Normothermic machine perfusion (NMP) has emerged as an innovative organ preservation technique. Developing an understanding for the donor organ immune cell composition and its dynamic changes during NMP is essential. We aimed for a comprehensive characterization of immune cell (sub)populations, cell trafficking and cytokine release during liver NMP. Single-cell transcriptome profiling of human donor livers prior to, during NMP and after transplantation shows an abundance of CXC chemokine receptor 1(+)/2(+) (CXCR1(+)/CXCR2(+)) neutrophils, which significantly decreased during NMP. This is paralleled by a large efflux of passenger leukocytes with neutrophil predominance in the perfusate. During NMP, neutrophils shift from a pro-inflammatory state towards an aged/chronically activated/exhausted phenotype, while anti-inflammatory/tolerogenic monocytes/macrophages are increased. We herein describe the dynamics of the immune cell repertoire, phenotypic immune cell shifts and a dominance of neutrophils during liver NMP, which potentially contribute to the inflammatory response. Our findings may serve as resource to initiate future immune-interventional studies.

Brain death (BD) has been associated with an immunological priming of donor organs and is thought to exacerbate ischemia reperfusion injury (IRI). Recently, we showed that the essential nitric oxide synthase co-factor tetrahydrobiopterin (BH4) abrogates IRI following experimental pancreas transplantation. We therefore studied the effects of BD in a murine model of syngeneic pancreas transplantation and tested the therapeutic potential of BH4 treatment. Compared with sham-operated controls, donor BD resulted in intragraft inflammation reflected by induced IL-1ß, IL-6, VCAM-1, and P-selectin mRNA expression levels and impaired microcirculation after reperfusion (p < 0.05), whereas pretreatment of the BD donor with BH4 significantly improved microcirculation after reperfusion (p < 0.05). Moreover, BD had a devastating impact on cell viability, whereas BH4-treated grafts showed a significantly higher percentage of viable cells (p < 0.001). Early parenchymal damage in pancreatic grafts was significantly more pronounced in organs from BD donors than from sham or non-BD donors (p < 0.05), but BH4 pretreatment significantly ameliorated necrotic lesions in BD organs (p < 0.05). Pretreatment of the BD donor with BH4 resulted in significant recipient survival (p < 0.05). Our data provide novel insights into the impact of BD on pancreatic isografts, further demonstrating the potential of donor pretreatment strategies including BH4 for preventing BD-associated injury after transplantation.

AimsEpithelial cell adhesion molecule (EpCAM) is a widely used immunohistochemical marker for epithelial human malignancies. Antibodies to target EpCAM are usually directed against its ectodomain (EpEX), but do not detect the intracellular domain (EpICD). The aim of this study was to compare membranous EpEX versus EpICD expression by immunohistochemistry.Methods and resultsConcurrent EpEX and EpICD expression was investigated retrospectively in cancerous and matched non-neoplastic tissue samples from patients with pancreatic adenocarcinoma. In total, 317 paired samples of pancreatic tissue from 88 patients were analysed and correlated with clinicopathological parameters. In non-cancerous tissue, a high concordance of membranous EpEX and EpICD expression was observed and defined as the expression of the full-length EpCAM (EpEX(+)/EpICD(+) phenotype, EpCAM(MF)), which was highly predominant. In contrast, while most tumour samples were EpEX positive, loss of membranous EpICD expression (EpEX(+)/EpICD(-) phenotype, EpCAM(MT)) was observed in one-third of cases, and these patients had a significantly shortened disease-free and overall survival.ConclusionsThis study demonstrates for the first time that loss of membranous EpICD expression is a frequent event and predicts poor prognosis in patients with pancreatic cancer. Additional studies evaluating the predictive and prognostic value of the expression of different membranous EpCAM variants are warranted in epithelial cancers.

Introduction: Tetrahydrobiopterin has already been shown to efficiently abrogate ischemia-reperfusion injury following murine pancreas transplantation. However, whether the beneficial effect of tetrahydrobiopterin relies on its co-factor activity of the nitric oxide synthase reaction or of one of the other four known tetrahydrobiopterin-dependent reactions, or whether it is based on its antioxidative capacity has still to be elucidated. Herein we compared tetrahydrobiopterin with tetrahydroneopterin which has similar biochemical properties but shows no nitric oxide synthase co-factor activity, and with the antioxidants 5-methyltetrahydrofolate and vitamin C. Methods: Male syngeneic C57BL6 mice were used as size-matched donor and recipient pairs. Cervical vascularized pancreas transplantation was performed with a modified no-touch technique. Donor mice were pretreated with tetrahydrobiopterin (50 mg/kg b.w.), tetrahydroneopterin (50 mg/kg b.w.), vitamin C (350 mg/kg b.w.) or 5-methyltetrahydrofolate (50 mg/kg b.w.) immediately before organ retrieval. Subsequently, pancreatic grafts were subjected to 16 h prolonged cold ischemia time and 45 min warm ischemia time. Untreated and non-transplanted animals served as controls. Following 2 h graft reperfusion microcirculation was assessed by confocal intravital fluorescence microscopy and quantified by the functional capillary density (FCD). The extent of parenchymal damage was analyzed by histology using hematoxylin and eosin stain as well as nitrotyrosine immunostaining, an indirect marker for peroxynitrite formation. Tetrahydrobiopterin tissue levels were determined by high performance liquid chromatography. Since occurrence of graft pancreatitis in this model is not compatible with long-term survival, recipient survival served as ultimate readout. Additionally, the capacity of the tested substances to scavenge peroxyl-radicals was evaluated by the Oxygen Radical Antioxidant Capacity (ORAC) assay and their influence on intracellular reactive oxygen species (ROS) levels was tested in HepG2 cells. Results: Compared to non-transplanted controls prolonged cold ischemia time resulted in a significant breakdown of the FCD (p< 0.01). Tetrahydroneopterin as well as 5-methyltetrahydrofolate and vitamin C, which were both shown to have equal antioxidative potential, failed to preserve the capillary net post reperfusion (p=ns). In contrast, tetrahydrobiopterin pretreatment succeeded in significantly abolishing capillary breakdown of the graft compared to untreated controls (p< 0.01). Along with increased intragraft tetrahydrobiopterin levels, only pretreatment with tetrahydrobiopterin led to significant reduction of ischemia-reperfusion injury-related parenchymal necrotic lesions and nitrotyrosine staining. Finally, tetrahydrobiopterin was the only treatment agent that enabled long-term recipient survival. Conclusion: The observed abrogation of ischemia reperfusion injury associated graft pancreatitis by tetrahydrobiopterin but neither by the related compound tetrahydroneopterin nor by antioxidants supports the hypothesis that the beneficial action of this compound depends on its nitric oxide synthase co-factor activity rather than on its antioxidative capacity.

BACKGROUND P150, a 150 kDa protein, was isolated from virally and oncogene-transformed mouse cell lines, partially purified and cloned. P150 is part of the large subunit of the eukaryotic translation initiation factor 3 with sequence homology to centrosomin A. A significant correlation between p150 expression and malignancy in breast, cervical and esophageal cancer have recently been demonstrated. MATERIALS AND METHODS Here, 110 colorectal carcinomas of different grades and stages, including lymph node and liver metastases were compared to adjacent normal mucosa by immunohistochemistry of P150. Western blot analysis of selected cases confirmed the expression levels determined by immunohistochemistry. Additionally, immuno-electron and laser scanning microscopy (LSM) was performed. RESULTS All investigated carcinomas revealed high levels of p150 protein compared to normal adjacent mucosa. The staining intensity was slightly heterogeneous, and positivity was correlated to the tumor grade with statistically significant differences of p150 expression between normal and neoplastic mucosa (p<0.0001, Kruskal-Wallis test). Western blots confirmed higher expression levels of p150 in the tumor. Immunogold labelling and LSM investigation showed high expression levels of p150 on the rough endoplasmic reticulum and polyribosomes, indicating that p150 is translationally active in these tumors. CONCLUSION Thus, we propose that p150 plays an important role in development and growth of colorectal carcinomas. Furthermore, p150 expression might provide us with reliable information on the biological behaviour of tumors and the clinical course of the disease.

Oberhuber, R.; Maglione, M.; Cardini, B.; Hermann, M.; Obrist, P.; Schneeberger, S.; Margreiter, R.; Pratschke, J.; Werner, E.; Brandacher, G. Author Information

Depletion of the nitric oxide synthase cofactor tetrahydrobiopterin (H4B) during ischemia and reperfusion is associated with severe graft pancreatitis. Since clinically feasible approaches to prevent ischemia reperfusion injury (IRI) by H4B-substitution are missing we investigated its therapeutic potential in a murine pancreas transplantation model using different treatment regimens. Grafts were subjected to 16 h cold ischemia time (CIT) and different treatment regimens: no treatment, 160 μM H4B to perfusion solution, H4B 50 mg/kg prior to reperfusion and H4B 50 mg/kg before recovery of organs. Nontransplanted animals served as controls. Recipient survival and endocrine graft function were assessed. Graft microcirculation was analyzed 2 h after reperfusion by intravital fluorescence microscopy. Parenchymal damage was assessed by histology and nitrotyrosine immunohistochemistry, H4B tissue levels by high pressure liquid chromatography (HPLC). Compared to nontransplanted controls prolonged CIT resulted in significant microcirculatory deterioration. Different efficacy according to route and timing of administration could be observed. Only donor pretreatment with H4B resulted in almost completely abrogated IRI-related damage showing graft microcirculation comparable to nontransplanted controls and restored intragraft H4B levels, resulting in significant reduction of parenchymal damage (p < 0.002) and improved survival and endocrine function (p = 0.0002 each). H4B donor pretreatment abrogates ischemia-induced parenchymal damage and represents a promising strategy to prevent IRI following pancreas transplantation.

Transplantation I . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Transplantation II . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Transplantation III . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Freie Vorträge I . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Freie Vorträge II . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Onkologie I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Onkologie II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Background: Tetrahydrobiopterin (H4B) is both an essential co-factor of nitric oxide synthases (NOS) and a strong antioxidant. Under oxidative stress-associated H4B depletion NOS is known to produce oxygen radicals rather than nitric oxide (NO) by uncoupling, a phenomenon thought to contribute to endothelial dysfunction in a variety of vascular diseases. Recent findings showed also dramatically reduced ischemia/reperfusion injury (IRI) following murine pancreas transplantation after H4B substitution. To differentiate the antioxidant effect from uncoupling-prevention we compared in this study H4B supplementation previous to organ retrieval with supplementation of tetrahydroneopterin (H4N), a pterin with equal antioxidant potential but no co-factor activity. Material and Methods: Male syngeneic C57BL6 (H-2b) mice, 10–12 weeks old, were used as size-matched donor and recipient pairs. Murine cervical vascularized pancreas transplantation was performed with a modified no-touch technique. Pancreatic grafts were subjected to 16h prolonged cold ischemia time (CIT) as well as to 45 min warm ischemia time (WIT) and different treatment regiments: untreated animals (I), H4B 50 mg/kg i.m. prior to organ retrieval (II) and H4N 50 mg/kg i.m. prior to organ retrieval (III). Non transplanted animals served as controls (IV). After 2h of reperfusion intravital fluorescence microscopy was used for analysis of graft microcirculation by means of functional capillary density (FCD) and capillary diameters (CD). Quantitative assessment of parenchymal damage was analyzed by histology (H&E) and by performing nitrotyrosine-immunostaining to determine peroxynitrite formation. Intragraft pteridin levels were measured by HPLC. Results: Following prolonged CIT and 45 minutes of WIT only pancreatic grafts treated with H4B prior to organ explantation (II) displayed markedly higher values of FCD and CD compared to non treated animals (I) (p<0.01 and p<0.05 respectively). In contranst, pre-treatment of donor animals with H4N did improve neither FCD nor CD levels. Compared to non treated animals H4B and H4N pre-treatment significantly attenuated peroxynitrite formation (p<0.05). However, parenchymal damage like acinar, fat and hemorrhagic necroses after after 2 h graft reperfusion were significantly reduced only by H4B pre-treatment (p<0.05). Partially oxidized H4B was substantially decreased in the H4B treatment group. Conclusions: The protective effect of H4B donor pre-treatment may rely not only on its antioxidative activity but also on its NOS co-factor capacity and might therefore be a novel, promising agent in clinical pancreas transplantation preventing early parenchymal damage after graft reperfusion.

We investigated the role of secretory leukocyte protease inhibitor (SLPI) in ischemia/reperfusion injury in cardiac transplantation. SLPI-/- mouse hearts and wild-type (WT) controls were transplanted immediately or after 10 h of cold ischemia (CI). Recombinant SLPI (rSLPI) was added to the preservation solution or given systemically. After evaluation of myocardial performance, grafts were investigated for histology, SLPI, TNF-alpha, TGF-beta, NF-kappaB and protease expression at indicated time points. Early myocardial contraction was profoundly impaired in SLPI-/- hearts exposed to CI and associated with high intra-graft protease expression. Systemic administration of rSLPI had no effect, however, when SLPI was added to the preservation solution, myocardial contraction was restored to normal. At 10 days, inflammation, myocyte vacuolization and necrosis were significantly more severe in SLPI-/- hearts. SLPI gene expression was detected in WT mice at 12 and 24 h and was significantly higher after CI. SLPI protein was observed at 24 h and 10 days. High intra-graft concentrations of SLPI after administration of rSLPI were inversely correlated with protease levels early and TGF-beta expression late after reperfusion. SLPI plays a crucial role in early myocardial performance and postischemic inflammation after cardiac transplantation. A dual inhibitory effect on protease and TGF-beta expression might be the underlying mechanism.

AIMS:Pancreatic adenocarcinoma is an aggressive gastrointestinal malignancy with only a few long-term survivors even after radical surgery. Patients with ampullary cancer have a better prognosis but adjuvant therapy needs further improvement. Epithelial cell adhesion molecule (Ep-CAM) is strongly expressed in a variety of epithelial cancers and represents a promising target for immunological tumour therapy. Thus, the aim of this study was to investigate Ep-CAM expression and its potential prognostic impact in pancreatic and ampullary carcinomas.METHODS:Ep-CAM expression was investigated retrospectively by immunohistochemistry in paraffin-embedded primary tumour tissue samples from a series of consecutive patients with pancreatic (n = 153) and ampullary cancer (n = 34).RESULTS:Ep-CAM overexpression was observed in 85 of 153 pancreatic cancer specimens (56%) and in 29 of 34 ampullary cancer samples (85%). Overall, Ep-CAM failed to be an independent prognostic marker. However, subgroup analyses showed that Ep-CAM overexpression correlated with shorter overall survival among patients with ampullary cancer and advanced stage pancreatic cancer. In the latter subgroup, survival gradually worsened with increasing Ep-CAM scores. Furthermore, in ampullary cancer, Ep-CAM overexpression was found to correlate with tumour stage.CONCLUSIONS:Ep-CAM overexpression was detectable in the majority of cases with pancreatic and ampullary cancer. Therefore, Ep-CAM represents an attractive target for immune-based therapeutic interventions in these tumour entities. However, the prognostic value of Ep-CAM overexpression remains undetermined.

Background. Lipocalin-2 (Lcn-2) is associated with ischemia=reperfusion injury (IRI) in different organs. Data on Lcn-2 expression during allograft rejection have been missing so far. The main focus of this study was to analyze the possible implication of Lcn-2 during acute rejection following solid organ transplantation. Methods. We investigated the expression of Lcn-2 in two different settings: (1) Hearts from male inbred C3H mice were transplanted into C57BL=6 wildtype and Lcn-2 = recipients. Hearts were recovered at different time points (0min, 2, 4, 6 and 8 d) and Lcn-2 expression was analyzed by conventional histology, Western blot and immunohistochemistry. (2) Serum of 40 patients undergoing orthotopic liver transplantation was collected preoperatively and postoperatively from day 1 to 15. Lcn-2 was analyzed by ELISA and expression levels were correlated with parameters of allograft rejection. Results. (1) Lcn-2 expression already increased in early stages of acute rejection (2 d) and significantly correlated with the amount of infiltrating polymorphonuclear cells. Coincidental expression of migration molecules like MIP2 or MMP-9 with Lcn-2 upregulation was observed. (2) Serum levels of NGAL following liver transplantation were elevated 3 to 7-fold immediately after transplantation due to IRI and also increased prior to clinically apparent acute rejection closely related to elevated laboratory parameters. Conclusions. Our data suggest a chemoattractant function of Lcn-2 in the allograft due to infiltrating immune competent cells. Lcn-2 is an inflammatory marker upregulated during acute graft rejection and its expression prior to clinical parameters of acute rejection might help to identify possible targets for therapeutic intervention.