Daniel Candinas

Role of endothelial cells in transplantation

Endothelial cell activation with accompanying vascular inflammatory changes is considered central to the experimental manifestations of both hyperacute and delayed xenograft rejection responses. Natural xenoreactive antibodies directed at alpha-galactosyl residues of xenogeneic glycoproteins and glycolipids, with associated complement activation via the classical pathway, are considered major immediate mediators of graft endothelial cell injury in the clinically relevant discordant swine to primate combinations. In delayed xenograft rejection processes, where recipients are treated prophylactically to ameliorate these initial events, activation of infiltrating mononuclear phagocytes and natural killer cells are associated with ongoing endothelial cell activation processes, procoagulant generation and vascular thrombosis. Allograft hyperacute rejection is observed when vascularised organs are transplanted to sensitized individuals with high levels of cytotoxic antibodies. Less dramatic forms of humoral allograft rejection (termed accelerated or vascular rejection) and the more common cell-mediated endothelialitis are associated with significant graft damage. Endothelial cell activation is also linked with graft preservation injury, forms of chronic rejection and delayed graft loss. Experimental work is currently being directed at the control of hyperacute rejection, the close understanding of endothelial cell thromboregulation in both transplanted xeno- and allografts and the development of novel therapeutic agents including gene therapy and the possible use of organs from transgenic animals.

Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation

Abstract: The binding of human xenoreactive antibody (XNA) to porcine endothelium with complement (C) activation via the classical pathways is considered the major event triggering hyperacute rejection (HAR) with microvascular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of complement by endothelial cells results in xenogeneic platelet activation in vitro.

Inhibition of platelet GPIIbIa in an ex vivo model of hyperacute xenograft rejection does not prolong cardiac survival time

Abstract: Discordant cardiac xenografts are rapidly rejected in a process characterized by platelet activation with microvascular thrombosis, termed hyperacute rejection (HAR). The fibrinogen receptor GPIIbIIIa is crucial for the formation of platelet aggregates and potentiates platelet adhesion to subendothelial matrix. We have studied the effects of a specific GPIIbIIIa antagonist (GPI 562) in an ex vivo working heart model using discordant porcine hearts perfused with fresh, heparinized human blood. Stable plasma GPI 562 inhibitory levels were confirmed by inhibition of human platelet aggregation in vitro. Biopsies from the left ventricle of rejected hearts were analyzed by immunopathology. Control porcine hearts (n=8) underwent HAR and ceased functioning at around 60 min. Hearts perfused with human blood containing GPI 562 at 0.5 μM (n=5) appeared to show an initial increase in coronary blood flow relative to controls, but neither this difference nor survival times of the hearts reached significance. Mean cardiac output values were 7.3 ml/g (SEM 2.5) in the experimental group and 5 ml/g (SEM 0.6) in the control group following 5 min of working mode and were comparable at other timepoints. Platelet counts in the perfusate were maintained in the presence of GPI 562, unlike the reduction of over 50% in control samples. Immunohistochemistry suggested decreased platelet vascular plugging as determined by P‐selectin staining in the GPI 562 group, with associated reduction in neutrophil adherence and fibrin deposition. The use of GPI 562 in this ex vivo model conferred no marked benefits with respect to cardiac function and explant survival despite some positive differences on histological comparison. Further studies of this agent, in association with modalities of complement inhibition, are warranted in other models of discordant xenograft rejection.

Thrombin Inhibition in discordant xenograft rejection

Abstract: Microvascular thrombosis and the associated platelet and endothelial cell activation are prominent observations in xenograft rejection. This pathological picture could be related to the excessive generation of thrombin in the context of either inflammation or putative inter‐species molecular incompatibilities between activated coagulation factors and their natural anticoagulants. Relatively selective thrombin Inhibition with the serine protease inhibitor SDZ MTH 958 (MTH‐958) are independent of heparinoids and anti‐thrombin III. MTH‐958 has been shown to significantly prolong porcine cardiac function during perfusion with human blood in an ex vivo model. The aim of this study was to validate the role of thrombin generation in a rodent model of discordant xenograft rejection in vivo.

Xenograft accommodation is accompanied by intragraft Th2 cytokines and vascular expression of protective genes

Abstract: We have studied the accommodation (survival of an organ graft in the presence of anti‐graft antibodies and complement) of heterotopic Golden Syrian hamster heart xenografts transplanted to Lewis rat recipients. The rats were treated with cyclosporine A (15 mg/kg/day i.m.) for the duration of the experiment and for 11 days with cobra venom factor. This regimen resulted in long‐term xenograft survival in approximately 75% of cases. Analysis of endothelial cells (and smooth muscle cells) in long‐surviving grafts showed expression of “protective” genes: A20, Bcl‐xL, Bcl‐2, and hemoxygenase, which we define as genes that prevent endothelial cells from undergoing responses that might lead to graft rejection. Surviving xenografts were also associated with intragraft Th2 cytokine expression. Rejected grafts did not express the protective genes and had a Thl pattern of cytokine expression. These studies indicate a potential mechanism linking molecular and cellular responses to development of xenograft accommodation.

Externalized percutaneous stent vs. internal double J stent: Short- and long-term complications after kidney transplantation

BACKGROUNDnIn patients undergoing kidney transplantation, ureteral stents are an established technique to reduce major urologic complications such as leakage and stenosis of the ureter. However, the best technique for ureteral stenting remains unclear. The aim of this study was to compare the outcome of percutaneous ureteral stents (PS) with internal double J stents (JJS) after kidney transplantation.nnnMETHODSnAll patients undergoing kidney transplantation between 2005 and 2014 were retrospectively analyzed. After excluding patientsxa0<18 years old, patients without stenting, and patients who underwent multiorgan transplantation, a total of 308 patients were included in the study. Two consecutive cohorts of patients were compared. In the cohort transplanted between 2005 and 2010, stenting was routinely performed using PS (216 patients), and in the second cohort, those transplanted after 2011, stenting was routinely performed using JJS (92 patients). For ureteric anastomosis, the Lich-Grégoir technique was used in all patients.nnnRESULTSnThere was no statistical difference in postoperative urinary tract infections (Pxa0=xa0.239) between the 2 cohorts. In patients with PS, the incidence of major urologic complications (11.6% vs 3.3%; Pxa0= .018), vesicoureteral reflux (14.3% vs 2.2%; Pxa0< .001), and urologic reinterventions (14.4% vs 5.4%; Pxa0= .031) was significantly higher when compared with JJS patients. Multivariable logistic regression revealed increased incidence of major urologic complications (odds ratio [OR] 3.66, 95% confidence interval [CI] 1.07-12.55, Pxa0= .039) and vesicoureteral reflux (OR 5.29, 95% CI 1.21-23.10, Pxa0= .027) in patients with PS compared with JJS.nnnCONCLUSIONnStenting of ureterovesical anastomosis using JJS is associated with reduced complications compared with PS after kidney transplantation.

Pancreatic Neuroendocrine Tumors: How Much Surgery is Safe?

Background: Neuroendocrine tumors of the pancreas (pNET) nare rare. Often the patients are asymptomatic for a long time and npresent late with metastasized disease. Although there are guidelines nfor the treatment of these tumors, there is no clear consensus whether nthe resection of liver metastases may be combined with the primary ntumor. nMethod: We retrospectively analyzed the patients operated at our ninstitution between 1/2003 and 12/2012. The patients were analyzed nfor demographic and clinical data, surgical treatment, tumor size and nstage, histology, complications, survival and tumor recurrence over ntime. nResults: We analyzed 53 patients, 23 females, 30 males. Patients nwith a one-step surgical approach to pancreas and liver had similar nmorbidity and mortality compared to patients with disease confined nto the pancreas. The primary tumors were smaller in tumors confined nto the pancreas. Angioinvasion as well as positive lymph nodes nwere strongly correlated with synchronous or metachronous liver nmetastases. Progression free survival was shorter in patients with nprimary metastasized disease. nConclusion: The treatment of pNET is challenging. The surgical napproach should be tailored to the patient’s general condition. nPatients benefit from extended and combined resections even in nmetastasized or locally advanced situations. Combined pancreatic and nhepatic surgery may be performed safely.

Lebertransplantation – wer, wann, wie?

Die orthotope Lebertransplantation ist mittlerweile ein standardisiertes Verfahren mit geringer Morbiditat und Mortalitat und fuhrt bei korrekter Indikationsstellung zu einer ausgezeichneten Uberlebensrate und verbesserter Lebensqualitat.

89 DELETION OF TRAIL ON NK CELLS IS ASSOCIATED WITH EXCESSIVE HEPATIC ISCHEMIA-REPERFUSION INJURY IN MICE

Background and Aims: Ischemia-reperfusion injury (IRI) is a key factor that contributes to early and late dysfunction of liver grafts. Recent studies reveal that natural killer (NK) cells play an important role in liver injury post IRI. We hypothesized that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand with high expression on NK cells significantly impacts IRI. Methods: C57/BL6 wild-type and TRAIL knock-out mice (TRAIL−/−) were subjected to hepatic IRI for one hour. Adoptive transfer of wild-type and TRAIL−/− NK cells was performed into RAG2/common gamma null mice that lack T, B and NK cells. Liver injury was assessed by hepatic neutrophil infiltration, alanine aminotransferase (ALT), aspartate transaminase (AST), hepatic neutrophil activation by myeloperoxidase (MPO) activity. NK cell subsets of the ischemic liver lobe were analysed by flow cytometry. NK cell cytotoxicity and interferon gamma secretion were performed in vitro studies. Results: TRAIL−/− mice exhibit significantly more hepatic damage assessed by AST and ALT levels 24 hours post IRI compared to wildtype mice. Adoptive transfer of TRAIL−/− NK cells to Rag2/common gamma-null mice was associated with significantly increased IRI compared to transfer with wild-type NK cells. Hepatic neutrophil activation was significantly increased in TRAIL−/− compared to wild-type mice. Staining for CD107a, a marker of degranulation and cytotoxicity on NK cells was significantly elevated in ischemic liver lobes of TRAIL−/− compared to wild-type mice. In vitro NK cell cytotoxicity to a Yac-1 cancer cell line was significantly increased in sorted TRAIL−/− NK cells compared to wild-type NK cells. Systemic cytokine levels (TNF alpha, IL-1beta, IL-6) and interferon gamma secretion of NK cells after stimulation with IL-12/IL-18 in vitro were not significantly different between the groups. Conclusions: These results show that expression of TRAIL on NK cells is protective in a murine model of hepatic IRI via the modulation of NK cell mediated cytotoxicity.

Eine neue Technik der orthotopen Lebertransplantation vom Meerschweinchen auf die Ratte

Aufgrund des enormen Wissenszuwachses auf dem Gebiet der Xenotransplantation und der Moglichkeit, diskordante xenogene Organe als „bridging“ bei fulminantem Leberversagen einzusetzen, werden dringend entsprechende in vivo-Forschungsmodelle fur die diskordante Lebertransplantation benotigt.