Graft Protection Research Articles

Preemptive Donor Apoptotic Cell Infusions Induce IFN-γ-Producing Myeloid Derived Suppressor Cells for Cardiac Allograft Protection.

We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid derived suppressor cells (MDSCs) in the graft protection provided by infusion of such apoptotic donor cells is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b+ cells in the spleen that phenotypically resemble monocytic-like (CD11b+Ly6CHI) and granulocytic-like (CD11b+Gr1HI) MDSCs. Both populations suppress T cell proliferation in vitro, and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b+Gr1HI cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ dependent mechanisms. These findings reveal a novel link between apoptotic donor cell infusions and induction of MDSCs as a mechanism for transplant graft protection, and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.

CD8 Intrinsic Defects in Myd88-/- Recipients Determine the Effective Control of Effector T Cell Accumulation in Kidney Allografts.

Previous reports indicate that MyD88 plays distinct roles in transplantation by regulating DC maturation and T cell priming. The Purpose of this study is to determine whether Myd88 signaling in T cells is required in kidney allograft rejection in mice. Methods: Orthotopic kidney transplants were performed between MHC fully mismatched mice, in which B6 kidneys were transplanted into binephrectomized wild type (WT) or MyD88-/- BALB/c mice (allografts). To determine whether Myd88 expression in T cells is required for accumulation of effector T cells in the kidney allografts, we used a dual adoptive transfer approaches in which 1:1 mixed naïve CD8 T cells from both Myd88-/- and WT mice were infused into BALB/c RAG1-/- mice. Kidney grafts were examined by histology and phenotypes of cellular infiltrates were identified by immunohistochemistry and flow cytometry between POD7 and 14. T cell function was assessed by a mixed lymphocyte reaction. Results: Recipient MyD88 is not required for the initiation of acute allograft rejection featured by cellular infiltration and tubulitis, however, recipient MyD88 deficiency results in spontaneous clearance of the rejection response and subsequent restoration of renal graft function, leading to long-term allograft survival compared to MyD88 sufficient (WT) recipients. The graft protection was associated with a reduced intragraft accumulation of CD11b-Gr-1+ cells and activated CD8+ T cells in the MyD88-/- mice. T cells, specifically CD8 T cells, from the MyD88-/- recipients failed to mount a donor specific recall response as indicated by diminished proliferation and cytokine production upon donor antigen re-stimulation in vitro. Interestingly, exogenous IL-6 restored the proliferation of T cells from MyD88-/- recipients to that of T cells from WT recipients. Furthermore, adoptively transferred MyD88-/- CD8 T cells in RAG1-/- recipients exhibited a diminished expression of chemokine receptors, specifically CCR4 and CXCR3. This was accompanied by an impaired ability to accumulate in the kidney allografts, even in a MyD88 sufficient environment. Conclusion: This study provides a novel mechanism linking the lack of intrinsic MyD88 signaling in T cells to compromised effector T cell expansion and accumulation in the kidney allografts, thereby leading to the spontaneous resolution of initial cellular infiltration of kidney allografts and long-term graft acceptance.

Preemptive donor apoptotic cell infusions induce IFN-γ-producing myeloid-derived suppressor cells for cardiac allograft protection.

We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid-derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b(+) cells in the spleen that phenotypically resemble monocytic-like (CD11b(+)Ly6C(high)) and granulocytic-like (CD11b(+)Gr1(high)) MDSCs. Both populations suppress T cell proliferation in vitro and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b(+)Gr1(high) cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ-dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction.

Real-time immune cell interactions in target tissue during autoimmune-induced damage and graft tolerance

Real-time imaging studies are reshaping immunological paradigms, but a visual framework is lacking for self-antigen-specific T cells at the effector phase in target tissues. To address this issue, we conducted intravital, longitudinal imaging analyses of cellular behavior in nonlymphoid target tissues to illustrate some key aspects of T cell biology. We used mouse models of T cell-mediated damage and protection of pancreatic islet grafts. Both CD4(+) and CD8(+) effector T (Teff) lymphocytes directly engaged target cells. Strikingly, juxtaposed β cells lacking specific antigens were not subject to bystander destruction but grew substantially in days, likely by replication. In target tissue, Foxp3(+) regulatory T (Treg) cells persistently contacted Teff cells with or without involvement of CD11c(+) dendritic cells, an observation conciliating with the in vitro "trademark" of Treg function, contact-dependent suppression. This study illustrates tolerance induction by contact-based immune cell interaction in target tissues and highlights potentials of tissue regeneration under antigenic incognito in inflammatory settings.

Efficacy of DHMEQ, a NF-κB Inhibitor, in Islet Transplantation

Pancreatic islet transplantation (PITx) is an attractive treatment option for restoring appropriate glucose homeostasis in type 1 diabetes patients. Although islet grafts can successfully engraft after PITx, large numbers of islet grafts are required mainly because immune reactions, including inflammation, destroy islet grafts. In these processes, nuclear factor (NF)-κB plays a central role. We hypothesized that the inhibition of NF-κB activation would ameliorate inflammatory responses after PITx and aid successful engraftment. To test this hypothesis, a newly developed NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), was used on a syngeneic mouse PITx model. One hundred seventy-five islets from C57BL/6 (B6) mice were transplanted into streptozotocin-induced diabetic B6 mice. The recipient mice were administered DHMEQ for 1, 2, or 3 days after PITx. The underlying mechanisms of DHMEQ on islet graft protection were investigated in an in vitro coculture model of pancreatic islets and macrophages. With a vehicle treatment, only 11.1% of the islet-recipients achieved normoglycemia after PITx. In sharp contrast, DHMEQ treatment markedly improved the normoglycemic rate, which was associated with the suppression of serum high mobility group complex-1 (HMGB1) and proinflammatory cytokines, including tumor necrosis factor-α, monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, interleukin-1β, and interleukin-6, after PITx. In a murine macrophage-like cell line, DHMEQ inhibited HMGB1-driven activation and proinflammatory cytokine secretion and further prevented death isolated islets after coculture with these activated macrophages. Inhibition of NF-κB activation by DHMEQ after PITx reduces the HMGB1-triggered proinflammatory responses and results in engraftment of transplanted islets even with fewer islet grafts.

Protection of islet grafts through transforming growth factor-β-induced tolerogenic dendritic cells.

In type 1 diabetes, the insulin-producing β-cells are destroyed by the immune system. One way of restoring glucose control is to transplant β-cells from a donor. Although this procedure may restore endogenous insulin production, immunosuppressive treatment is needed to prevent the recipient from rejecting the donor-derived islets. We investigated the possibilities of transient expression of the immunosuppressive cytokine transforming growth factor (TGF)-β within islets to achieve long-term graft tolerance. We found that brief expression of TGF-β prevented rejection of syngeneic islets, that there was reduction of dendritic cell (DC) activation in the graft, and that there was reduced reactivation of T cells in the graft-draining lymph nodes. In vitro exposure of bone marrow–derived DCs to TGF-β reduced expression of costimulatory molecules CD80 and CD86, as well as production of proinflammatory cytokines such as interleukin-12 p70 in DCs, but did not alter levels of major histocompatibility complex classes I and II. Furthermore, the capacity of TGF-β–treated bone marrow–derived DCs to activate both CD4+ and CD8+ T cells was reduced. Adding TGF-β–conditioned tolerogenic DCs to the grafted islets led to long-term survival of the graft, demonstrating that TGF-β–induced tolerogenic DCs can provide an effective means to restore immune tolerance in an already established autoimmune disease.

Initial topical cooling followed by backtable Celsior flush perfusion provides excellent early graft function in porcine single lung transplantation after 24 hours of cold ischemia

Topical in situ cooling of the donor lungs is a prerequisite for procurement of non-heart-beating donor lungs and may be of interest for living related lung donation. Twenty-four single lung transplants were performed in 4 groups of Landrace pigs (6 per group). Control LPD, control Celsior and topical cooling in situ, followed by LPD (exLPD) or Celsior (exCel) ex situ flush, were employed. All lungs were perfused antegrade with 1 liter of solution at 4°C. Lungs were stored immersed in preservation solution for 24 hours at 4°C. After transplantation of the left lung, the right recipient bronchus and pulmonary artery were clamped. Four of 6 animals each in the LPD and Celsior groups and all 6 animals in both the exLPD and the exCel groups survived the 7-hour reperfusion. The mean oxygenation index was favorably preserved in the exCel group at 7 hours after reperfusion (417 ± 81) over all other groups (LPD 341 ± 133, Celsior 387 ± 86, exLPD 327 ± 76; p < 0.0001). Pulmonary vascular resistance showed significantly lower values in the Celsior and exCel groups (LPD 1,310 ± 620, Celsior 584 ± 194, exLPD 1,035 ± 361, exCel 650 ± 116 dyn/s/cm(5) at 7 hours after reperfusion; p < 0.0001). Consistently, the wet-to-dry lung weight ratio also indicated beneficial graft protection in the exCel group (LPD 8.1 ± 0.8, Celsior 8.4 ± 0.8, exLPD 7.5 ± 1.0, exCel 3.1 ± 0.9; p < 0.0001). Initial topical cooling followed by backtable perfusion is a sufficient technique for pulmonary graft preservation providing excellent post-transplant function. Celsior subsequent to in-situ topical cooling revealed the most beneficial results in this setting. This combined technique could advance non-heart-beating, living related lung lobe donation and, potentially, regular heart-beating lung donation.

Are Nonesterified Fatty Acids Protective in Chronic Allograft Nephropathy?

Chronic allograft nephropathy is understood to be a multifactorial process that involves synergistic and time-dependent effects of both immunologic and nonimmunologic factors (1). The characteristic structural abnormalities of glomerulosclerosis, tubulointerstitial fibrosis, and arteriosclerosis are similar to chronic nephron loss in native kidneys, suggesting that the factors responsible for the progression of chronic kidney disease may be generic (1). Apart from the dissection of the role of ischemia, the identification of other nonimmunologic factors underlying chronic allograft loss has received little attention. The role of lipids in mediating the progression of chronic allograft loss is not well understood. Hyperlipidemia is a common feature in the renal transplantation population, but based on a small number of studies, statins do not appear to affect graft function (2). Nonesterified (or free) fatty acids (NEFAs) are the main building blocks of triglycerides, which consists of three fatty acids linked to a glycerol backbone. Structurally, NEFAs are carboxylic acids with long-chain hydrocarbon groups and may be classified into various categories (essential vs. nonessential; mono/polyunsaturated vs. saturated). NEFAs serve as a key metabolic fuel for the body and are released from adipose tissue in response to systemic hormones (glucagon, corticosteroids, adrenocorticotropic hormone, catecholamines) acting through hormone-sensitive lipase (present in the cytosol of adipocytes). Besides this, NEFAs are likely to have pathologic roles because they are increased in obesity, type 2 diabetes, and many other chronic inflammatory diseases (3). Moreover, certain types of NEFAs are precursors of bioactive eicosanoids, such as prostaglandin E2 (3), have antibacterial properties (4), and all probably act as secondary messengers capable of upregulating or downregulating proinflammatory transcription factor (nuclear factor-κB) and transcriptomes (tumor necrosis factor α interleukin 1β, interleukin 6, monocyte chemotactic protein 1), depending on the type of NEFA (5). In this issue, Klooster et al. (6) started with a hypothesis that NEFAs have a detrimental role in chronic allograft dysfunction. Because 99.9% of NEFAs are albumin bound, the hypothesis was based on previous experimental data suggesting that the glomerular ultrafiltration of NEFA-bound albumin leads to luminal toxicity, promoting tubulointerstitial inflammation in experimental models of proteinuric renal disease. The results, however, were converse to the authors’ hypothesis and expectations. Using a longitudinal study design, they showed that posttransplantation serum levels of NEFAs (from appropriately collected samples and using a colorimetric assay that measured both “free” and albumin-bound NEFAs) are inversely correlated with better graft function in 461 renal transplant recipients with an average follow-up of 7.1 years (hazard ratio of 0.61 using multivariate analysis). These results were independent of body mass index, proteinuria, anti–human leukocyte antigen antibodies, and type of rejection. One of the main limitations of these data is that it is based on a single measurement of NEFA. NEFAs are a biomarker of negative energy balance, and levels can be highly variable and influenced by systemic hormones and nutritional status, limiting their routine use in clinical practice. Nevertheless, further investigation of these intriguing observations is warranted. Perhaps the most vexing questions are what could be the mechanisms of graft protection and which type of NEFA is important. These questions have in part been addressed by a previous study (7) and interestingly mirror the data of Klooster et al. (6). In a carefully undertaken study, Baker et al. (7) reported an inverse relationship between pretransplantation serum levels of NEFA (for the omega-6 and omega-3 polyunsaturated families measured using high-performance liquid chromatography) and graft outcome in 130 nonconsecutive renal transplant recipients, and revealed that an inverse relationship was present for the n-6 family (arachidonic acid and γ-linoleic acid) but not for the n-3 family (α-linoleic, eicosapentaenoic, and docosahexaenoic). For arachidonic acid, the highest quartile was associated with an 80% reduction in graft failure. These data raise the hypothesis that the protective effects of NEFAs against graft loss may relate to the direct immunomodulatory effects of specific NEFAs (such as arachidonic acid) rather than the hypothesized nonspecific luminal nephrotoxicity of all NEFAs-coupled albumin involving glomerular ultrafiltration (6). Other potential mechanisms of the renoprotection include the possibility of the effects of NEFAs on either infection or on drug handling of immunosuppressants (6). Clearly, there are many complexities to resolve. If indeed levels of NEFA are found to prevent graft loss, then what impact will this have on cardiovascular disease and posttransplantation diabetes, given that obesity and insulin resistance are positively correlated with NEFAs in renal transplant recipients (8)? Meanwhile, the task of understanding the role of common factors in the progression of chronic native and transplant kidney disease remains important.

Histological and ultrastructural findings of corneal tissue after failed descemet membrane endothelial keratoplasty

To investigate in a retrospective review the histological and ultrastructural findings after failed primary and early Descemet membrane endothelial keratoplasty (DMEK), propose possible pathomechanisms of graft failure and give clinical implications. The explanted grafts underwent light- and electromicroscopical investigations in eight failed DMEK cases. Haematoxylin - Eosin, periodic acid Schiff and Alcian blue stainings were performed. Special note was given to any residual stromal remnants, absence of endothelial cells, lamellar structure and 'activation' of keratocytes. Of the eight cases, six were re-DMEKs and two penetrating keratoplasties. Partial graft separation was seen in six and no graft separation in two of the cases. The average time-interval to the re-DMEK or penetrating keratoplasty was 4.6 months. Light and electron microscopy of the two explanted stromal specimens showed varying degrees of keratocyte activation. Endothelial cell loss was observed in essentially all explants with varying degrees and positive correlation with intraoperative difficulty. Assumed upside-down situations showed large areas of intact endothelial cells. In addition, a new layer, situated between the endothelial cell layer and the posterior nonbanded layer, was observed with loose intercellular structure. A loss of the endothelial cell layer of varying degrees and positive correlation with intraoperative difficulty are the prominent feature of primary and early DMEK graft failure. Of note is the upside-down situation, in which in some cases, the endothelial cell layer not only remains intact but also demonstrates metabolical activity in forming a novel cellular layer.

Addition of Vardenafil into Storage Solution Protects the Endothelium in a Hypoxia-Reoxygenation Model

Based upon the well known protective effect of intracellular cyclic guanosine monophosphate (cGMP) accumulation, we tested the hypothesis that storage solution enriched with optimal concentration of the phosphodiestherase-5 inhibitor vardenafil could provide better protection of vascular grafts against reperfusion injury after long-term cold ischaemic storage. Isolated thoracic aorta obtained from rats underwent 24-h cold ischaemic preservation in physiological saline or vardenafil (10(-11) M)-supplemented saline solution. Reperfusion injury was simulated by hypochlorite (200 μM) exposure for 30 minutes. Endothelium-dependent vasorelaxation was assessed, and histopathological and molecular-biological examination of the aortic tissue were performed. Compared with the control group, the saline group showed significantly attenuated endothelium-dependent maximal relaxation (Rmax) to acetylcholine after hypoxia-reoxygenation, which was significantly improved by vardenafil supplementation (Rmax control: 98 ± 1%; saline: 48 ± 6%; vardenafil: 75 ± 4%; p < .05). Vardenafil treatment significantly reduced DNA strand breaks (control: 10.6 ± 6.2%; saline: 72.5 ± 4.0%; vardenafil: 14.2 ± 5.2%; p < .05) and increased cGMP score in the aortic wall (control: 8.2 ± 0.6; saline: 4.5 ± 0.3; vardenafil: 6.7 ± 0.6; p < .05). Our results support the view that impairment of intracellular cGMP signalling plays a role in the pathogenesis of the endothelial dysfunction induced by cold storage warm reperfusion, which can be effectively reversed by pharmacological phosphodiesterase-5 inhibition.

Targeting Purinergic Receptors in Islet Transplantation

Transplanted islets are exposed to a distinctively hostile immunological environment. Grafted islets typically encounter a combination of innate, allogeneic and anti-islet immune responses—the relative contributions of which are not fully understood. The relatively low success rate of islet transplantation compared with other organs suggests that current immunomodulatory protocols are inadequate to provide long-term islet graft protection. As the negative consequences of long-term immunosuppression can arguably outweigh the benefits of islet transplantation, there is intense focus on the development of strategies to induce immune tolerance toward grafts, thereby avoiding the requirement of chronic immunosuppression. In this issue of Diabetes , Vergani et al. (1) provide evidence that targeting purinergic receptor signaling is a viable strategy for islet allograft protection. Extracellular ATP modulates many aspects of the immune system. For example, ATP signaling is able to promote the differentiation of proinflammatory Th17 cells (2), which may play a role in islet rejection and recurrent autoimmunity. ATP signaling can also suppress the differentiation and function of regulatory T cells (Tregs) (3), which are immunosuppressive T cells that play a role in immune tolerance (4). Thus, ATP signaling acts to shift the immune response toward a more inflammatory, less tolerant state (Fig. 1). FIG. 1. Schematic summary of the proposed mechanism of action of ATP during islet allograft rejection. A : Extracellular ATP activates P2X receptors and promotes T-cell …

Correction: Combined Strategy of Endothelial Cells Coating, Sertoli Cells Coculture and Infusion Improves Vascularization and Rejection Protection of Islet Graft

Correction: Combined Strategy of Endothelial Cells Coating, Sertoli Cells Coculture and Infusion Improves Vascularization and Rejection Protection of Islet Graft

Influence of pharmacological immunomodulatory agents on CD4+CD25highFoxP3+ T regulatory cells in humans

T regulatory cells (Tregs) play a critical role in the immunologic tolerance to the graft in transplantation. Thus, due to their immunosuppressive capability, ex vivo expanded Tregs may be used as a cellular therapy and an attractive novel strategy to control chronic rejection and eliminate need for lifelong pharmacological immunosuppression. Since Treg therapy is still in its infancy, initially Tregs still need to be applied in combination with pharmacological agents to prevent rejection. Fortunately, some of the medications have been shown to enhance the function and number of Tregs. In the clinic, different immunosuppressive regimens are used for individual patients for different types of organ transplantation. In this review, we present the most commonly used pharmacological agents for immunosuppression and discuss how they affect the Treg population. It is extremely difficult to dissect the effect of single agent on Tregs population in clinical settings since usually the combination of several medications is applied at the same time for graft protection. Nevertheless, experimental and clinical data indicate that thymoglobulin as immunosuppressive induction and mTOR inhibitors as immunosuppressive maintenance agents have the most beneficial effect on Treg population in the blood. Among supplemental agents promoting Tregs, anti-TNFα preparations have been in clinical use (in autoimmune diseases) for many years, so they are optimal candidates for testing in transplant settings in combination with Treg based cellular therapy.

Combined Strategy of Endothelial Cells Coating, Sertoli Cells Coculture and Infusion Improves Vascularization and Rejection Protection of Islet Graft

Improving islet graft revascularization and inhibiting rejection become crucial tasks for prolonging islet graft survival. Endothelial cells (ECs) are the basis of islet vascularization and Sertoli cells (SCs) have the talent to provide nutritional support and exert immunosuppressive effects. We construct a combined strategy of ECs coating in the presence of nutritious and immune factors supplied by SCs in a co-culture system to investigate the effect of vascularization and rejection inhibition for islet graft. In vivo, the combined strategy improved the survival and vascularization as well as inhibited lymphocytes and inflammatory cytokines. In vitro, we found the combinatorial strategy improved the function of islets and the effect of ECs-coating on islets. Combined strategy treated islets revealed higher levels of anti-apoptotic signal molecules (Bcl-2 and HSP-32), survival and function related molecules (PDX-1, Ki-67, ERK1/2 and Akt ) and demonstrated increased vascular endothelial growth factor receptor 2 (KDR) and angiogenesis signal molecules (FAk and PLC-γ). SCs effectively inhibited the activation of lymphocyte stimulated by islets and ECs. Predominantly immunosuppressive cytokines could be detected in culture supernatants of the SCs coculture group. These results suggest that ECs-coating and Sertoli cells co-culture or infusion synergistically enhance islet survival and function after transplantation.

Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold ischemia reperfusion injury

BackgroundThe dramatic shortage of organs leads to consider the steatotic livers for transplantation although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) at low and non-toxic dose for fatty liver graft protection against cold IRI. ExperimentalSteatotic and non-steatotic livers have been stored in UW enriched with BRZ (100nM) or MG132 (25μM), for 24h at 4°C and then subjected to 2-h normothermic reperfusion (37°C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity (ChT), and UPS (19S and 20S5 beta) protein levels have been measured. ResultsChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers. ConclusionMG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications in the prevention of IRI.

High graft protection and low incidences of infections, malignancies and other adverse effects with intra-operative high dose ATG-induction: A single centre cohort study of 760 cases

In 1990 we introduced the intra-operative single high-dose induction (HDI) with ATG-Fresenius as a novel renal sparing concept. The aim of this analysis was to compare both the long-term patient and graft survival and the incidences of adverse effects in recipients treated with standard triple-drug therapy (TDT) alone or with an additional HDI with ATG-F. A total of 760 renal transplant recipients receiving either TDT, consisting of steroids, azathioprine and cyclosporine (n=238) or TDT + 9mg/kg ATG-F intra-operatively (n=522) were included in this retrospective analysis. Compared to the TDT cohort the graft and patient survival over the entire ten year period was significantly prolonged in the TDT+HDI cohort. In contrast, main adverse effects (TDT+HDI vs. TDT) such as malignancies (4.4 vs. 2.1%), PTLD (0.4 vs. 0.4%), CMV diseases (18.6 vs. 15.5%), Herpes zoster infections (2.9 vs. 1.3%), bacterial pneumonias (3.1 vs. 1.3%) and post-operative thrombocytopenia <50×10³/µl (0.5 vs. 1.3%) did not significantly differ between the two immunosuppressive regimens. Only CMV-IgM seroconversions occurred significantly more in the HDI cohort (39.3 vs. 23.5%). The absolute numbers of CD3, CD4 and CD8 cell counts were significantly reduced in TDT+ATG-F HDI cohort only over a time period of about five days. This world-wide largest single-centre cohort analysis clearly shows the superiority of the HDI with ATG-F compared to TDT alone in improving long-term graft survival without increasing the risk for infections, malignancies or other adverse effects.

P115 Induction Immunosuppression (IS) with Antithymocyte Globulin (ATG) Followed by Mycophenolic Acid (MPA) Significantly Reduces Risk of Acute Cellular Rejection (ACR) But May Increase the Risk of Post-Transplant Lymphoproliferative Disease (PTLD) Post Lung Transplantation (LTx)

IntroductionThe ideal IS post LTx is unknown and the balance between graft protection against alloimmune injury vs the risks of infection and malignancy is a delicate one. Our unit adopted...

How Safe Is Conversion from Twice-Daily Tacrolimus to Once-Daily Prolonged Formulation? Single Centre Experience

Introduction: The calcineurin inhibitor tacrolimus, is widely available as a twice- daily formulation (Tacrolimus BID). A once- daily prolonged- release formulation (Tacrolimus QD) has been developed to improve the adherence and provide the long- lasting graft protection. Methods: The objective of this retrospective single centre study was to analyze 589 kidney transplant recipients who underwent conversion from Tacrolimus BID to Tacrolimus QD in 11/2007-09/2010. The aim of the study was to compare the renal function in patients before and after conversion and to determine the incidence of acute rejection after switch to Tacrolimus QD during the 12 month follow-up. The rejection was histologically verified in indication biopsies performed due to graft function deterioration. Maintenance immunossuppresive therapy was based on tacrolimus, mycophenolate mophetil and corticosteroids in majority of patients. Results: The median time of conversion was 4.1 years after transplantation. We observed no graft function deterioration (eGFR-MDRD before 52.8 ± 17.4 ml/min/1.73m2 vs. after 46.2 ± 19.2 ml/min/1.73m2, p = n.s.,) nor the significant change in Tacrolimus trough level (5.07 ± 1.22 μg/L vs. 4.93 ± 1.01 μg/L, p = n.s.) after switch to Tacrolimus BID. Rejection episodes occurred at median time 10 months after conversion. We assessed the rejections with regard to time to drug switch and observed higher rate of late rejection (acute rejection 2 %, chronic rejection 1.6 %) compared to early rejections within 3 months after conversion (0.5 % acute and 0.3 % chronic rejection). No new-onset of diabetes mellitus after transplantation was recorded. 1- year graft survival was 97 %. 49 out of 589 patients (8 %) didn't finish the follow-up due to various reasons (graft failure, intolerance of Tacrolimus QD, version to mTOR inhibitors due to malignancy or severe arteriolopathy). Conclusions: No differences in renal function, tacrolimus level was found after conversion from Tacrolimus BID to Tacrolimus QD and incidence of acute rejection was low. These findings suggest that a once-daily morning dosing regimen maximizes the potential for adherence and may improve the long- term outcomes.

Small-For-Size Liver Transplantation Increases Pulmonary Injury in Rats: Prevention by NIM811

Introduction: Pulmonary complications occurring after liver transplantation frequently lead to mortality. Transplantation of small-forsize liver grafts increases the risk of graft failure. Accordingly, the aim of this study was to investigate whether acute pulmonary injury occurs after small-for-size liver transplantation. Moreover, we reported that NIM811, an inhibitor of the mitochondrial permeability transition (MPT), prevents dysfunction of small-for-size liver grafts. Here, we investigated whether protection of liver grafts by NIM811 also attenuated acute pulmonary injury after small-for-size liver transplantation. Methods: Livers were reduced to 50% of original size, stored in cold UW solution for 6 h and then implanted into recipients of the same or about twice the donor body weight, resulting in half-size and quarter-size liver grafts, respectively. NIM811 (5 μM) was added to the storage solution. Lungs and livers were harvested at 5, 18 and 38 h after implantation. Results: Hepatic necrosis and alanine aminotransferase release were higher after transplantation of quarter-size than full-size and half-size grafts, indicating more severe liver injury. Liver regeneration increased in half-size grafts but was suppressed in quarter-size grafts. NIM811 blocked liver injury and promoted regeneration of quarter-size grafts, as observed previously. Pulmonary histological alterations were minimal at 5-18 h after liver transplantation. After 38 h, infiltration of neutrophils and monocytes/macrophages to pulmonary alveoli occurred with septal thickening and increased cellularity in quarter-size graft recipients. These changes were mild in lungs of full-size and half-size graft recipients. NIM811 decreased alveolar septal thickening, monocyte/macrophage infiltration and neutrophil infiltration by 52, 44 and 51%, respectively. Infiltrating leukocytes can produce reactive oxygen and nitrogen species, leading to cell death. 4-Hydroxynonenal and 3-nitrotyrosine adducts increased markedly in pulmonary leukocytes, vascular endothelial cells and alveolar epithelial cells in quarter-size graft recipients, indicating oxidative/nitrosative stresses. TUNEL-positive vascular endothelial/alveolar epithelial cells were 12- and 8- fold higher in lungs of quarter-size compared to full-size and half-size graft recipients, indicating apoptosis. NIM811 blunted 4-hydroxynonenal and 3-nitrotyrosine adduct formation and attenuated apoptosis by >80% after quarter-size liver transplantation. After transplantation, hepatic IL-1β and TNFα mRNA were 3.3 and 2.5-fold higher in quarter-size than full-size grafts. Pulmonary ICAM-1 expression was also markedly higher in quarter-size graft recipients. NIM811 decreased hepatic IL-1β and TNFα as well as pulmonary ICAM-1 expression. Conclusion: Together, failing small-for-size grafts produce toxic cytokines, leading to pulmonary inflammation and injury. NIM811 decreased hepatic injury and toxic cytokine formation, thus attenuating acute pulmonary injury after small-for-size liver transplantation (NIDDK).

Intragraft CD11b+IDO+ Cells Mediate Cardiac Allograft Tolerance by ECDI-Fixed Donor Splenocyte Infusions

We have previously shown that pre- and post-transplant infusions of donor splenocytes treated with 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide (ECDI-SPs) provide permanent donor-specific protection of islet allografts. The efficacy of donor ECDI-SPs in protecting vascularized cardiac allografts and mechanism(s) of protection are unknown. In this study, we show that infusions of ECDI-SPs significantly prolong cardiac allograft survival concomitant with an impressive accumulation of CD11b(+) IDO(+) cells in the cardiac allograft, and that the presence of this population is dependent on Gr1(+) cells. Consequently, depletion of Gr1(+) cells or inhibition of indoleamine 2,3 dioxygenase (IDO) activity abrogates graft protection by ECDI-SPs infusions. In addition, T cells from ECDI-SPs treated recipients secrete high levels of interleukin 10 and interleukin 13 upon in vitro restimulation, which are also dampened in recipients treated with the IDO inhibitor. Furthermore, combination of donor ECDI-SPs with a short course of rapamycin provides indefinite cardiac allograft survival in 100% of the recipients. These findings reveal a novel mechanism of donor ECDI-SPs in inducing cardiac transplant tolerance and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.