swissknife spezial 06 12.06.2006 13:39 Uhr Seite 44 Results: Eight of fifteen patients completed the trial (7 stages III, 8 stages IV). Drop outs occurred because of progressive disease (3), patient request (2), skin toxicity related to the GM-CSF (1) and a cerebrovascular insult not related to the trial. Overall, 11 of 15 patients (73%) doubled their CTLp against one or more melanoma epitopes. Seven of the 8 patients (86%) completing the trial responded to 2 or 3 melanoma epitopes. The average CTLp achieved was 3.2, 8.5 and 1<strong>4.</strong>1 CTLp/10e8 CTL for the Gp100, Mart and tyrosinase epitopes, respectively. Interestingly, a positive correlation (p
swissknife spezial 06 12.06.2006 13:39 Uhr Seite 45 factor-1). The anti-apoptotic protein A20 was first described as a TNF-responsive gene in epithelial cells. Furthermore it is a potent inhibitor of the transcription factor NF-B. In the liver, it has been shown that A20 is up-regulated by pro-inflammatory stimuli and protects from apoptosis and limits cell-damage. In previous work from our group, we have observed that A20 mRNA is induced in livers of mice un<strong>der</strong> hypoxic conditions. The aim of this study was to determine whether hypoxia has a direct effect on the expression of A20 and to determine if the mechanism of its induction is dependent on HIF-1. Methods: Primary human hepatocytes (n=10) were isolated from resected liver tissue obtained from consented patients from our clinic. Cells were cultured either un<strong>der</strong> normoxic (21%, 02) or hypoxic (1.5%, 02) conditions for 6 hours. To stabilize HIF-1 un<strong>der</strong> normoxic conditions, cells were treated with Dimethyloxaloylglycine (DMOG, 125 M), desferoxamin (DFO, 100 M) or cobalt chloride (CoCl2, 100 M) for 6 hours. Furthermore, as a positive control, cells were treated with TNF (10ng/ml), a known inducer of A20 mRNA and protein. Total RNA was extracted, cDNA was synthesized and mRNA was quantitated by real-time PCR (ABI 7700) using fam-labelled MGB probes. Protein extracts were analyzed by Western blotting for HIF-1 and A20 protein expression. Results: In the primary human hepatocytes, TNF predictably lead to a 9.1-fold induction of A20 mRNA un<strong>der</strong> normoxic conditions. In cells cultured un<strong>der</strong> hypoxia, A20 mRNA was significantly increased after 6 hours with an average 37.7-fold increase compared to normoxic values. In agreement with RNA data, protein was also increased. Cells treated with either DMOG, CoCl2 or DFO lead to a stabilization of HIF-1 protein un<strong>der</strong> normoxic conditions, but did not lead to an up-regulation of A20 mRNA or protein. Conclusion: We demonstrate for the first time that the anti-apoptotic protein A20 is up-regulated by hypoxia. Although HIF-1 is known to be a master regulator for transcription in hypoxic environment, our data show, that it is not directly involved in the hypoxic up-regulation of A20. 26.07 R.M. Baertschiger 1 , D. Bosco 1 , P. Morel 2 , M. Armanet 1 , A. Wojtusciszyn 1 , V. Serre-Beinier 3 , T. Berney 1 , L. Bühler 3 , C. Gonelle-Gispert 3 1 Surgery, University Hospital Geneva, Cellular Isolation and Transplantation Laboratory, 1211 Genève/CH, 2 Chirurgie Viscérale, Hôpital Cantonal de Genève, Genève/CH, 3 Surgery, University Hospital Geneva, Surgical Research Unit, 1211 Genève/CH Human exocrine pancreas-<strong>der</strong>ived mesenchymal stem cells and their potential to differentiate into beta cells Objective: Transplantation of in vitro generated islets or insulin producing cells represents an attractive option for treatment of type 1 diabetes. Therefore, stem or progenitor cells with the capacity to differentiate into beta cells have to be identified. In this study we isolated and expanded mesenchymal stem cells (MSC) obtained from human exocrine pancreas and we investigated their potential to differentiate into beta cell. Methods: We have cultured human exocrine pancreatic tissue obtained after isolation and purification of pancreatic islets for clinical transplantation in expansion media for human MSC (IMDM + 10% FCS + PDGF-BB). After 2 8 passages, these cells were characterized by FACS and compared to human MSC isolated from bone marrow. Mesenchymal differentiation potential was tested by culturing these cells in adipogenic and chondrogenic differentiation media. In or<strong>der</strong> to induce endocrine differentiation, these cells were cultured at high density on non-adherent plastic in differentiation medium (high glucose DMEM containing nicotinamide, activin A and HGF). Differentiation was assessed by RT-PCR for insulin and early endocrine markers. Results: In 9 of 10 human pancreatic exocrine fractions with a purity of 99%, adherent fibroblast-like cells appeared and could be expanded. Cells were grown up to 40 population doublings (19 passages). These cells displayed a similar antigen surface expression as bone marrow MSC, i.e. they were negative for CD31, CD34, CD45, CD106, MHC class 1, CD54low, and positive for CD44, CD90, CD105. Culture of these cells in adipogenic and chondrogenic differentiation media allowed differentiation into adipocyte-like and chondrocyte-like cells, demonstrating mesenchymal phenotype and multipotentiality. Pancreatic MSC, when cultured in differentiation medium formed pseudo-islet-like clusters and after 14 days of differentiation expressed Nkx2.2, Nkx6.1, NeuroD, Isl-1, insulin and glucagon in 4 out of 7 experiments. However, these islet-like clusters were negative for Glucokinase and Glut2. Conclusion: Our data show that MSC are present in the exocrine fraction of human pancreas and that they can be expanded extensively. These cells have the potential to form islets-like clusters expressing several early and late beta-and alpha-cell genes. 26.08 P. Georgiev 1 , W. Jochum 2 , F. Dahm 3 , A. Nocito 1 , R. Graf 4 , P. Clavien 5 1 Swiss Hpb Center, Dept. of Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH, 2 Dept. of Pathology, University Hospital of Zurich, 8091 Zurich/CH, 3 Dept. Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH, 4 Dept. of Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH, 5 Swiss Hpb Center, Dept. Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH Common bile duct ligation in mice: a model revisited Objective: Experimental ligation of the common bile duct (CBDL) has been performed for decades to study cholestatic liver disease, fibrosis, and the impact of cholestasis on remote organs. To date, a description of the ensuing morphological and molecular changes in mice is lacking with respect to several important parameters. It is therefore unclear which time point after CBDL should be chosen to answer specific questions related to cholestasis. The aim is to assess time related changes in mice after CBDL. Methods: C57BL/6 mice un<strong>der</strong>went CBDL (n=6 per time point) or sham laparotomy (n=4 per time point) for 8h, 1d, 2d, 3d, 5d, 7d, 14d, 28d, and 45d and serum and tissues were analyzed. Results: A. Hepatocellular injury and regeneration: ALT release and biliary infarcts peaked on days 2 and 3, respectively, followed by a specific peak of hepatocellular proliferation (Ki67+ hepatocytes) at day 5. At day 7, these parameters stabilized at a slightly elevated level over baseline. B. Cholangiocellular reaction: Release of alkaline phosphatase peaked at day 2 followed by a steady increase from day 7 on. The peak of cholangiocellular proliferation (Ki67+ cholangiocytes) differed between large bile ducts (2d) and ductules (5d). Bile duct proliferation (cytokeratin+ tubular structures/portal field) steadily increased up to day 14 with no further rise thereafter. C. Immune cell infiltration and cytokine expression: Biliary infarcts, detectable 8h after CBDL, were accompanied by infiltrating granulocytes through day 5. From day 7 on, portal fields contained a mixture of B220+ cells, CD3+ cells, and granulocytes. Expression of TNF-alpha and IL-6 followed a biphasic pattern with a first peak at day 1 and a second peak at day 1<strong>4.</strong> D. Fibrogenesis: Expression of alpha-SMA, collagen (I) and TGF-beta1 displayed a first peak at day 3 and a second peak at day 14 followed by stable expression thereafter. Collagen content (Sirius red staining) remained low up to day 7, increased markedly until day 14 without further increase up to day 45 and without reaching histological criteria for liver cirrhosis. Conclusion: CBDL elicits specific time related changes in C57BL/6 mice. The minute chronological dissection and quantification of these molecular and morphological changes enhances the un<strong>der</strong>standing of cholestatic liver injury and presents a basis for the thorough design of future studies. 26.09 A. Nocito 1 , P. Georgiev 1 , F. Dahm 2 , R. Graf 3 , W. Moritz 4 , W. Jochum 5 , B. O<strong>der</strong>matt 6 , P. Clavien 7 1 Swiss Hpb Center, Dept. of Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH, 2 Dept. Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH, 3 Dept. of Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH, 4 Dept. of Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/ CH, 5 Dept. of Pathology, University Hospital of Zurich, 8091 Zurich/CH, 6 Institute for Clinical Pathology, UniversiätsSpital Zürich, 8091 Zurich/CH, 7 Swiss Hpb Center, Dept. Visceral and Transplant Surgery, University Hospital of Zurich, 8091 Zurich/CH Impact of platelets in the pathogenesis of normothermic ischemia/reperfusion injury in the mouse liver Objective: One of the hallmarks of hepatic ischemia and reperfusion (I/R) is the formation of leukocyte-platelet aggregates. Upon activation, platelets generate reactive oxygen species and release proapoptotic and proinflammatory mediators as well as growth factors. In cold hepatic ischemia adhesion of platelets to endothelial cells mediates sinusoidal endothelial cell apoptosis. Furthermore, serotonin, which is almost exclusively stored in platelets, mediates liver regeneration. We therefore hypothesized that platelets might be mediators of reperfusion injury after normothermic hepatic ischemia. The aim of this study was to investigate the role of platelets in normothermic hepatic I/R injury in a model of platelet dysfunction and in immune thrombocytopenia. Methods: Inhibition of platelet aggregation in mice was achieved by Clopidogrel feeding. Immune thrombocytopenia was induced by intraperitoneal injection of anti-CD41 antibody. All mice were subjected to sixty minutes of partial hepatic ischemia and various timepoints of reperfusion. Hepatic injury was determined by aspartate aminotransferase and histological analysis of necrotic area and leucocyte infiltration. Furthermore, in platelet depleted animals and in mice lacking peripheral serotonin (Tph1-/-), liver regeneration was determined by immunohistochemistry. Results: Neither inhibition of platelet aggregation nor platelet depletion led to an improvement of I/R injury. In contrast, liver regeneration and remodelling were significantly impaired in platelet depleted animals, whereas mice lacking peripheral serotonin were only deficient in liver regeneration. Conclusion: Platelets have no direct impact on the pathogenesis of normothermic I/R-injury, however they mediate tissue remodelling and liver regeneration. In addition, platelet <strong>der</strong>ived serotonin is a specific mediator of regeneration in the postischemic liver. 27 27.01 B. Perrin 1 , D. Delay 2 , M. Hurni 3 , V. Argitis 4 , L.K. von Segesser 5 1 Department of Cardio-vascular Surgery, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne/CH, 2 Department of Cardiovascular Surgery, CHUV, 1011 Lausanne/CH, 3 Chirurgie Cardio-vasculaire, Centre Hospitalier Universitaire Vaudois (CHUV), 1011 Lausanne/CH, 4 Chirurgie Cardiovasculaire, CHUV, 1011 Lausanne/CH, 5 Chirurgie Cardiovasculaire, CHUV, 1011 Lausanne/CH Late reoperations after surgical repair of type A aortic dissection Objective: Type A aortic dissections usually need surgical treatment. Short-term outcome is generally good, but sometimes serious late unfavourable evolutions may appear leading to reoperation. The objective of the present study was to review our experience with the long term evolutions after surgical treatment of type A aortic dissections. Methods: A retrospective review of 189 patients operated on for type A aortic dissection during the last 10 years requiring late redo surgery. Results: 189 consecutive patients un<strong>der</strong>went surgical repair of type A aortic dissection between 1995 and 2005. 12 patients (6,3%) were operated for late evolutions between 1999 and 2005. There were 9 men and 3 women averaging 57 years of age (range 50 to 72). Patients were reoperated a mean of <strong>4.</strong>2 years (range 9 months to 10 years) after type A aortic dissection surgery. Indications for surgery were aortic roof aneurysm (n=1), peri-prosthetic false aneurysms associated with aortic valve insufficiency (n=2), acute retrograde dissection (n=1), aortic arch aneurysm (n=1), arch and descending thoracic aortic aneurysm (n=4), thoraco-abdominal aneurysms (n=3), and renal artery occlusion (n=1). Operative procedures included ascending aortic replacement with aortic valve replacement (n=1), Bentall procedure (n=4), arch replacement (n=4), descending thoracic aortic repair by endoprothesis, descending thoracic aortic replacement (n=4), thoraco-abdominal aortic replacement (n=1) and aortoiliac reconstruction (n=1). Three patients have been operated twice and one three times. Thirty days mortality was 17% (2/12). Causes of death were cardiac failure in one case, and massive cerebral embolism in the other. Conclusion: In our serie, only a small percentage of patients were diagnose with evolutions implicating all segments of the aorta and requiring surgery. Results of reoperation are satisfying with acceptable perioperative mortality. Postoperative follow-up is important for detection of potential severe evolution after type A aortic dissection surgery. 27.02 F. Schoenhoff 1 , H. Burger 2 , J. Triller 3 , E. Delacrétaz 4 , T. Carrel 5 , P.A. Berdat 6 1 Department of Cardiovascular Surgery, University of Berne, 3010 Bern/CH, 2 Department of Pathology, University of Berne, Bern/CH, 3 Department of Radiology, University of Berne, Bern/ CH, 4 Department of Cardiology, University of Berne, Bern/CH, 5 Department of Cardiovascular Surgery, University of Berne, Bern/CH, 6 Cardiovascular Surgery, University Hospital Bern, 3010 Bern/CH swiss knife 2006; special edition 45
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