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