterally nephrectomized LEW rats following a prolonged cold ischemic period (6 h). Tolerance was induced either by application of non-depleting anti-CD4 mAb RIB 5/2 (10 mg/kgx5d; day –1 to 4) or application of CTLA-4Ig (2.5 mg/kg, day 2 post Tx). In both groups adoptive transfer experiments were performed at day 100 into sublethally irradiated naive LEW receiving DA kidneys without further immunosuppression. Animals were followed for an additional 20 days (n = 7/group). All animals receiving RIB 5/2 survived the first observation period compared to only 64 % in the CTLA-4Ig group. Surviving animals showed a normal graft function and moderate structural changes. The percentage of CD4+ T cells of transferred splenocytes was significantly reduced in the CTLA-4Ig group (p < 0.05), while frequencies of CD4+CD25+ T cells were comparable. 20 days after cell transfer, animals initially treated with CTLA-4Ig showed an increased T cell alloreactivity (ELISPOT; p = 0.06) and significantly elevated levels of IFN-J and IL-10 (p < 0.05). Furthermore, frequencies of CD4+CD25+ T cells were reduced in blood, lymph nodes and graft, and significantly decreased in the spleen (8.9 ± 2.4 vs. 15.1 ± 2.4 %, p = 0.001). The rate of activated donor-derived cells within the dendritic cell population (OX62+ DC) was significantly lower in the CTLA-4Ig group (RT1A ab +CD86+ DC, graft: p < 0.01; blood: p < 0.05). At the same time this cell population had significantly increased in spleens and lymph nodes (p < 0.01). In a model of strong histoincompatibility and prolonged cold ischemia an adoptively transferable tolerance could be induced by use of different induction protocols. However, the immune response after adoptive cell transfer was more pronounced in the CTLA-4Ig tolerance induction protocol. Einleitung Im Tiermodell wurden zahlreiche Strategien zur Induktion einer Transplantattoleranz entwickelt. Die Analyse zugrundeliegender Mechanismen unterschiedlicher Toleranzinduktionsprotokolle ist entscheidend fur eine klinische Adaptation. In der vorliegenden Arbeit wurde die Immunantwort nach Modifizierung von Signal 1 (anti-CD4 mAK RIB 5/2) mit den Ereignissen nach Blockade kostimulatorischer Signale (CTLA-4Ig, Signal 2) verglichen. Methodik Nieren von DA Spenderratten wurden nach einer kalten Ischamiezeit von 6 Std. in bilateral nephrektomierte Lewis Empfanger transplantiert. Die Toleranz wurde entweder durch Verabreichung eines

The majority of transplants are derived from donors who suffered from brain injury. There is evidence that brain death causes inflammatory changes in the donor. To define the impact of brain death, we evaluated the gene expression of cytokines in human brain dead and ideal living donors and compared these data to organ function following transplantation. Hepatic tissues from brain dead (n = 32) and living donors (n = 26) were collected at the time of donor laparotomy. Additional biopsies were performed before organ preservation, at the time of transplantation and one hour after reperfusion. Cytokines were assessed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and cytometric bead array. Additionally, immunohistological analysis of tissue specimens was performed. Inflammatory cytokines including IL-6, IL-10, TNF-alpha, TGF-beta and MIP-1alpha were significantly higher in brain dead donors immediately after laparotomy compared to living donors. Cellular infiltrates significantly increased in parallel to the soluble cytokines IL-6 and IL-10. Enhanced immune activation in brain dead donors was reflected by a deteriorated I/R injury proven by elevated alanin-amino-transferase (ALT), aspartat-amino-transferase (AST) and bilirubin levels, increased rates of acute rejection and primary nonfunction. Based on our clinical data, we demonstrate that brain death and the events that precede it are associated with a significant upregulation of inflammatory cytokines and lead to a worse ischemia/reperfusion injury after transplantation.

Previously, we described an enhanced early immune response associated with advanced chronic allograft nephropathy after the engraftment of elderly donor organs. In this study we investigated detailed the early immune response in a model of strong histoincompatibility.

Brain death (BD) of the donor, a risk factor uniquely relevant for organs derived from cadaver donors, influences organ quality by induction of various inflammatory events. Consequently ischemia/reperfusion injury is deteriorated and acute and chronic rejections accelerated. Donor treatment might be an approach to improve the quality of the graft. The induction of heme oxygenase 1 (HO-1) has been shown to exert beneficial effects in living-donor transplantation models. Therefore, we examined the impact of donor treatment with the selective inducer of HO-1, cobalt protoporphyrin (CoPP), on organ quality and transplant outcome in a standardized BD model in a F344-->LEW kidney transplant rat model. Immediately after BD induction, donor animals were administered a single dose of CoPP (5 mg/kg) and in control groups, HO-1 activity was blocked with zinc protoporphyrin (ZnPP, 20 mg/kg). Recipients of organs from brain-dead donors treated with CoPP survived significantly better than those from untreated brain-dead donors (p < 0.05) and intra-graft analysis showed improved histology (p < 0.05). Blockade of HO-1 with ZnPP decreased the survival rates (p < 0.05) comparable to untreated brain-dead donors. Our results demonstrate that HO-1 induction by one single treatment of CoPP in brain-dead donors leads to enhanced allograft survival.

O43* Aims: Experimentally tolerance induction leads to long-term acceptance of grafts. Yet all published experiments describing tolerance were performed with healthy living donors, clinical data are disappointing. It is shown that the brain death of the donor leads to a upregulation of proinflammatory cytokines and therefor activates the donor graft and leads to significant organ dysfunction after transplantation. It is unknown whether brain death interferes with tolerance inducing mechanisms. We investigated the influence of brain death in a established model of tolerance induction with CD4 antibodies. Methods: A standardized model of kidney transplantation (F344 to Lew) with brain dead donors was used. CD4 antibody was given for 5 days (2.5 mg/kg/d), low dose CyA for 10 days (1.5mg/kg/d)(Gp1) in the recipient. Controls received a monotherapy with CyA (Gp2) for 10 days without antibodies. Further controls were living donors which were teated in the same fashion (Gp3 CyA+CD4, Gp4 CyA) To assess functional deterioration, proteinuria was examined every 4 weeks after transplantation up to 52 weeks (n=24/group/time point). To determine the effects of brain death on tolerance induction, grafts (n=5/time point) were examined morphologically by semiquantitative analysis up to 52 weeks after transplantation by histology and immmunhistology (ED1, CD4, CD5, CD8, CD25, MHCII). Alloreactivity was assessed by Elisspot and flowcytometric investigations. Results: Functional abnormalities correlated with the structural changes in all groups. Tolerance induction was sucessful only in the living donor group (Gp3) with stable kidney function, minor morphologic changes and low cellular infiltrates. At 52 weeks both groups with brain dead donor organs showed comparable proteinuria (NS), indicating no beneficial effects of tolernce induction in the brain dead group (Gp1 and 2). Leukocyte infiltration, tubular injury, development of interstitial fibrosis, arteriosclerosis and glomerulosclerosis were comparable between Gp2 and chronically rejecting grafts in Gp1 at all time-points (p=NS). Cellular infiltrates in Gp1 were more marked than in Gp2 begining at 24 weeks (ED1 74 13 vs. 81 18, CD4 160 17 vs. 190 17, CD5 41 7 vs. 63 11*, CD8 37 4 vs. 62 4*, MHCII 197 14 vs. 194 15 cells/view/x40, *p<0.05). Alloreactivity was highest in Gp2, lowest in Gp3. Conclusions: Our data demonstrate that the risk factor donor brain death influences significantly tolerance induction. These results stress the importance of combining tolerance inducing protocols with graft associated risk factors, as well as the importance of donor treatment as a potential approach to diminish organ injury before transplantation.

Introduction: Experimentally tolerance induction protocols lead to long-term acceptance of grafts without morphologic changes and further need of immunosuppression. Yet all published experiments describing tolerance were performed with healthy living donors, clinical data are disappointing. It is shown that the brain death of the donor leads to a significant upregulation of proinflam-matory cytokines and therefore activates the donor graft and leads to significant organ dysfunction after transplantation. It is unknown whether the central injury of the organ donor interferes with tolerance inducing mechanisms. We investigated the influence of brain death in a established model of tolerance induction with CD4 antibodies. Methods: A standardized model of kidney transplantation (F344 to Lew) with BD-donors was used. CD4 antibody was given for 5 days (2.5 mg/kg/d), low dose CyA for 10 days (1.5 mg/kg/d)(Gpl) in the recipient. Controls received a monotherapy with CyA (Gp2) for 10 days. To assess functional deterioration, proteinuria was examined every 4 weeks after Tx up to 40 weeks (n = 24/group/time point). To determine the effects of BD on tolerance induction, grafts (n = 4 - 6/time point) were examined morphologically by semiquantitative analysis up to 40 weeks after Tx by histology and immunhistology (ED 1, CD4, CD5, CD8, MHCII, HO-1). Results: Functional abnormalities correlated with the structural changes in both groups. At 24 weeks both groups showed comparable proteinuria (34 ± 11 vs. 28 ± 5 mg/dl/24 h, p = NS). Leukocyte infiltration, tubular injury, development of interstitial fibrosis, arteriosclerosis and glomerulosclerosis were comparable between Gp2 and chronically rejecting grafts in Gpl (p = NS). Cellular infiltrates in Gpl were more marked than in Gp2 (EDI 74 ± 13 vs. 81 ± 18, CD4 160 ± 17 vs. 190 ± 17, CD5 41 ± 7 vs. 63 ± 11*, CD8 37 ± 4 vs. 62 ± 4*, MHCII 197 ± 14 vs. 194 ± 15 cells/view/x40, *p < 0.05). Conclusions: Our data suggest that the risk factor donor brain dead may influence tolerance induction, due to an unspecific injury prior to transplantation. These results stress the importance of donor treatment as a potential approach to improve donor organ quality and to diminish brain death induced injury.