Rupert Oberhuber

Effects of brain death on organ quality and transplant outcome

The inferiority of organs from brain dead donors is reflected by impaired graft survival and patient outcome. Brain death effects hemodynamic stability, hormonal changes, and neuroimmunologic effects and unleashes a cascade of inflammatory events. Despite considerable efforts in experimental and clinical research, most of the mechanisms linked to brain death are only appreciated on a descriptive level. This overview presents our current understanding of the pathophysiology and consequences of brain death on organ injury and summarizes available therapeutic interventions.

IDO and Regulatory T Cell Support Are Critical for Cytotoxic T Lymphocyte-Associated Ag-4 Ig-Mediated Long-Term Solid Organ Allograft Survival

Costimulatory blockade of CD28-B7 interaction with CTLA4Ig is a well-established strategy to induce transplantation tolerance. Although previous in vitro studies suggest that CTLA4Ig upregulates expression of the immunoregulatory enzyme IDO in dendritic cells, the relationship of CTLA4Ig and IDO in in vivo organ transplantation remains unclear. In this study, we studied whether concerted immunomodulation in vivo by CTLA4Ig depends on IDO. C57BL/6 recipients receiving a fully MHC-mismatched BALB/c heart graft treated with CTLA4Ig + donor-specific transfusion showed indefinite graft survival (>100 d) without signs of chronic rejection or donor specific Ab formation. Recipients with long-term surviving grafts had significantly higher systemic IDO activity as compared with rejectors, which markedly correlated with intragraft IDO and Foxp3 levels. IDO inhibition with 1-methyl-dl-tryptophan, either at transplant or at postoperative day 50, abrogated CTLA4Ig + DST-induced long-term graft survival. Importantly, IDO1 knockout recipients experienced acute rejection and graft survival comparable to controls. In addition, αCD25 mAb-mediated depletion of regulatory T cells (Tregs) resulted in decreased IDO activity and again prevented CTLA4Ig + DST induced indefinite graft survival. Our results suggest that CTLA4Ig-induced tolerance to murine cardiac allografts is critically dependent on synergistic cross-linked interplay of IDO and Tregs. These results have important implications for the clinical development of this costimulatory blocker.

Intracellular signaling pathways control mitochondrial events associated with the development of ischemia/ reperfusion-associated damage.

Ischemia (I) and reperfusion (R) trigger a series of events, which culminate in severe injury to the transplanted organ. Cell death resulting from the formation of mitochondrial reactive oxygen species (ROS) coupled with the perturbation of mitochondrial Ca2+ homeostasis is central to the development of IR‐associated tissue damage. We and others have shown recently that intracellular signaling pathways critically control these mitochondrial changes, making them potential targets for therapeutic intervention. Using a heterotopic murine heart transplant model as well as primary and immortalized cardiomyocyte cells we established the activity patterns of mitogen‐activated protein kinases (MAPKs) ERK, JNK, and p38 during IR, and probed into their role in the perturbation of mitochondrial ROS and Ca2+ homeostasis, which are necessary for cardiomyocyte death. Our results showed a strong activation of all three MAPKs as well as a rise in mitochondrial ROS and Ca2+ during early reoxygenation. Inhibiting p38 kinase most efficiently prevented ROS production, Ca2+ overload and cell death, suggesting that targeting this signaling molecule may provide a possible strategy to limit the effects of IR.

T-regulatory cell treatment prevents chronic rejection of heart allografts in a murine mixed chimerism model

Background The mixed chimerism approach induces donor-specific tolerance in both pre-clinical models and clinical pilot trials. However, chronic rejection of heart allografts and acute rejection of skin allografts were observed in some chimeric animals despite persistent hematopoietic chimerism and tolerance toward donor antigens in vitro. We tested whether additional cell therapy with regulatory T cells (Tregs) is able to induce full immunologic tolerance and prevent chronic rejection. Methods We recently developed a murine “Treg bone marrow (BM) transplantation (BMT) protocol” that is devoid of cytoreductive recipient pre-treatment. The protocol consists of a moderate dose of fully mismatched allogeneic donor BM under costimulation blockade, together with polyclonal recipient Tregs and rapamycin. Control groups received BMT under non-myeloablative irradiation and costimulation blockade without Treg therapy. Multilineage chimerism was followed by flow cytometry, and tolerance was assessed by donor-specific skin and heart allografts. Results Durable multilineage chimerism and long-term donor skin and heart allograft survival were successfully achieved with both protocols. Notably, histologic examination of heart allografts at the end of follow-up revealed that chronic rejection is prevented only in chimeras induced with the Treg protocol. Conclusions In a mouse model of mixed chimerism, additional Treg treatment at the time of BMT prevents chronic rejection of heart allografts. As the Treg-chimerism protocol also obviates the need for cytoreductive recipient treatment it improves both efficacy and safety over previous non-myeloablative mixed chimerism regimens. These results may significantly impact the development of protocols for tolerance induction in cardiac transplantation.

Combined pancreas-kidney transplantation for patients with end-stage nephropathy caused by type-2 diabetes mellitus.

Background Simultaneous pancreas-kidney (SPK) transplantation is widely accepted as an optimal therapeutic option for patients with type 1 diabetes mellitus (T1DM) and end-stage renal disease, but the indication for patients with type 2 diabetes mellitus (T2DM) is still controversially discussed. Methods Twenty-one T2DM recipients of a first combined pancreas-kidney graft performed at our center during a 9-year period were retrospectively analyzed with regard to demographic characteristics; cardiovascular risk factors; surgical, immunological, and infectious complications; and patient and graft survivals and compared with T1DM recipients (n=195) and 32 T2DM patients who received a kidney transplant alone (KTA) during the same period. Results Patient survival at 1 and 5 years was 96.9% and 91.6% for the T1DM group, 90.5% and 80.1% for the T2DM group, and 87.1% and 54.2% for the T2DM KTA group, respectively (P<0.001). Actuarial pancreas graft survival for SPK recipients at 1 and 5 years was calculated to be 92.6% and 80.7% for the T1DM group and 81.0% and 75.9% for the T2DM group, respectively (P=0.19). Kidney allograft survival at 5 years was 83.6% for T1DM, 80.4% for T2DM, and 52.7% for T2DM KTA (P<0.0001). Multivariate analysis adjusting for donor and recipient age, secondary complications of diabetes, body mass index, waiting time, cold ischemic time, delayed graft function, and coronary risk factors showed that differences did not remain statistically significant. Conclusion Favorable results can be achieved with SPK transplantation in type 2 diabetics with a low coronary risk profile. A high cardiac death rate impacts results of KTA and calls for stringent selection.

Tetrahydrobiopterin protects the kidney from ischemia–reperfusion injury

Tetrahydrobiopterin (BH4) is an essential cofactor for the nitric oxide (NO) synthases and represents a critical determinant of NO production. BH4 depletion during ischemia leads to the uncoupling of the synthases, thus contributing to reperfusion injury due to increased superoxide formation. To examine whether BH4 supplementation attenuates ischemia-reperfusion injury, we clamped the left renal arteries of male Lewis rats immediately following right-side nephrectomy. BH4 tissue levels significantly decreased after 45 min of warm ischemia compared with levels in non-ischemic controls. Histopathology demonstrated significant tubular damage and increased peroxynitrite formation. Intravital fluorescent microscopy found perfusion deficits in the microvasculature and leakage of the capillary mesh. Supplemental BH4 treatment before ischemia significantly reduced ischemia-induced renal dysfunction, and decreased tubular histologic injury scores and peroxynitrite generation. BH4 also significantly improved microcirculatory parameters such as functional capillary density and diameter. These protective effects of BH4 on microvasculature were significantly correlated with its ability to abolish peroxynitrite formation. We suggest that BH4 significantly protects against acute renal failure following ischemia reperfusion. Whether BH4 has a therapeutic potential will require more direct testing in humans.

Donor age-specific injury and immune responses.

Utilization rates of organs from elderly donors have shown the highest proportional increase during the last decade. Clinical reports support the concept of transplanting older organs. However, the engraftment of such organs has been linked to accelerated immune responses based on ageing changes per se and a proinflammatory environment subsequent to compromised injury and repair mechanism. We analyzed the clinical consequences of transplanting older donor organs and present mechanistic aspects correlating age, injury repair and effects on host immunoresponsiveness.

Enteroscopic biopsies in the management of pancreas transplants: a proof of concept study for a novel monitoring tool.

Background. Although percutaneous biopsies are considered to be the gold standard in diagnosing pancreas graft rejection, they are not performed routinely because of their association with severe complications. On the other hand, correct diagnosis of rejection is essential but may be difficult in cases of enteric drainage, particularly in patients with a pancreas transplant alone or a pancreas after kidney transplant. Methods. Pancreas recipients who underwent enteroscopy between May 2005 and September 2009 were included in this retrospective analysis. Biopsies were graded 0 to 4 for interstitial and vascular changes. Results. During the study period a total of 65 simultaneous pancreas-kidney transplants, 13 pancreas after kidney transplants and 4 pancreas transplants alone were performed. Sixty-three patients underwent a single enteroscopy, 10 had two, and 6 had three or more. Indications were protocol graft monitoring (n=73), graft dysfunction (n=17), enteric hemorrhage (n=9), or other (n=3). The duodenal segment was accessed in 76 instances (75%) with abnormal findings in 23. A total of 69 biopsies were obtained and revealed normal mucosa in 49 cases (71%). Histology showed signs of acute rejection in 11 cases. The upper gastrointestinal tract was also assessed, and, in 13 cases, additional pathologies were identified including gastroduodenitis (n=10), gastric/duodenal ulcer (n=2), and hemorrhagic esophagitis (n=1). No procedure-related complication occurred. Conclusions. This series of enteroscopies demonstrates that the duodenal segment of a pancreatic graft is accessible using our implant technique, and thus permitting biopsies to be obtained and endoscopic interventions to be performed.

The faster the better: anastomosis time influences patient survival after deceased donor kidney transplantation

Despite a continuously growing knowledge of the impact of factors on kidney graft function, such as donor age, body mass index, and cold ischemia time, few data are available regarding anastomosis time (AT) and its impact on long‐term results. We investigated whether surgical AT correlates with patient and graft survival after kidney transplantation performing a retrospective analysis of 1245 consecutive deceased donor kidney transplantations between 01/2000 and 12/2010 at Innsbruck Medical University. Kaplan–Meier and log‐rank analyses were carried out for 1‐ and 5‐year patient and graft survival. AT was defined as time from anastomosis start until reperfusion. Median AT was 30 min. Five‐year survival of allografts with an AT >30 min was 76.6% compared with 80.6% in the group with AT <30 min (P = 0.027). Patient survival in the group with higher AT similarly was inferior with 85.7% after 5 years compared with 89.6% (P < 0.0001) [Correction added on February 18, 2015, after first online publication: the percentage value for patient survival was previously incorrect and have now been changed to 89.6%]. Cox regression analysis revealed AT as an independent significant factor for patient survival (HR 1.021 per minute; 95% CI 1.006–1.037; P = 0.006). As longer AT closely correlates with inferior long‐term patient survival, it has to be considered as a major risk factor for inferior long‐term results after deceased donor kidney transplantation.

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor‐specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti‐CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor‐specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC‐mismatched/minor antigen‐matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non‐MHC antigens cause graft rejection and split tolerance.