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Langenbecks Arch Surg (2005) 390: 448494 DOI 10.1007/s00423-005-0580-5 ABSTRACTS # Springer-Verlag 2005 Organizers and Chairmenship Professor Dr. I. Marzi Professor Dr. T. Schmitz-Rixen Professor Dr. K.W. Jauch (SCF) Organization: Christina Larson Dr. M. Lehnert Dr. T. Tonn Priv.-Doz. Dr. F. Adili Date of conference: 1921 September 2005 Conference venue: Klinikum der Johann Wolfgang Goethe-Universität Frankfurt am Main Theodor-Stern-Kai 7 60590 Frankfurt/Main, Germany Contents: Accepted abstracts of lectures and posters from all surgical specialities with emphasis on tissue engineering and molecular biology in surgery. 1 S-100 B measurement during carotid endarterectomy under local anaesthesia Marko Aleksic, Jörg Heckenkamp, Michael Gawenda, Jan Brunkwall Division of Vascular Surgery, Department of Visceral- and Vascular Surgery, University Clinic of Cologne, Cologne, Germany Background: The neuronal protein S-100 B has been found to be an early indicator of cellular brain damage. In carotid endarterectomy (CEA), performed under general anaesthesia, during carotid cross- clamping, an increase of 120% has been seen in the jugular vein. The following study evaluates whether CEA under local anaesthesia shows the same S-100 B release as during general anaesthesia. Patients and methods: In 45 consecutive patients, 23 were asymptomatic, 14 had had TIA and 8 had a stroke. There were 33 males and 12 females. The median age was 70 years. CEA was performed under local anaesthesia and serume S-100 B measure- ments were taken before surgery (T1), before carotid cross clamping (T2), before cerebral reperfusion (T3), after reperfusion but before the end of surgery (T4) and 6 hours postoperatively (T5). At T1 and T5 blood samples were drawn only from the radial artery. Intraoperatively (T2-4) samples were additionally collected from the internal jugular vein. The S-100 B levels were determined using a luminometric immunoassay (LIAISON ® Sangtec ® 100). Results: An intraluminal shunt was used in 8 cases because signs of cerebral ischemia like unconsciousness or hemiplegia occurred during carotid cross-clamping. All patients returned to normal during shunting and none sustained a permanent deterioration of their neurological condition postoperatively. The median baseline (T1) and postoperative (T5) S-100 B levels were identical (77 pg/ml). Before cross-clamping the level measured in the jugular vein was significantly increased compared to the arterial blood samples (121 vs. 82 pg/ml). During cross-clamping only a further slight increase of 13% and 18% (137 vs. 97 pg/ml) was measured in the venous and arterial samples. Conclusion: CEA can be performed safely under local anaesthesia and there is no significant increase of S-100 B, in contrary to the results when CEA is done under general anaesthesia. 9th Annual Meeting on Surgical Research / 9. Chirurgische Forschungstage 2005, 1921 September 2005, Frankfurt am Main, Germany

9th Annual Meeting on Surgical Research / 9. Chirurgische Forschungstage 2005, 19-21 September 2005, Frankfurt am Main, Germany

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Page 1: 9th Annual Meeting on Surgical Research / 9. Chirurgische Forschungstage 2005, 19-21 September 2005, Frankfurt am Main, Germany

Langenbecks Arch Surg (2005) 390: 448–494DOI 10.1007/s00423-005-0580-5 A B S T RACT S

# Springer-Verlag 2005

Organizers and ChairmenshipProfessor Dr. I. MarziProfessor Dr. T. Schmitz-RixenProfessor Dr. K.W. Jauch (SCF)

Organization:Christina LarsonDr. M. LehnertDr. T. TonnPriv.-Doz. Dr. F. Adili

Date of conference:19–21 September 2005

Conference venue:Klinikum der Johann Wolfgang Goethe-UniversitätFrankfurt am MainTheodor-Stern-Kai 760590 Frankfurt/Main, Germany

Contents:Accepted abstracts of lectures and posters from all surgicalspecialities with emphasis on tissue engineering andmolecular biology in surgery.

1S-100 B measurement during carotid endarterectomyunder local anaesthesiaMarko Aleksic, Jörg Heckenkamp, Michael Gawenda,Jan BrunkwallDivision of Vascular Surgery, Department of Visceral- and VascularSurgery, University Clinic of Cologne, Cologne, GermanyBackground: The neuronal protein S-100 B has been found to be anearly indicator of cellular brain damage. In carotid endarterectomy(CEA), performed under general anaesthesia, during carotid cross-clamping, an increase of 120% has been seen in the jugular vein. Thefollowing study evaluates whether CEA under local anaesthesiashows the same S-100 B release as during general anaesthesia.Patients and methods: In 45 consecutive patients, 23 wereasymptomatic, 14 had had TIA and 8 had a stroke. There were 33males and 12 females. The median age was 70 years. CEA wasperformed under local anaesthesia and serume S-100 B measure-ments were taken before surgery (T1), before carotid cross clamping(T2), before cerebral reperfusion (T3), after reperfusion but beforethe end of surgery (T4) and 6 hours postoperatively (T5). At T1 andT5 blood samples were drawn only from the radial artery.Intraoperatively (T2-4) samples were additionally collected fromthe internal jugular vein. The S-100 B levels were determined using aluminometric immunoassay (LIAISON® Sangtec® 100).Results: An intraluminal shunt was used in 8 cases because signs ofcerebral ischemia like unconsciousness or hemiplegia occurredduring carotid cross-clamping. All patients returned to normal duringshunting and none sustained a permanent deterioration of theirneurological condition postoperatively. The median baseline (T1)and postoperative (T5) S-100 B levels were identical (77 pg/ml).Before cross-clamping the level measured in the jugular vein wassignificantly increased compared to the arterial blood samples (121vs. 82 pg/ml). During cross-clamping only a further slight increase of13% and 18% (137 vs. 97 pg/ml) was measured in the venous andarterial samples.Conclusion: CEA can be performed safely under local anaesthesiaand there is no significant increase of S-100 B, in contrary to theresults when CEA is done under general anaesthesia.

9th Annual Meeting on Surgical Research /9. Chirurgische Forschungstage 2005,19–21 September 2005,Frankfurt am Main, Germany

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2Induction of angiogenesis by VEGF-transfected fibroblastsreduces necrosis in random pattern flapsMichaela Amon1, Yves Harder1, Bence Bucsky2, Hans-GüntherMachens2, Michael D. Menger11Institute for Clinical & Experimental Surgery, University ofSaarland, Homburg/Saar, Germany2Clinic for Plastic and Hand Surgery, Burn Center, UKSH CampusLübeck, Lübeck, GermanyBackground: Inadequate nutritive perfusion after elevation of skin-flaps may result in ischemia and, finally, tissue necrosis. Theincreased morbidity of the affected patients may necessitate furtheroperations, resulting in poor aesthetical outcome and increasedmortality. VEGF is known to possess strong angiogenic properties.The aim of the presented study was to evaluate whether VEGF-transfected fibroblasts are capable of improving microcirculation andinducing angiogenesis in ischemic random pattern flaps.Animals and methods: A random pattern flap was elevated in theback of anaesthetised C57/BL6-mice, including the panniculuscarnosus. Subsequently, the flap was fixed into a dorsal skinfoldchamber. Either VEGF-transfected fibroblasts (5x106/100 mlDMEM) or non-transfected fibroblasts were injected into thepanniculus carnosus directly after elevation of the flap. Flap elevationwithout further treatment served as control operation. Elevation of theflap was performed at day 0, followed by repetitive intravital micro-scopic analyses at day 1, 3, 5, 7, und 10. We quantified the amount ofnecrotic tissue, functional capillary density of pre-existing vessels inthe distal, most critical part of the flap as well as angiogenesis,reflected by the density of newly formed blood vessels.Results: Nutritive perfusion was significantly improved in the distalpart of the flap by injection of transfected fibroblasts while applicationof non-transfected cells did not change functional capillary density ascompared to sham-treated animals (sham: 24±17 cm/cm2; VEGF:147±33 cm/cm2; fibroblasts: 56±38 cm/cm2; p<0.05; day 10). Dif-ferences between groups were obvious already at day 1 after elevationof the flap and there were no major changes until the end of exper-iments at day 10. Injection of non-transfected cells into the panniculusinduced a minor angiogenic response, however, the expression ofVEGF significantly augmented the formation of new vessels (fibro-blasts: 28±15 cm/cm2; VEGF: 111±18 cm/cm2; p<0.05; day 10). Incontrols, no signs of angiogenesis could be observed. Furthermore,development of necrosis was only slightly diminished by injection ofnon-transfected fibroblasts, while VEGF-transfection significantlyreduced the area of necrotic tissue (sham: 47±4% of total flap sur-face; fibroblasts: 37±10%; VEGF: 12±4%; p<0.05; day 10).Conclusion: The application of VEGF-transfected fibroblastsreduced the development of necrosis in this model of random patternflaps both by the formation of new blood vessels and the maintenanceof nutritive perfusion from the onset of ischemia. As a singleinjection of cells during elevation of the flap was sufficient to elicitthese effects and no long-term pre-treatment was necessary, thistherapeutic approach may be an option in clinical practice for thereduction of tissue necrosis in flap surgery.

3Injection of human preadipocytes within fibrin glue, analternative for volume augmentation in plastic surgery?Niklas Baerlecken, Nestor Torio-Padron, Arash Momeni, G. BjörnStark, Jörg BorgesDepartment of Plastic and Hand Surgery, University of FreiburgMedical Center, Freiburg, GermanyBackground: Correction of soft tissue deficits and contourirregularities represents a challenge in plastic surgery. Currently anideal filler for tissue augmentation does not exist. By using allogenicmaterials currently available or surgical techniques such as

lipostructure, complications such as allergic reactions, granulomaformation as well as unpredictable results can be observed. In thisstudy, a potential alternative to the commonly used filler materials ispresented. By tissue engineering techniques, autologous humanpreadipocytes can be isolated, cultured and subcutaneously injectedat the same patient in order to achieve the desired volumeaugmentation.Animals and methods: Human preadipocytes were isolated fromadipose tissue obtained during elective procedures like abdomino-plasties or dermolipectomies. After cultivation and cell expansion,human preadipocytes diluted in fibrin were subcutaneously injectedin different cell concentrations into the back of athymic nude mice.The implants were harvested after 1, 3 and 6 months, respectively,and histological analysis was performed to determine the presenceand quality of the newly formed adipose tissue. Fibrin glue lackingpreadipocytes was injected as a control using the same procedure.Results: The fat graft could be macroscopically recognized at theinjection site on the back of the animals as an elevation, even after 6months. Histological analysis after 1, 3 and 6 months demonstratedthe presence of newly formed adipose tissue with a homogeneousstructure without signs of tissue necrosis, oil cyst formation orinflammatory response. In the control group the construct wasreabsorbed within the first 4 weeks following implantation and nosigns of adipose tissue formation were observed.Conclusion: In conclusion, we demonstrate that the injection ofhuman preadipocytes within fibrin glue into athymic nude mice leadsto formation of newly adipose tissue resembling normal fatty tissuefound in humans. This approach can represent an alternative to thecurrently filler materials as well as surgical techniques used for tissueaugmentation in the near future.

4Analysis of genetic alterations and aneuploidyin preneoplastic lesions in chronic pancreatitisMario Baumgart1, Meike Werther2, Ernst Heinmöller2,Joseph Rüschoff2, Heinz Becker1, B. Michael Ghadimi11Klinik für Allgemeinchirurgie, Georg-August-UniversitätGöttingen, Göttingen, Germany2Institut für Pathologie, Klinikum Kassel, Kassel, GermanyBackground: Chronic pancreatitis (CP) is a predisposing disease forpancreatic cancer (PC). Precise molecular mechanisms of cancerdevelopment in the background of CP are disease defined. We haveapplied genetic analysis of TP53, p16INK4 and DPC4 genes andfluorescence in situ hybridization (FISH) experiments on lasermicrodissected material of pancreatic intraductal lesions (PanIN) intissues of CP.Patients and methods: Microdissected PanINs (PanIN1A toPanIN3) and normal ducts from formalin-fixed paraffin-embeddedtissues of a patient suffering from CP were pretreated with proteasesfor isolation of interphase nuclei for FISH or by whole genomeamplification (I-PEP-PCR) followed by microsatellite PCR for LOHanalysis. We performed FISH with centromeric probes for chromo-somes 7, 8 and 17 to determine the levels of aneuploidy in lesions ofdifferent stages. Furthermore, PanINs were analysed for mutations inTP53 and p16INK4 by ABI-sequencing and immunohistochemicalexpression analysis of p53, p16INK4 and DPC4 protein wasperformed.Results:We found an increase of aneuploidy for the chromosomes 7,8 and 17 with rising PanIN level, ranging from 3% to 6% in normaltissue, 10% to 17% in PanIN-1, 18% to 21% in PanIN-2 and 18% to25% in PanIN-3 lesions. In multiple PanIN-3 lesions, p53 proteinoverexpression and loss of protein expression for p16INK4 and DPC4protein was seen. Here, LOH was found at the TP53, p16INK4 andDPC4-locus. A heterozygous mutational status without LOH wasfound in some early PanINs (PanIN1A) suggesting the presence of

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base mutations early in carcinogenesis with subsequent clonal ex-pansion and final gene inactivation by LOH.Conclusion: Ploidy changes of different chromosomes can be foundin early PanIN, indicating an early onset of chromosomal instability.Heterozygous mutations of tumor suppressor genes TP53 andp16INK4 occur early in CP and are clonally expanded, but finalinactivation by LOH occurs later in pancreatic carcinogenesis.

5Hyperbaric oxygen enhances wound lactateproduction in ratsStefan Beckert1,2, Rummana Aslam2, Farshid Farrahi2,Heinz Scheuenstuhl2, Harriet Hopf2, Alfred Königsrainer1,Stephan Coerper1, Thomas Hunt21Department of General and Transplant Surgery, Universityof Tübingen, Tübingen, Germany2Department of Surgery, University of California, San Francisco,CA, USABackground: Healing wounds are characterized by high lactatelevels in a relatively hypoxic microenvironment. In the past, the shiftto an anaerobic energy metabolism was thought to be the main sourcefor increased lactate concentration. Recently, it has been discoveredthat lactate is also generated by an NADPH-linked oxygenase usingoxygen as one of its substrates, a mechanism called aerobic glyco-lysis. We hypothesize that hyperbaric oxygen therapy by increasingoxygen availability enhances wound lactate production.Material and methods: Four wire mesh wound cylinders (n=142)were implanted underneath the dorsal skin in each of 36 maleSprague-Dawley rats (312+11 g). Animals were randomized to 2groups: The first group (n=18) received 100% oxygen with 2.1 ATAfor 90 minutes twice a day for a total of 7 days. The second group(n=18) was treated with 21% oxygen with 1 ATA for the same timeschedule. Hyperbaric treatments were administered in a hyperbaricchamber designed for small animals (Model 100; Western Hyperbar-ic Services, Union City, California). Wound fluid from the cylinderswas aspirated between the two treatment cycles at day 2 and 5 andanalysed for lactate using a lactate analyser (model 2700; Yellowsprings Instruments, Ohio). Additionally, at day ten (after two days oftreatment with 21% oxygen in 1ATA in both groups) lactateconcentrations in wound fluid were measured again. Data areexpressed as mean+SD. A p-value <0.05 was considered significant.Results: Wound lactate concentration was significantly increasedboth in control and hyperbaric rats at day 5 and day 10 compared today 2(controls 4.5+1, 5.9+0.8, 6.7+1.2 mmol/l; p=0.04 andhyperbarics 4.4+1.2, 6.5+1.3, 8.2+1.3 mmol/l; p=0.01).At day 2 and day 5 there was no significant difference in lactatelevels between control and hyperbaric rats (p>0.05). However, at dayten lactate concentration was significantly higher in the hyperbaricgroup compared to the control group (6.7+1.2 vs. 8.2+1.3 mmol/l;p=0.01).Conclusion:Hyperbaric oxygen therapy enhances lactate productionin wounds. This investigation provides evidence for the mechanismof hyperbaric oxygen action since lactate is known to stimulatecollagen production and angiogenesis.

6First time assessment of whole blood RNA expressionimmediately after successful cardiopulmonaryresuscitationMartina Beißer, Jürgen Landes, Henning Laven, Christian Schütz,Viktoria Bogner, Julia Stegmaier, Chlodwig Kirchhoff, WolfMutschler, Peter BiberthalerChirurgische Klinik und Poliklinik, Klinikum der UniversitätMünchen – Innenstadt, München, Germany

Background: The mortality of successfully resuscitated patients issubstantially influenced by the development of systemic inflamma-tory response syndrome and consecutive multiple organ failure. Inthis context, measurement of RNA encoding for immunologicalrelevant factors is considered an early technique to assess immunesystem imbalance. However, the measurement of blood cell RNAwas handicapped by complex isolation techniques. Recently, thePAXgene Blood RNA System was introduced as a potential new toolproviding snap-shot RNA preservation out of whole blood andcontainig cells. However, it is unclear whether this system allows forreproducible and valid RNA assessment in patients after cardiacresuscitation. Hence the aim of this study was first to analyze thefeasibility of analyses using the PAXgene system under preclinicalemergency conditions and second to evaluate the RNA expression ofimmunological relevant mediators in resuscitated patients after returnof spontaneus circulation (ROSC).Patients and methods: In this pilot study, 7 patients were enrolledafter successful out-of-hospital resuscitation and ROSC. Bloodsamples (2 PAXgene tubes) were drawn immediately after ROSC(0h) as well as 6, 12, 24, 48 and 27 hours after successful car-diopulmonary resuscitation. RNA expression of TNFa, IL-1ra andIL-10 were measured using RT-PCR (Light-Cycler) after RNA-Isolation and cDNA synthesis. Data are given in mean±SEM andstatistics were performed by ANOVA followed by SNK-test.Results: TNFa expression was not significantly increased ascompared to initial baseline levels immediately after ROSC. Incontrast, IL-1ra expression was significantly (p< 0.05) elevated at 6 hafter ROSC and returned to baseline from 12h until end ofobservitation period. Additionally, IL-10 expression was signifi-cantly increased already at 6 and 12 h.Conclusion: We demonstrated for the first time in successfullyresuscitated patients at i) assessment of initial RNA expression usingPAXgene Blood RNA System is feasable under preclinicalemergency conditions and ii) that both, a pro- and anti-inflammatorymediator were elevated after 6 and 12 h respectively. Further studiesare under evaluation to correlate RNA expression to the systemiccytocine level and to detect differences of RNA expression betweendifferent clinical outcome.

7Effect of oxygenated perfluorocarbons on pancreatic isletsin long-term cultureH. Bergert1,2, K.-P. Knoch2, S. Kersting1,2, R. Meisterfeld2,J. Ouwendijk2, H.D. Saeger1, M. Solimena2,31Department of Surgery and 2Department of ExperimentalDiabetology, University of Technology Dresden, Medical School,Dresden, Germany3Max Planck Institute for Molecular Cell Biology and Genetics,Dresden, GermanyBackground: Perfluorocarbons are known for their high oxygen-solubility coefficients and for their maintenance of high-oxygenpartial pressures for extended time. They serve also as oxygen“reservoirs” for harvested organs in pancreas organ transplantation.The aim of our study was to assess the influence of oxygen saturatedperfluorohexane (PFH) on isolated pancreatic islets at differentculture times.Methods: The islets were isolated by collagenase digestion. Afterpurification by density gradient centrifugation and pooling they weredivided in aliquots in Petri dishes and cultured with RPMI-1640medium with or without oxygen saturated PFH at 37°C. Duringcultivation medium change and oxygen saturation was performedevery second day. For assessment of islet function we measuredinsulin secretion by RIA. Secretory granules were examined byimmunoelectronmicroscopy, while the levels of polypyrimidinetract-binding protein (PTB), a factor promoting secretory granule

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biogenesis, were measured by western blotting. Apoptosis wasassessed by PARP-immunostaining, necrosis with trypan blue-staining, and expression of insulin and ICA512 (markers of secretorygranules) mRNAs by quantitative real-time PCR.Results: After 1 and 7 days in culture insulin secretion wassignificantly decreased in PFH treated islets. We also detected atendency to a higher rate of apoptosis and necrosis, a reducedexpression of PTB and of insulin and ICA512 mRNAs compared toislets grown for the same length of time in the absence of PFH.Conclusions: PFH has a detrimental effect on the viability of purifiedrat islets. Our data show that PFH, while advantageous for thetransport of harvested organs, is not indicated during the purificationand culture of islets for transplantation.

8Development of a molecular assay for islet cell qualitybefore transplantation in a diabetic animal modelHendrik Bergert1, Klaus-Peter Knoch2, Stephan Kersting1,Ronny Meisterfeld2, Michele Solimena2, Hans Detlev Saeger11Klinik für Visceral-, Thorax- und Gefäßchirurgie and 2AbteilungExperimentelle Diabetologie, Universitätsklinikum Carl GustavCarus, Technische Universität Dresden, Dresden, GermanyBackground: Since the recent introduction of the Edmontonexperience by Shapiro et al. the transplantation of insulin producingtissue has become an attractive therapeutic approach for the treatmentof type 1 diabetes. In order to transfer this therapy into the clinicalroutine, the currently available pancreatic tissue must be used,however, more efficiently. An essential criterion for that is the isletquality which can vary dramatically dependently on the donor andthe isolation procedure. However, up to now there is no possibilitysurely to predict the metabolic performance of islets aftertransplantation.Material and methods: Pancreatic islets were cultured for 24 hoursafter isolation. Then samples of islets were exposed to 2 hoursglucose stimulation (25 mM). After the stimulation the insulinsecretion index were measured as well as the binding activity ofpolypyrimidine tract-binding protein (PTB) onto the m-RNA ofselected markers of secretory granules (ICA-512, PC1). In oursyngenic transplantation model the islets were transplanted then ineach case into diabetic Lewis-rats as 1500 islet equivalents (IEQ),1800 IEQ, 2000 IEQ and 2500 IEQ under the kidney capsule. Themetabolic performance of the transplanted islets were controlled withdaily measurements of blood glucose values and body weight for atleast 3 months postoperatively.Results: We were able to show the stabilization of insulin-mRNAand further markers of secretory granules through a RNA bindingprotein (PTB). The stabilization effect occurs after translocation ofPTB through phosphorylation from the nucleus into the cytoplasmand binding of this protein on the specific mRNA’s. A statisticallysignificant correlation was observed between the ability to activatePTB and to release insulin according to glucose stimulation in vitro.Additionally, a significant correlation between the transplantationsuccess (normoglycemia) and the measured parameters kept on beingable to be proved in vivo. Diabetic receipients, which weretransplanted with islets of high PTB-activation indices needed lessIEQ and became faster normoglycemic postoperatively.Conclusion: With this designed solid phase assay on mRNA - levelwe were able to predict the effectiveness of islet cell transplantation.With a high islet quality based on our marker the islet yield,necessary for a successful transplantation, can be reduced. Theobserved results must be reproduced now in the human transplanta-tion and tested under study conditions.

9VEGF as a potent stimulator of liver regeneration afterpartial hepatectomyMaximilian Bockhorn1, Phillip Dammann1, Dennis Prokofiev1,Michal Goralski1, Petra Grünewald2, Jörg F. Schlaak2,Andreja Frilling1, Christoph E. Broelsch11Department of General Surgery and Transplantation Surgery,University Hospital Essen, Essen, Germany2Department of Gastroenterology and Hepatology,University Hospital Essen, Essen, GermanyBackground: After partial hepatectomy (PH), clinical outcome,morbidity and mortality of the patients depend on an efficientregeneration of the liver. Angiogenesis is essential for theregeneration of the liver and Vascular Endothelial Growth Factor(VEGF) belongs to the most potent angiogenic factors. The aims ofthe study were to determine the effects of exogenous VEGFadministration in rats after 2/3 hepatectomy on angiogenesis byintravital microscopy, on regeneration by immunohistochemistry andon expression of angiogenic genes by cDNA arrays.Methods: Adult male Lewis rats (n=5/group) were subjected to70% PH and split in 3 groups. Group A (VEGF), group B (anti-VEGF), and group C (Control) were administered VEGF (100 ng/µl),anti-VEGF (4 µg/µl) or NaCl i.v. at 0h, 36h, and 96h. At 0h, 24h, 48h,72h, 120h, 168h postoperatively 5 rats each underwent intravitalmicroscopy of the exposed liver remnant. Recorded parameters were:Vessel density (VD), vessel diameter (VDi) and vessel flow (VF).Liver regeneration was monitored by measuring liver body weightratio (LBR) and immunohistochemically by proliferating cell nuclearantigen (Ki-67). Subsequently, specimens from regenerating liverwere harvested, and angiogenic gene expression profiles weredetermined by an inhouse cDNA macroarry including 70 genesinvolved in liver regeneration.Results: In VEGF treated rats (group A), VD was significantlyincreased compared to group B or C (p<0,05). VDi was significantlyincreased through 24–72h in group A (p<0,05) compared to group Aand C. In the VEGF treated animals, LBR was significantly higherafter 48h, 72h and 120h (p<0,025). PCNA immunostaining showeda significantly higher labelling index of hepatocytes at 24h afterPH with 82% compared to the control groups (3%). cDNA macro-arrays showed a complex modulation of genes involved in liverregeneration.Conclusion: Exogenous VEGF administration leads to increasedangiogenesis, which subsequently results in faster liver regeneration.This effect can be blocked by anti-VEGF. Therefore, VEGFtreatment may provide a novel strategy for optimization of liverregeneration in patients after PH.

10NF-KB plays a critical role in human mesenchymalstem cell invasion in vitroWolfgang Böcker, Emmanouil Pappou, Virginia Egea-Alonso*,Denitsa Docheva, Vera Krump-Konvalinkova, Christian Ries*,Oliver Roßmann, Wolf Mutschler, Matthias SchiekerExperimental Surgery and Regenerative Medicine(www.ExperiMed.de), Department of Surgery,University of Munich (LMU), Munich, Germany*Division of Clinical Chemistry and Biochemistry,Department of Surgery, University of Munich (LMU),Munich, GermanyBackground: Fracture healing may require undifferentiated plu-ripotential mesenchymal stem cells (MSCs), which have been found

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in fracture callus after systemic injection. Migration is initiated bycytokines like TNF-α and very high levels of TNF-α have been foundafter bone injury. Several recent reports suggest that TNF-α isinvolved in the mediation of both fracture and bone repair. However,the impact of TNF-α and the exact molecular mechanisms on MSCsinvasion/migration are currently unknown. TNF-α leads to activationof the NF-κB pathway and NF-κB-inducible genes play an importantrole in cell invasion/migration and proliferation. IKK-2 is the ratelimiting step in the activation of the NF-κB signal transductionpathway and it has been shown that the dominant-negative mutant ofIKK-2 effectively blocks the activation of NF-κB signalling afterstimulation with TNF-α. We hypothesized, that the NF-κB signaltransduction pathway plays a critical role in hMSCs invasion/migration.Methods: We have created a lentivirus which expresses a dominant-negative mutant of the IκB kinase 2 (dn-IKK-2). hMSCs have beentransduced with this construct using a green fluorescent protein(eGFP) as negative control. Over-expression of dn-IKK-2 wasverified by Northern blot. Nuclear translocation of NF-κB (p65) wasstudied by immunohistochemistry and Western blot. Invasion/migration assays using human extracellular matrix were carried outunder stimulation with 50 ng/ml human recombinant TNF-α.Results: 60% to 80% of hMSCs were initially transduced with thelentiviral constructs. After Blasticidine-selection, more than 98%hMSCs were expressing the transgene. Stimulation with TNF-αcaused a NF-κB (p65) translocation from cytoplasma to nucleoplas-ma within 30 minutes. This effect was completely blocked by over-expression of dn-IKK-2 in hMSCs. We found that in the presence ofTNF-α hMSCs efficiently transmigrated through extracellular matrixwhich was significantly inhibited by overexpression of dn-IKK-2 inthese cells.Conclusion: This study shows for the fist time that NF-κB isexpressed in human mesenchymal stem cells. This pathway isregulated by TNF-α through activation of IKK-2. Furthermore, theNF-κB signal transduction pathway is implicated in TNF-α mediatedmigration of hMSCs. Therefore, our results support the idea that NF-κB may be involved in hMSC migration to the fracture site.

11Predicting delirium after vascular surgery - resultsfrom a prospective trialHinrich Böhner1, Ute Habel2, Christian Ohmann3, Ralf Friedrichs4,Wilhelm Sandmann5, Frank Schneider21Department of Surgery I, Lukaskrankenhaus Neuss, Neuss,Germany2Department of Psychiatry and Psychotherapy,University of Aachen, Aachen, Germany3Coordination Center for Clinical Trials, University of Düsseldorf,Düsseldorf, Germany4Department of Anesthesia, University of Düsseldorf, Düsseldorf,Germany5Department of Vascular Surgery and Kidney Transplantation,University of Düsseldorf, Düsseldorf, GermanyBackground: The aim of the study was to determine pre- andintraoperative risk factors for the development of postoperativedelirium among patients undergoing aortic, carotid, and peripheralvascular surgery in order to predict the risk for postoperativedelirium.Patients and methods: Pre-, intra-, and postoperative data wereprospectively collected including the first seven postoperative dayswith daily follow-up by a surgeon and a psychiatrist of 153 patientsundergoing elective vascular surgery. Delirium (DSM IV) wasdiagnosed by the psychiatrist. Multivariate linear logistic regressionand a cross validation analysis were performed to find a set ofparameters to predict postoperative delirium.

Results: Sixty patients (39.2%) developed postoperative delirium.The best set of predictors included the absence of supraaorticocclusive disease and hypercholesterinemia, history of a majoramputation, age over 65 years, a body size of less than 170 cm,preoperative psychiatric parameters and intraoperative parameterscorrelated to increased blood loss. The combination of theseparameters allows the estimation of an individual patients’ risk forpostoperative delirium already at the end of vascular surgery with anoverall accuracy of 69.9%.Conclusions: Postoperative delirium after vascular surgery is afrequent complication. A model based on pre- and intraoperativesomatic and psychiatric risk factors allows prediction of the patient’srisk for developing postoperative delirium.

12Generation of soluble mediators and peri-implant cellactivation after adherence of leukocytes to biomaterialsDenise Bogdanski, Thomas A. Schildhauer, Gert Muhr,Manfred KöllerSurgical Research, Department of Surgery, BG KlinikenBergmannsheil, Ruhr-University Bochum, Bochum, GermanyBackground: Blood leukocytes belong to the first cells which willimmediately get into close contact with implant surfaces. Physiolog-ically, these cells are necessary for a controlled tissue integration.Additionally, they provide growth factors necessary to initiate andaccelerate wound healing, tissue repair and regeneration. Besidetissue remodeling soluble leukocyte mediaters are key signals forhost defense and/or inflammatory reactions. Thus, we analyzedleukocyte functions in the presence of biomaterial conditionedmedia.Methods: Conditioned media (CM) were obtained by interactions ofleukocytes with implant biomaterial samples for 24 h using cellculture conditions. Following materials (10 mm diameter discs) weretested: polished solid metals (nickel titanium-NiTi, tantalum) orporous metal samples (tantalum, titanium) or calcium phosphatecoated NiTi. Polymorphonuclear neutrophil leukocytes (PMN) andperipheral blood mononuclear cells (PBMC) were isolated fromEDTA-anticoagulated peripheral blood. The influence of CM onleukocyte functions was analyzed (regulation of adhesion molecules,chemotaxis, phagocytosis, apoptosis, bacterial killing) using flowcytometric assay and microbiological assays. The cytokine pattern ofrespective CMs was analyzed by ELISA and protein array technique.Results: In contrast to CM obtained by polished solid materials,conditioned media obtained by coated or porous materials led to cellcluster formation and upregulation of adhesion molecules (CD11b,CD66, ICAM-1), to an increase in chemotaxis, to an increase inphagocytosis and to an increase in bacterial killing of freshly isolatedleukocytes or whole blood. Cytokine analysis revealed significantlyelevated concentrations of IL-1ra, IL-6, IL-8, TNF and GM-CSF ofCM obtained from coated/porous material compared to CM frompolished solid materials. In contrast, concentrations of IL-2 or IFN-γwere not significantly different in both CMs.Conclusions: Our data demonstrate that the interaction of leukocyteswith implant material led to the release of soluble mediators (such ascytokines) which may influence functional activities of cells in theperi-implant microenvironment. Among the analyzed cytokines thosefactors released from myeloid cells (PMN and monocytes within thePBMC) were elevated which indicates that these cells are predomi-nantly activated during cell surface contact whereas lymphocytes arenot activated under these experimental conditions. The materialsurface topology (e.g. roughness, porosity, total surface area)influences leukocyte mediater generation more than the nature ofthe material. Whether a microenvironment enriched with cytokinesfrom activated leukocytes will also provide an area of enhancedlocal host defense in vivo has to be proven by further studies.

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13Implementation of Interleukin-6 serum level measurementas a routine diagnostic tool in multiple injured patientsV. Bogner, J. Stegmaier, C. Kirchhoff, W. Mutschler, P. BiberthalerChirurgische Klinik und Poliklinik Innenstadt, Ludwig MaximiliansUniversität, München, GermanyBackground: Many experimental investigations have not onlyapproved the impact of the pro-inflammatory cytokine Interleukin-6(IL-6) in the context of posttraumatic inflammation, but more im-portantly its significant role within the induction of a consecutiveMultiple Organ Dysfunction Syndrome or even multiple organfailure. However, as IL-6 measurement presupposed money and timeconsuming ELISA experiments so far, any integration of this pa-rameter into the clinical routine diagnostic has not been practicableyet. Therefore, the intent of this study was to determine whether theuse of a completely automated, quantitative cytokine analysis systemreveals valid cytokine concentrations under clinical routine condi-tions in the management of multiple injured patients.Patients and methods: 54 patients presenting with blunt multipleinjury defined by an Injury Severity Score > 16 points were includedinto the study. Blood samples were drawn on admission of the patientand at 6h, 12h, 24h, 48h, and 72h after the traumatic event. Aftercentrifugation, interleukin-6 serum levels were determined using thecompletely automated chemiluminescence-assay (IMMULITE,DPC-Biermann GmbH, Bad Nauheim, Germany). With respect toclinical outcome, the patient collective was divided into two clinicalgroups, survived (n=42) and deceased individuals (n=12). Statisticalanalysis of the time dynamic of IL-6 expression was performed byANOVA on Ranks, followed by Dunn’s method, a SNK-test wasused to detect group-specific differences.Results: Survived patients exhibited significantly elevated IL-6serum levels at 6h, 12h, 24h and 48h as compared to the admissionvalue (p<0.05). IL-6 levels in deceased patients also significantlyincreased at 6h 3524±1528 pg/ml (p<0.05) and at 12h 6216±4645 pg/ml (p<0.05) as compared to the admission value (218±62 pg/ml).Furthermore, deceased patients showed significantly higher IL-6expression at 6h (3524±1528 pg/ml vs. 610±108 pg/ml, p<0.007) andat 12h (6216±4645 pg/ml vs. 995±377 pg/ml, p<0.01) in comparisonto those patients, who survived the traumatic event.Conclusions: These data demonstrate for the first time, that i) theIMMULITE technology reveals valid and representative IL-6 serumconcentrations both under physiological and under pathophysiolog-ical conditions, ii) there are significantly different concentrations inclinical relevant groups and iii) the determined IL-6 levels correlatewith the patients clinical course. This system represents a completelyautomated, sensitive technology, which allows the implementation ofIL-6 measurement into the clinical routine diagnostic as an additionaltool for the assessment of the inflammatory status, thereby implying afirst step into more individualized treatment strategies managingmultiple injured patients.

14Microvascular endothelial cell growth influencedwith pre-adipocyte mediumA.M. Boos, G.B. Stark, G. FelmererDepartment of Plastic and Hand Surgery, University of FreiburgMedical Center, Freiburg, GermanyBackground: Hypertrophy of adipose tissue is a characteristicfeature of chronic lymphedema. Interactions between blood capillaryendothelial cells and pre-adipocytes are well known. The ratio oflymph and blood capillary endothelial cells was examined in pre-adipocytes (PAC) conditioned medium.

Methods:Human dermalmicrovascular endothelial cells (HDMECs),a mixture of blood endothelial cells (BECs) and lymphatic endothelialcells (LECs), rather then pure LECs were used. HDMECs wereextracted from human skin usingmagnetic isolation (DynaBeads) andan antibody against CD31. The ratio of BECs and LECs wasinvestigated. By using endothelial cell basal medium cells werecultivated for 3 days under 8 different conditions. To this 10% fetalcalf serum (FCS) was added and in addition as controls 25 ng/mlVEGF–A, 150 ng/ml VEGF–C and 10% medium, which wereconditioned 2 days by pre-adipocytes. In another 4 preparations theendothelial cell basal medium with 10 % FCS and 10 %medium froma pre-adipocytes culture were used. In every preparation one of thefollowing growth factor receptor was inhibited: VEGFR- 1/2 or Tie 2receptor. After a follow up of 3 days a CD 31/LYVE 1 double stainingwas conducted and 3 fields of vision were counted.Results: It could be observed that the amount of LECs that werestimulated with conditioned pre- adipocytes–medium was augmentedin comparison to BECs. This effect was obvious on the firstobservation day; but longer incubation times did not increase theeffect. On the 2nd and 3nd day a decrease of the effect was observed,which can be explained by administration of growth factors whichwere in the pre-adipocytes –medium. Only a minimal variance of theratio were seen when VEGF-Awas added. This had less influence onthe growth of the two cell populations then the conditioned medium.By adding VEGF-C no effect take place. In the conditions with pre-adipocytes –medium and inhibition of receptor VEGFR 1, 2 and Tie 2the same stimulation effect on the LECs compared to pure conditionedmedium could be observed. The growth factor receptors VEGF-A/B/Eand Ang 1/2 are not involved in the observed effects.

Culture condition Percentage of LEC

Endothel cell basal medium EBM 1,9 %EBM + PAC conditioned medium 7,7 %EBM + VEGF-C 1,3 %EBM + VEGF-A 2,8 %EBM + PAC conditionedmedium + VEGFR 1 inhibited 9,6 %EBM + PAC conditionedmedium + VEGFR 2 inhibited 8,8 %EBM + PAC conditionedmedium + Tie 2 inhibited 7,5 %

Conclusion: It seems that LEC and pre-adipocytes are in multi-factorial interaction which cannot be reduced to a single vasculargrowth factor. In order to shed new light on this phenomenon andfind out if there are also cell-cell interactions a larger amount ofassays has to be performed. The functional verification of a stim-ulation of the pre-adipocytes on the growth of the LEC may provideimportant information on development and therapies of adiposehypertrophy in chronic lymphedema.

15High cyclooxygenase-2 expression following neoadjuvantradiochemotherapy is associated with minorhistopathologic response and poor prognosisin esophageal cancerJan Brabender, Daniel Vallböhmer, Ralf Metzger, Stephan E. Baldus,Ute Warnecke-Eberz, Arnulf H. Hölscher, Paul M. SchneiderDepartment of Visceral and Vascular Surgery, Institute of Pathology,University of Cologne, Cologne, GermanyBackground: High expression of cyclooxygenase-2 (COX-2) wasshown to inhibit chemo- and radiotherapy induced apoptosis. We

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analyzed the association of COX-2 mRNA and protein expressionwith histomorphologic response to neoadjuvant radiochemotherapyin esophageal cancer.Material and methods: 52 patients with resectable esophagealcancers (cT2-4, Nx, M0) received neoadjuvant radiochemotherapy(cisplatin, 5-FU, 36 Gy) followed by transthoracic en bloc esopha-gectomy. Histomorphologic regression was defined as majorresponse when resected specimens contained less than 10% ofresidual vital tumor cells. RNA was isolated from endoscopicbiopsies (paired tumor and normal tissue) prior to neoadjuvanttreatment and quantitative real-time reverse transcriptase PCR (RT-PCR, TaqMan™) assays were performed to determine COX-2mRNA expression levels standardized for ß-actin. COX-2 proteinexpression in pretreatment biopsies and posttherapeutic resectionspecimens was analyzed by immunostaining of tumor cells.Results:Median COX-2 mRNA expression levels were significantly(p < 0.0001) different between paired tumor (median 2.2) and normaltissues (median 0.159). Comparison of pre- and posttherapeuticspecimens showed a significant difference (p<0.006) in COX-2protein expression. 12/52 tumors showed down-regulation and 3/52upregulation of COX-2 protein expression during neoadjuvantradiochemotherapy. High COX-2 protein expression in postther-apeutic resection specimens was significantly associated with minorhistopathologic response (p<0.04) and poor prognosis (5-yearsurvival probabilities: 26.3±8.2% for minor and 58.6%±12.9% formajor histopathologic response, p<0.01).Conclusion: High COX-2 protein expression following neoadjuvantradiochemotherapy in resection specimens is significantly associatedwith minor histopathologic response to neoadjuvant therapy and verypoor prognosis.

16After a severe trauma, serum procalcitonin concentrationis dependent upon total severity and anatomical siteof injury and correlates to the development of systemicinflammatory response syndrome (SIRS), sepsisand multiple organ dysfunction syndrome (MODS)Kevin Brauns, Thomas Thuemmel, Csaba Biro, Mark Lehnert,Dirk Henrich, Felix Walcher, Ingo MarziDepartment of Trauma Surgery,Johann Wolfgang Goethe-University, Frankfurt/Main, GermanyBackground: Procalcitonin (PCT) shows a sound correlation to theactivity of inflammation within critically injured patients. It appearsto be able to distinguish between SIRS and sepsis and is furthercapable of controlling how systemic inflammation and organdysfunction is treated. Previous data shows that an early andincreasing induction of PCT could indicate complications in a laterstage. The purpose of the study was to describe the course of the PCTserum concentration after the trauma, as well as to assess thedependance of the serum PCT on the anatomical site and severity ofthe injury over a 14 day period after the initial trauma.Methods: 12 months prospective clinical research, 57 patients,Criteria for inclusion: Admission into the emergency room,minimum age 18 years. Blood samples were taken in the emergencyroom and subsequently daily for the 14 day period. Measurementparameter: PCT and IL-6 serum concentration of the patient wasdetermined. Patients were subdivided into 5 groups depending on theseverity of the injury by means of Injury Severity Score (ISS) as wellas into 3 groups depending on the anatomical site of the injury bymeans of Abbreviated Injury Scale (AIS). Statistics: Wilcoxon test,Spearman-Rang correlation, p<0.05 significance.Results: Despite the severity of the injuries (I: ISS 1–8, n=5; II: ISS9-15, n=13; III: ISS 16-24 n=11; IV: ISS 25-40, n=16; V: ISS 41-75,n=12) all patients demonstrated a significant increase from day 0 today 1 of PCT serum concentration in comparison with the controlgroup (n=8) (p<0.05). During the first 5 days after the trauma the

concentration of PCT and IL-6 also increased significantly incomparison with the control group. On day 1 the level of PCTreached its peak in group I-IV (mean<2 ng/ml) after that time itdecreased steadily. After 8 days the results in group V showed anincrease for a 2nd time (mean>2 ng/ml) and remained high for theentirety of the research. IL-6 acted similarly during the course;however a second increase of IL-6 in critically injured people on day8 was not recognised. Significant differences between the groupscould be identified (PCT: III vs. IV, p<0.05) such that the level ofPCT and IL-6 concentration can be seen to be dependent on the totalseverity of injuries. The division into different injury patterns (I: AIS0-1; II: AIS 2-3; III: AIS 4-6) always showed a significant increase inPCT concentration when abdomen and pelvis were affectedcompared to similar injuries without damage to abdomen and pelvis.IL-6 could not be distinguished prominently here. During theexamination of isolated traumatic injuries of head, thorax andextremities a slight influence on the level of PCT could be seen. ThePCT level had the greatest increase on single thorax traumas, whilstsingle head traumas had the most significant impact on the IL6 level.There were no incidents of isolated abdominal injuries. PCT and IL6significantly revealed the existence of SIRS, however no parameterdifferentiated significantly between SIRS and sepsis. The concentra-tion of PCT and IL6 correlated tentatively, but not significantly whenlevels of Multiple Organ Dysfunction Score (MODS) und LogisticOrgan Dysfunction System (LOD) are increased. Further theydisplay an altogether large mean variation.Conclusion: A high PCT concentration at the first day after thetrauma is always accompanied by serious injuries and can indicatefrequent occurrences of shock and MODS. Abdominal and pelvictraumas in particular influence the level of PCT significantly. Ingroup V, which includes serious injuries and injuries without achance of survival, PCT shows a second peak after 8 days that couldbe seen as an expression of the systemic inflammation reaction orsepsis. That is why PCT in combination with other parameters can beused as an effective marker for the judgement of risk-profile ofpolytrauma patients.

17C3a is significantly increased in free flapsafter prolonged ischemia-reperfusion injuryA. Brüggemann1, A. Noltze1, M. Kaun1, S. Görg2, J. Gliemroth3,P. Mailänder1, H.G. Machens11Department of Plastic Surgery and Hand Surgery, Burn Care Center,2Department of Immunology, 3Department of Neurosurgery,UKSH/Campus Lübeck, Lübeck, GermanyBackground: Free microvascular tissue transplantation (FMTT)represents a well established reconstructive surgical technique andyet a clinical model to measure ischemia-reperfusion injury (IRI),which is responsible for occurrence of the no-reflow phenomenonafter complicated FMTT. Microdialysis has been used as an in-situmonitoring device for different metabolic, but not yet immunologicsubstances < 20 kDa after FMTT. This prospective, controlled studywas conducted to detect substances, relevant for IRI after FMTT.Methods: Nineteen patients were monitored by means of theMicrodialysis technique. Eighteen free myocutaneous latissimusdorsi muscles and one free radial forearm flap were used for lower legreconstruction. Each 1 catheter was placed into the flap target tissueand 1 catheter was implanted into healthy reference tissue. Weanalysed biochemical substances such as glucose, pyruvate, lactateand glycerol and – for the first time – immunologic substances such asinterleukin 8, C3a and RANTES. We collected samples duringischemia and changed the microvials every 90 minutes afterreperfusion. Each patient was monitored for at least eighteen hoursand classified as group A (not prolonged IRI) or group B (prolongedIRI). In addition, clinical monitoring was performed after each FMTT.

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Results: All free flaps healed without major complications (nopartial/complete loss of flap tissue). Minor complications prolongedIRI and included intraoperative and postoperative revisions of themicrovascular anastomoses due to hematoma or thrombus formation,which significantly increased total flap ischemia time in group Acompared to group B (p<0.001).We detected no significant differencebetween the concentrations of biochemical substances duringreperfusion in target and control tissue of both group A (n=12) andgroupB (n=7). Interleukin 8 andC3awere found in both groupsA andB, whereas the concentration of RANTES was below threshold inmost cases. For both groups A and B separately, we found higherconcentrations of C3a in target tissue compared to control tissue.Comparing the target tissue of groups A and B, we found a highlysignificant increase of C3a (P<0.001) only in group B during the first90 minutes of reperfusion.Conclusions: C3a seems to be a highly sensitive early indicator ofischemia-reperfusion damage. This result could, at least in part,contribute to explain the “no-reflow-phenomenon” occurring aftercomplicated FMTT.

18The interaction of apoptosis and angiogenesisin a tumor cell environmentPeter Čamaj1, Michael Brückel1, Enrico DeToni2, Markus Guba1,Karl-Walter Jauch1, Christiane J. Bruns11Department of Surgery, University of Munich, KlinikumGroßhadern, Munich, Germany2Department of Internal Medicine II, University of Munich,Klinikum Großhadern, Munich, GermanyBackground: Equilibrium between cell proliferation and cell deathshifted to enhancement of cell proliferation is necessary forprogression of tumour growth. Tumour cells are characterized by ahigh proliferation activity and very often also by impaired apoptosis.Quickly proliferating and metabolically active tumour cells have anincreased demand for oxygen and nutrients. Only cells that are wellsupplied by both sufficient amount of oxygen and nutrients are ableto survive. Therefore vascularization is a crucial process influencingthe development of the growing neoplastic lesion. It would have a lotof advantages for tumour cells if both processes (regulation ofapoptosis and angiogenesis) would be coordinated.Methods and experimental design: We have transfected a p53mutant pancreatic cancer cell line MiaPaCa and a p53 wild typecolon cancer cell line LS513 with plasmids harbouring genes such asCrmA, Bcl-2, its mutant variant lacking the BH4 domain and the Bcl-2 variant with point mutation G145A (unable to interact with pro-apoptotic protein Bax), under the control of both a constitutive CMVpromoter and a tetracycline inducible expression system, respec-tively. We have determined the rate of apoptosis and proliferationusing FACS after propidium iodide staining. Furthermore, we haveassayed VEGF production by ELISA in the conditioned medium ofthe above mentioned transfectants and demonstrated their pro-angiogenic properties on HUVECs by a MTT proliferation assay.The in vivo properties of these cells were demonstrated byimmunohistochemistry on sections obtained from orthotopic tumoursgrowing in nude mice following injection of the transfectantsdescribed above. These sections were stained by anti-VEGFantibody.Results: Our experiments show, that decreased apoptosis due to theexpression of wild type Bcl2 or CrmA leads to increased productionof VEGF and consequently stimulates tumour angiogenesis.Interestingly, cells expressing the mutant variants of Bcl2 G145Aunable to interact with Bax exhibit very high level of apoptosis aswell as very high production of VEGF in the same time. In animalexperiment we have confirmed these in vitro findings. Tumours fromnude mice injected with cells transfected with mutant Bcl2 G145Aare smaller and produce high amount of VEGF as demonstrated byimmunohistochemistry.

Conclusion: These preliminary results showed that overexpressionof anti-apoptotic genes lead to increased angiogenesis both in vitroand in vivo. The finding that Bcl2 mutant G145A, unable to interactwith the pro-apoptotic protein Bax and therefore causing increasedapoptosis but also increased VEGF production in the same time,leads to the conclusion, that angiogenesis is not directly dependent byapoptosis but by interaction between Bcl2 and Bax. This conclusiondoes not contradict the generally accepted hypothesis aboutactivation of angiogenesis by inhibition of apoptosis, however, itre-considers the potential underlying mechanism.

19Effective therapy of human pancreatic cancer in SCIDmice with coagulation active Antithrombin IIIIlhan Celik1, Oguzkan Sürücü1, Carsten Dietz2, Elias Karakas2,Uwe Kalina3, Oliver Kisker21Institute of Theoretical Surgery, Philipps University, Marburg,Germany2Department of General Surgery, Philipps University, Marburg,Germany3ZBL Behring, Marburg, GermanyBackground: Tumor growth is dependent upon the balance ofpositive and negative regulators of angiogenesis. Antiangiogeniccompounds such as antiangiogenic Antithrombin III (aaAT III) in-hibit endothelial cell biology in vitro and angiogenesis in vivo. Untilnow it is unknown whether coagulation active Antithrombin (AT III)is effective as an antiangiogenic inhibitor. The aim of the study wasto determine the effect of coagulation active AT III (Kybernin©) onthe growth of human pancreatic cancer in a mouse model (SCID).Material and methods: A human pancreatic cancer cell line(BxPC3) was injected subcutaneously into the dorsa of male,immunodeficient (SCID) mice. When tumor volume was 100 mm3,mice were randomized into five groups (n=8/group) receivingsystemic (s.c.) Kybernin© (10; 50; 100 mg/kg /day with n=8 /group),latent AT III (antiangiogenic AT III) (100 mg/kg/day with n=4/group)injections or Buffer (n=5/group) as control for 20 days. Tumors weremeasured every third day and the ratio of treated to control tumorvolume (T/C) was determined for the last time point. Descriptive andexplorative statistical analysis was performed.Results: After 20 days of treatment the tumor volume in the groupthat re-ceived Buffer was 1741±557 mm3 versus 335±63 mm3 forthose being treated with Kybernin© 100 mg/kg/day; 166±25 mm3

when treated with Kybernin© 50 mg/kg/day ; 538±40 mm3 whentreated with Kybernin© 10 mg/kg/day and 349±65 mm3 for treatmentwith latent AT III (100 mg/kg/day). The T/C ratio (treatment/control)decreased progressively during the experiments, and was 0.19 forKybernin© 100 mg/kg/day, 0.10 for 50 mg/kg/day, 0.31 for 10 mg/kg/day and 0.20 for latent AT III (100 mg/kg/day). The inhibition oftumor growth was statistically significant in all treatment groupscompared to placebo. All mice gained weight throughout the study.Conclusion: Coagulation active AT III (Kybernin©) was effective inthis mouse tumor model of pancreatic cancer. The most effectivedosage was 50 mg/kg/day. Based on this results and furtherpreclinical investigations, clinical trials with coagulation active ATIII (Kybernin©) in cancer patients might be of interest.

20Induction of lymphangiogenesis in human adultmesenchymal stem cell linesClaudius Conrad1, Ralf Huss2, Stefan Huber1, Hanno Nies1,Karl-Walter Jauch1, Christiane Bruns11Department of Surgery, University of Munich, Munich, Germany2Institute of Pathology, University of Munich, Munich, Germany

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Background: It was our intent to evaluate the mechanisms of stemcell differentiation during lymphangiogenesis and the possible role ofHuman Adult Mesenchymal Stem Cells (MSC) in this process.Materials and methods: We used a well characterized human adultstem cell line V54/2 derived from the peripheral blood of a healthydonor as well as the murine stem cell line balb/c. As a mean ofinduction we used supernatant from human dermal microvascularcells (HDMEC), supernatant from isolated lymphatic endothelialcells (LEC) as well as VEGF-C. As outcome parameters we usedmorphological changes and several molecular markers of lymphan-giogenesis such as Podoplanin, Lyve-1 VEGF-R II and III. Activemigration was evaluated using a modified Boiden chamber.Results: We were able to demonstrate that the morphology of thestem cells changed dramatically towards an endothelium-likephenotype after incubation with supernatant from isolated LEC.Moreover, we found expression of LYVE-1 in fluorescencemicroscopy. Using FACS-Analysis we were able to demonstratethe up regulation of Podoplanin as well as VEGF-R II and –RIIIhighest in the group incubated with supernatant of LEC. Incubationwith supernatant from HDMEC or with VEGF-C revealed a lessprominent effect. We were able to correlate those findings on RNA-level. There was a small subpopulation that expressed both receptorsVegf-R2 and –RIII on the surface. We used a modified Boidenchamber to evaluate migration of VEGF-C induced V54/2 vs. nativeV54/2 towards HDMEC. We found a significant higher migration ofVEGF-C induced V54/2 V54/2 in comparison to non induced stemcells.Discussion: The in vitro experiments show that MSC have po-tentially an important role in the process of lymphangiogenesis invivo. We present data that MSC have a significant higher migrationindex of VEGF-C induced vs. non-induced stem cells towardsHDMEC suggestive of an active homing mechanism. Moreover,these MSC have the capability of expressing characteristic markersof lymphangiogenesis. The more efficient lymphangiogenic induc-tion of MSC with supernatant vs. VEGF-C alone suggests thatalthough VEGF-C has been proclaimed as the predominant player inthe induction of lymphangiogenesis there must be factors that havethe same of even higher capability to do induce lymphangiogenicdifferentiation. It will be of crucial importance for the understandingof lymphangiogenesis to identify those factors.

21Tumor growth inhibition by a new angiogenesis inhibitor:FSAP (Factor VII activating protease)Carsten Dietz1, Sandip M. Kanse2, Oguzkan Sürücü3,Sebastian Hennig3, Thomas Weimer4, Ilhan Celik31Department of Visceral-, Thoracic- und Vascular Surgery,Philipps-University, Marburg, Germany2Institute for Biochemistry, Justus-Liebig-University, Giessen,Germany3Institute of Theoretical Surgery, Philipps-University, Marburg,Germany4ZBL Behring GmbH, Marburg, GermanyBackground: Factor VII activating protease (FSAP), a serineprotease from human plasma, is involved in hemostasis due to itspropensity to activate single-chain plasminogen activators andcoagulation factor VII. It was reported that FSAP has inhibitoryeffects on migration and proliferation of smooth muscle cells. Theaim of this study was to investigate the potential antiangiogenic andanti-tumor activity of FSAP in vitro and in vivo.Materials and methods: FSAP proenzyme isolated from humanplasma was used to treat a primary human pancreatic cancer (BxPC-3, 5x106 cells/mouse) implanted s.c. into the dorsa of immunodefi-cient (SCID) mice. After the tumor volume reached at least 100 mm3,

mice were randomized into two groups (n=4/group). The first groupreceived FSAP (daily, s.c. 8 mg/kg/day) for 20 days at sites distantfrom the tumors and the second received placebo (FSAP-buffer). Thevolume ratios of treated to control tumor (T/C) were determined.FSAP (0.2 – 40 µg/ml) was also tested in different in vitro experi-ments. For analysis of anti-proliferative and anti-migratory activity,FSAP was tested using endothelial cells (HUVEC and BREC).Results: FSAP significantly inhibited the proliferation and migrationof micro-endothelial (BREC) and macro-endothelial (HUVEC) cellsin a dose-dependent manner. In the tumor model, subcutaneousadministration of FSAP caused a 50% inhibition of BxPC-3 tumorgrowth compared to placebo. There were no apparent adverse sideeffects.Conclusion: For the first time to our knowledge, we have dem-onstrated a clear and significant antiangiogenic and anti-tumor effectof FSAP in vitro and in vivo. Administration of FSAP in tumorbearing mice led to a significant tumor inhibition. These resultsreveal a new function of FSAP as an inhibitor of angiogenesis andtumor growth and hence as a possible new candidate for antiangio-genic treatment of angiogenesis-associated diseases.

22Engineering of vascularized adipose tissue in vivofor reconstruction of soft tissue defects: longtermand transplantability resultsJürgen Dolderer1,4, Michael Findlay1, Justin Cooper-White3,Nicolas Trost2, Günter Germann4, Wayne Morrison11The Bernard O’Brien Institute of Microsurgery and Department ofPlastic and Reconstructive Surgery, St. Vincent’s Hospital,Melbourne, Australia2Department of Medical Imaging, St. Vincent’s Hospital,Melbourne, Australia3Department of Chemical Engineering, University of Melbourne,Melbourne, Australia4Department of Plastic and Reconstructive Surgery, BG-TraumaCenter, University of Heidelberg, Ludwigshafen, GermanyBackground: A high demand exists for flaps with large volumeswhich can reconstruct congenital or aquired soft tissue volumedefects. Tissue engineering offers the prospect of replacing missingor non-functioning body parts with newly created tissue. However,there are certain limitations in generation of three-dimensionalvascularised adipose tissue constructs. Especially complex three-dimensional tissue, in particular for breast reconstruction, requirevascularisation for its survival, with a view to transplanting it into thein vivo situation. The aim of the study was, to establish a techniquefor the de-novo generation of large amounts of vascularised, stableand transplantable adipose tissue using a growth chamber in ananimal model.Methods: In this study we examined the use of an adipofascial tissueflap and a matrix scaffold in a growth chamber in the rat (1.7 ml). Theadipofascial flap was 4% of the overall chamber volume at insertion.Subsequently, we used the method in the pig with a growth cham-ber of 78 ml volume and following pedicled transplantation of thenew generated adipose tissue to simulate in-situ breast reconstruc-tion or remote flap generation. Magnetic Resonance Angiography(MRA) was assessed for its ability to non-invasively monitor tissuegrowth and vascularisation within the chamber. Such monitoring isessential for application of these techniques in humans.Results: At 12 weeks post-insertion, the entire chamber was filledwith new tissue and the adipose tissue was grown up to 60% of thechamber volume. The MRA was capable of monitoring vesselpatency and could distinguish between the new tissue in thechamber and the matrix scaffold when compared with histologicalsections at harvest. Histology could confirm true hyperplasia of

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adipose tissue. Hypertrophic changes have not been seen. When thenew generated adipose tissue was transferred to an other site of thebody, the tissue stays stable in volume and showed no fibroticchange after 22 weeks.Conclusion: The study demonstrated a promising method ofproducing significant amounts of vascularised, stable and transfer-able adipose tissue that can be monitored non-invasively by MRA.This is an important step towards development of a permanentautologous soft tissue replacement and a clinical application.

23Influence of different synthetic meshes on humanfibroblasts and HeLa-cellsM. Duchrow1, S. Meder1, M. Friedrich1, S. Farke2,H.P. Bruch2, R. Broll21Surgical Research Laboratory and 2Surgical Clinic, UniversityClinic of Schleswig-Holstein, Campus Lübeck, Lübeck, GermanyBackground: Implantation of meshes leads to an acute and chronicinflammation. Recently, in an in vitro-model we could demonstrate asignificant increase in apoptosis in human fibroblasts after incubationwith polypropylene meshes (Prolene, Ethikon, Germany). Theapoptotic effect on fibroblasts was dramatically enhanced, whenthe meshes were re-sterilized by autoclaving at 121°C for 20 minutes.Scanning electron microscopy showed a disintegration of themeshes’ surface. After incubation of meshes in aqua dd we found agroup of soluble polymers by mass spectrometry with differences of44 m/z between each of them in these meshes. These polymers maybe responsible for the apoptotic effect of the meshes. In the currentstudy we investigated the influence of other synthetic meshes onhuman fibroblasts and HeLa-cells.Methods: The following meshes were used: (1) VyproII (Ethicon,partially resorbable multifilament mesh composed of Prolene andVicryl resp. poliglacine 910), (2) Vicryl (Ethicon, resorbable meshcomposed of polyglactine 910), (3) Ultrapro (Ethicon, partiallyresorbable monofilament mesh composed of Prolene und Monocrylresp. poliglecaprone), (4) TiMESH extralight (GfE, Nürnberg; titan-coated, non-resorbable monofilament polypropylene mesh). Aliquotsof the meshes (ca. 2 cm2) were incubated in 12-well plates with (a)MRC5-fibroblasts or (b) HeLa-cells under standard cell-cultureconditions. Cells without meshes served as negative controls. After72 h the proliferation indices and apoptosis indices of the cells wereestimated after staining with anti-pKi-67 or Annexin V, respectively,using flow cytometry.Results: Inkubation with VyproII elevated the apoptosis index offibroblasts from 4.8% at 0h to 7.9% after 72h and of HeLa-cells from4.4% to 8.0% while the apoptosis index of the controls decreasedafter 72 h from 4.8% to 4.1% for fibroblasts and 4.4% to 4.6% forHeLa-cells. The Vicryl mesh also enhanced the apoptosis index(fibroblasts: 5.3% to 7.5%; control: 5.3% to 2.9% / HeLa-cells: 4.4%to 7.8%; controls: 4.4% to 4.8%) while the apoptotic effect ofUltrapro mesh was lower. The TiMESH showed the lowest apopticeffect. The proliferation indices were unchanged or slightly loweredafter 72 h incubation with all meshes (ca. 1-2%).Conclusion: In summary our experiments demonstrate that somemeshes clearly have an influence on human cells regardingprogrammed cell death but not cell proliferation. The apoptoticeffect of ViproII and Ultrapro can be explained by their content ofProlene, that showed the same effect in a previous study. However,Vicryl also had a strong apoptotic effect, which could be explainedby other toxic components or metabolites. The less intensitive effectof TiMESH could be explained by the covering of polypropylenematerial by the metal titan. This coating may prohibit the release oftoxic substances from polypropylene.

24Efficiency of low-intensity pulsed ultrasound on distractionosteogenesis - a prospective, randomized studyMarcel Dudda, Axel Pommer, Friedrich Kutscha-Lissberg,Gert Muhr, Stefan A. EsenweinDepartment of Surgery, University Hospital Bergmannsheil,University of Bochum, Bochum, Germany and Helios HospitalWuppertal, University of Witten/Herdecke, Wuppertal, GermanyBackground: Low-intensity pulsed ultrasound has been proven toaccelerate fracture healing both clinically and experimentally. In thisstudy the influence of low-intensity pulsed ultrasound duringdistraction-osteogenesis of 40 patients was investigated.Patients and methods: 40 patients with distraction osteogenesis > 20mm could be included in this study. Both patient groups werecomparable regarding their demographic data. The average transpor-tation distance amounted to 669mm in the group using ultrasound andin the control

,s group 631 mm. The indication for the distraction

osteogenesis resulted in 9 patients in the case of primary substancedefect after fracture of lower extremity, in the case of 23 patients withosteitis it was accomplished a resection of bone before distraction. 20patients using low intensity pulsed ultrasound after instruction by aphysician. The adjunctive ultrasound treatment device was transcu-taneously applied (frequency 1.5 MHz, signal burst with 200 micro-seconds, signal repetition frequency 1.0 kHz, intensity 30 mW/cm2)with the transducer placed at the distraction zone for 20 minutesdaily. In all cases in-home treatment was performed. Evaluation wasdone by radiographic and sonographic controls of the distractionzone during examination of all patients at the outpatients’ departmentevery 3 – 4 weeks.Results: Because of low intensity pulsed ultrasound therapy themineralisation of the distraction zone was significantly faster than inthe control group. This was investigated with standardized x-rays anddigital image analysis. Ultrasound control investigations permittedgood control in the early phase of distraction osteogenesis. Twopatients of each group had to be amputated. Three other patientsneeded additional operative treatment including cancellous bonegrafts because of missing new bone formation. Negative effects oflow-intensity pulsed ultrasound during therapy could not be detected.Conclusion: We conclude that ultrasound treatment can acceleratebone maturation and formation in distraction osteogenesis, some-times even in states of poor callotasis. Our findings indicate that dueto the application of low-intensity pulsed ultrasound biomechanicalhealing after distraction-osteogenesis finally can be achieved faster. Itmay provide a method of great promise during distractionosteogenesis.

25Dynamics of PMN-Elastase in CSF of patientssuffering from severe traumatic brain injuryC. Egginger, C. Kirchhoff, U. Kreimeier, J. Landes, V. Bogner,W. Mutschler, P. BiberthalerChirurgische Klinik und Poliklinik-Innenstadt,Ludwigs-Maximilians-Universität München, München, GermanyBackground: Traumatic brain injury (TBI) is characterized by a highmortality, besides the initial traumatic damage, mainly caused bysecondary brain injury, determined by edema, inflammation andcerebral microcirculatory damage. We showed in recent studies thatpatients suffering from severe TBI show elevated CSF-levels ofimmunocompetent cells, such as granulocytes. However, thepathophysiologic role of this increase remains unclear so far. Asshown by multiple research projects concerning inflammatory braindiseases, like purulent meningitis, polymorphonuclear neutrophil

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(PMN) -elastase, as an indicator for cellular damage, originatingfrom neutrophils, shows a significant increase. Therefore the aim ofthis study was, to analyse intrathecal levels of PMN-elastase and theCD-15 granulocyte fraction in subject to intracranial pressure (ICP)in patients suffering from severe TBI.Patients and methods: In a prospective study we enrolled 12patients suffering from severe traumatic brain injury (initialGCS<8pts.). According to a serial protocol, CSF samples werecollected, directly after insertion of a ventricular pressure catheter aswell as 12, 24, 48 and 72 hrs post trauma. CSF from 8 healthypatients, which conceived spinal anaesthesia, served as controlgroup. For the analysis of granulocytes, CSF was stained using antiCD15-FITC (Caltag, Hamburg) and quantified using flow cytometry.PMN-elastase was monitored using ELISA (Milenia-Biotec, BadNauheim). Data of granulocytes are given as percentage of the CSFcell count. The ICP-values were monitored continuously at everysampling point. Statistics were performed using the ANOVAfollowed by SNK-test vs. baseline, and Mann-Whitney-U vs.controls.Results: The values of the PMN-Elastase in the CSF show asignificant increase at 48 hrs. 26.4±10.7 ng/ml (Mean±SEM) and 72hrs post trauma 54.2±27.8 ng/ml in comparison to the admission-value 7.6±2.2 ng/ml, as well as to the control group 7.4±1.2 ng/ml. Incontrast, granulocytes in patients are already significantly increasedat admission 4.8±0.7 compared to the control group 1.7±0.3. After 72hrs, there is a significant decrease 32.5±11.5. However, intracranialpressure was continuously increased, although no significant riseoccurred up to 72 hrs after trauma (n=12, p<0.05).Conclusion: In this study, we showed the dynamics of intrathecalPMN-Elastase in patients suffering from severe TBI for the first time.Although we observed a significantly increased level of CD15-positive granulocytes, PMN-elastase increased with a latency of48 hrs. Further studies are currently performed, to elucidate themechanisms leading to PMN-elastase in CSF.

26Identification and characterization of putative tumourstem-cells in pancreatic adenocarcinoma cell linesPatricia Alice Eisenach1, Hendrik Ungefroren, Heiner Schäfer2,Holger Kalthoff11Section Molecular Oncology, Clinic of General Surgery andThoracic Surgery, University Hospital of Schleswig-Holstein,Campus Kiel, Kiel, Germany2Clinic for Internal Medicine I, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, GermanyBackground: The cells of most tumours are heterogeneous withregard to their self-renewal capacity, their apoptosis-resistancemechanisms and their ability to induce new tumours aftertransplantation in vivo. The existence and distribution of tumorigenicstem cells in several solid and hematopoetic tumours, has recentlybeen proven and the latter is well described. These cells are suspectedto cause tumour recurrence after chemotherapeutical or irradiation-therapy. Pancreatic cancer is the 4th leading cause of cancer-relateddeath within the western world and among the most resistant tumoursto chemotherapy. The existence of tumour-stem cells in pancreaticcancer has not been proven so far. Normal and cancer stem cellsexpress high levels of ATP-binding cassette (ABC) drug transporterswhich mediate multi-drug resistance, such as ABCG2 (breast cancerresistance protein 1; BCRP1; MRX), a protein, originally identifiedin mitoxantrone-resistant MCF-7 cells. Despite these more universalstem cell markers, stem cells derived from solid tumours may alsoexpress organ-specific markers. The well characterized member ofthe ABC-transporter family ABCG2 confers resistance to variousanticancer drugs by mediating the efflux of diverse chemotherapeu-tics like mitoxantrone, topotecan, doxorubicin, daunorubcin, irino-tecan, imatinib and methotrexate. CD133 was initially shown to beexpressed on hematopoetic stem cells as well as on progenitor cells

and retinoblastoma cells. More recently, stem cell-like cells enrichedfor CD133+ in human brain tumours have been described. CD133 isalternatively spliced in two different isoforms whereas only the exon3-lacking AC133/2 variant is reported to confer stem cell capacity.Methods: Here, we quantified the mRNA expression of ABCG2 andCD133, both AC133/1 and AC133/2, in pancreatic cell linesrepresenting different grading (Panc1, Panc89, PancTuI, Colo357,A818-6) as well as in the ABCG2-positive breast carcinoma cell lineMCF-7, in the ABCG2-negative cell line Saos-2 (osteosarcoma) andin SKOV-3 cells (ovarian carcinoma) using real-time RT-PCR.Functional activity of ABCG2 was measured by the ability ofABCG2-positive cells to extrude the fluorescent Hoechst 33342 dyein the absence or presence of the ABC-transporter specific inhibitorverapamil in a flow cytometry assay. ABCG2 and CD133 proteinexpression was additionally detected with the BXP-34/BXP-21 andthe AC133 monoclonal antibody respectively, using flow cytometryas well as immunoblotting. The distribution and localization ofABCG2 was determined on cytospin preparations.Results: From previous data ABCG2 is known to be highlyexpressed in MCF-7 cells, amongst others, while Saos-2 was foundto be negative. In this study we identified, in addition, an increasedABCG2-expression in a subpopulation of all tested pancreatic celllines compared to non-malignant cells and to cells derived from othertumour entities respectively. On average, the relative gene expressionat the RNA-level was two times higher compared to fibroblasts withthe highest amount in A818-6 cells (4 fold increase). The drug-exportproperties conferred by the proteins belonging to the ABCtransporters are important stem-cell markers and could be used in afunctional detection assay. The fluorescent Hoechst 33342 dye isspecifically expelled by ABCG2 off the cells and could be inhibitedby verapamil. Hence, cells with stem cell properties could beidentified by showing a lower Hoechst 33342 concentration. Thesecells are referred to as “side population” cells. All tested pancreaticcell lines revealed a significant increase in Hoechst 33342fluorescence, which could be reversed by incubation with verapamil.About 10% out of all cells of each cell line could be identified as“side population” cells by these criteria. To compare the mRNAexpression levels and the functional Hoechst 33242 data with proteinlevels of ABCG2 in whole cell lysates, flow cytometry was per-formed. In accordance to the gene expression results, all pancreaticcell lines showed a high amount of ABCG2 on their surface.Immunoblotting of whole cell lysates revealed multiple signalsaccording to several potential glycosylation sites while ABCG2staining of cytospin preparations showed a considerable expressionat the apical cell membrane of a distinct cell-subpopulation. CD133is known to be a stem cell marker of the hematopoetic system as wellas leukemias and was recently shown in human glioblastomas. Inthree out of five pancreatic cell lines an increasing amount of theisoform AC133/2 could be detected. Except for the Saos-2 cells, thegene expression was about 4 times the amount of non-malignant cellsand cells of other tumour entities respectively. Compared to thesignificant increase of CD133 mRNA in pancreatic cell lines, onlyabout 1–2% of all cells were CD133 positive by flow cytometry. It isnoteworthy to mention that the monoclonal antibody used detectsboth isoforms AC133/1 and AC133/2.Conclusion: In this study we identified for the first time a distinctsubpopulation of cells highly enriched for ABCG2 expression in wellestablished pancreatic cell lines. All pancreatic cell lines used weretested positive on both RNA and protein level. An increasedexpression of ABC transporters contributes to the extensive multi-drug resistance of pancreatic tumours and may consequently be atarget to improve chemotherapeutical effects in patients withpancreatic cancer. In contrast, only a small amount of the welldescribed stem cell marker CD133 was expressed in pancreatic celllines. However, the existence of (more?) tumorigenic stem cells/sidepopulation cells as suggested by our results points at new targets fortherapeutic drug research, since these cells exhibit the most likelyorigin of tumour formation and recurrence.

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27Fluid-shear-stress-treatment is superior togrowth-factor-treatment in increasing arteriogenesisInka Eitenmüller, Alexander Kluge, Kerstin Broich, Matthias Heil,Thomas Schmitz-Rixen, Wolfgang SchaperMax-Planck-Institute for Heart and Lung Research, Bad Nauheim,GermanyDepartment of Vascular and Endovascular Surgery,J.W. Goethe-University, Frankfurt/Main, GermanyBackground: “Arteriogenesis” refers to the development offunctional collateral arteries based on pre-existent arteriolar connec-tions. Although in principle able to prevent loss of organ function,collateral growth is not sufficiently able to restore the full flowreserve of the normal femoral vascular bed. We hypothesize thatpremature normalisation of fluid shear stress (FSS) is the cause ofunsatisfactory adaptation. We therefore evaluated the effect ofchronically increased FSS on arteriogenesis by creating an arterio-venous shunt in a rabbit hind limb model. Furthermore, we comparedshunt-operated with growth-factor-treated animals.Methods: In the shunt-group the left femoral arteries of NZW rabbits(n=11) were ligated and an arterio-venous shunt was created by side-to-side anastomosis of the distal stump of the occluded artery and theleft femoral vein (shunt-side). The right femoral artery was onlyligated and served as control. Additional animals (n=18) underwentfemoral artery occlusion and were treated with continous infusion ofdifferent growth factors (MCP-1, FGF-2, PDGF and PLGF) directlyinto the collateral system via osmotic minipumps or treated withadenoviral intracollateral genetransfer (Ad5.1FGF-4). For furthercomparison, totally untreated rabbits were used. To quantifyarteriogenesis, measurements at rest were done using magneticresonance imaging (MRI) with phase-contrast imaging for assess-ment of absolute and relative blood flow in the iliac arteries and theaccompanying veins. Time of flight (TOF) MRI angiograms and 16row computed tomography (CT) served for collateral metering.Furthermore, maximum collateral conductances (Cmax) undermaximal vasodilatation were measured and postmortem angiogramswere prepared.Results: Seven days after femoral artery occlusion the shunt-treatedlegs showed Cmax (306,8±15,0 ml/min/100 mmHg), which werecomparable to those of legs without femoral occlusion (Cmax 345±14, n.s.) and significantly higher than in controls (155,2±27,0 ml/min/100 mmHg, p<0,01). Four weeks after surgery, Cmax of theshunt-treated legs were twice the maximum conductances ofuntreated rabbits. The number of visible collateral arteries confirmedthe differences between shunt- (42,2±2,0) and controls (18,0±0,9,p<0,01). The flow-ratio of the shunt-treated legs measured by MRIwas 5.8-fold that of controls after 7 days and increased up to 9.7-foldafter 4 weeks. In contrast, arteriogenesis (quantified via Cmax-measurements and postmortem angiographies) was significantly lessenhanced in growth-factor-treated or genetransfer-treated animals.Conclusion: High FSS is the most potent trigger of arteriogenesis.We conclude that complete restoration of vascular function can beachieved by increased FSS but not by growth-factor-therapy.

28Injectable liver: a novel approach using fibringel as a matrix for culture and intrahepatictransplantation of hepatocytesHenning C. Fiegel1, Helge Bruns1, Ulrich Kneser3,Stephanie Holzhüter1, Beate Roth1, Jantjeline Kluth1,Peter M. Kaufmann2, Dietrich Kluth11Department of Pediatric Surgery, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany2Department of Urology, MHH, Hannover, Germany

3Department of Plastic and Hand Surgery, University of Erlangen,Erlangen, GermanyBackground: Cell transplantation and tissue engineering with livercells are currently under investigation as experimental therapies forcertain liver diseases. In this study we evaluated a fibrin-based gel-matrix as carrier for hepatocyte transplantation into the liver in a ratmodel.Methods:Hepatocytes were harvested from rats. A fibrin-matrix wasgenerated using a modified fibrin sealant. Cells were seeded in a dropof fibrin-matrix on plastic culture dishes in a medium containingEGF and insulin. Cell numbers were assessed by DNA-content.Hepatocyte differentiation was evaluated by RT-PCR and immun-histology (IH) for CK-18 and albumin. PKH-26 labelled fibrin gel-immobilised hepatocytes were transplanted into the liver by directinjection underneath the capsule. Fluorescence-microscopy ofexplanted livers was performed to identify the pkh-26+- donor cells.The neotissue was characterized by IH for the markers CK-18, ED-1,and desmin.Results: Culture in a fibrin matrix allowed stable cell numbers and athree dimensional neo-tissue formation. RT-PCR and IH showedpreservation of liver specific markers CK-18 and albumin in vitro.Transplanted cells were identified by fluorescence microscopy after 2and 7 days. CK-18 and desmin staining showed integration of hepa-tocytes and hepatic stellate cells into the host liver.Conclusion: Fibrin-matrix is an appropriate environment forhepatocytes in culture. Direct intrahepatic injection of fibrin gel-immobilised hepatocytes is technically feasible. We conclude thatfibrin gel-immobilisation is an attractive tool for the development oftissue engineering-based liver support systems.

29Selective mobility enhancement for human vascularcell lines on peptide modified surfacesM. Fittkau, T. Fischlein, M. Weyand, P. Zilla*, N. Davies*Department for Cardiac Surgery, Friedrich-Alexander-UniversityErlangen, Erlangen, Germany*Cardiovascular Research Unit, Cape Heart Centre, University ofCape Town, Cape Town, South AfricaBackground: Spontaneous healing of artificial vascular implants interms of a fast and complete physiological tissue ingrowth includingendothelialisation of the luminal surface and establishment of alamina muscularis is a major goal in tissue engineering. Graftmodification by cell interactive peptides might provide not only celladhesion, but could also influence cell mobility within the healingscenario. A 2-D in vitro single cell migration model was used toinvestigate the motility of microvascular endothelial cells (MVEC)and vascular smooth muscle cells (SMC) on bioactive peptides.Mobility of these cells was examined on the peptides YIGSR(laminin derived) and PHSRN (fibronectin derived) when combinedwith the cell adhesive peptide RGD (derived from fibronectin). Theformer two peptides have had various activities ascribed to them suchas anti-angiogenicity and synergism for adhesion.Methods: Human cell lines were isolated from foreskin micro-vascular endothelial cells, (MVEC) and aorta (SMC). Peptides weresynthesized manually by solid phase Fmoc chemistry and linked toan inert polyethylene glycol (PEG) matrix. Computer-aided time-lapse video-microscopy was used to quantify single cell migration.Results: MVEC migration speed of 14.4 µm/h on RGD wasincreased by 25.8% (significant with p<0.05) on RGD combinedwith YIGSR, but unchanged on RGD combined with PHSRN. SMC(17.1 µm/h on RGD) did not show any significant differences inmobility on the peptide combinations.Conclusion: The mobility of MVEC in vitro can selectively beinfluenced by peptide modification of a 2-D matrix. This raises thepossibility of creating preferential ingrowth matrices for use in tissueengineering.

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30The influence of neuronal agrin on primary myoblastsevaluated by microarray methodV.T. Foerster1, J.P. Beier2, G.B. Stark11Department of Plastic and Hand Surgery, University of FreiburgMedical Center, Freiburg, Germany2Department of Plastic and Hand Surgery, University Hospital ofErlangen, Erlangen, GermanyBackground: In the field of skeletal muscle tissue engineeringmyoblasts need to be expanded in order to gain a sufficient number ofcells. After expansion of cells stimuli have to be established to inducein muscle precursor cells to induce differentiation into maturemyofibers. One possible candidate could neuronal agrin, sinceneuronal agrin is known to play an important role in developmentand maintenance of neuromuscular junctions and myogenesis. Theinfluence of neuronal agrin on mature musclefibers has been studiedextensively. Agrin induces an aggregation of acetylcholine receptors,acetylcholinesterase and other synaptic components on the musclecell surface. The influence of agrin on myogenic precursor cells hasnot been investigated yet.Methods: We examined the influence of neuronal agrin on primaryrat myoblasts in vitro. Cells were expanded over several passages andsubsequently treated with recombinant rat C-terminal agrin for 8hhours (concentration: 5 ng/ml). Expression of mRNA was assessedby a 26k-oligo-rat-Microarray, RT-PCR, realtime RT-PCR (Taq-man®) to determine the effect of agrin stimulation on myoblast geneexpression. Futhermore immunohistochemistry for desmin andmyogenin was performed.Results: We could determine expression for the muscle specificgenes actin, desmin, myogenin and MyoD by RT-PCR-analysisunder the influence of neuronal agrin, whereas negativer controlsrevealed only expression of early myogenic markers, such as desmin.We could confirm these PCR-results by positive immunohistochem-istry for proteins desmin and myogenin in agrin treated myoblastcultures. Quantitative analysis by realtime RT-PCR are still inprogress. Preliminary evaluation of microarray revealed significantregulation of at least 200 genes, with genes of interest still to beidentified.Conclusion: Preliminary results show an marked upregulation ofcommon myogenic genes under the influence of agrin stimulation.The identification of other agrin regulated muscle genes mightprovide further insight in agrin’s mechanism of action in muscleprecursor cells. Thus controlled induction of myogenesis in primarymyoblasts might once be rendered possible in order to recreateskeletal muscle tissue.

31Gene expression profiles of locally advanced rectal cancerbefore and after neoadjuvant radio-/chemotherapyB. von Gerstenbergk-Helldorff1, K. Horisberger1, M. Haak2,S. Post1, F. Willeke11Chirurgische Klinik, Universitätsklinikum Mannheim, Mannheim,Germany2Zentrum für Medizinische Forschung, UniversitätsklinikumMannheim, Mannheim, GermanyBackground: The response of locally advanced rectal carcinomas ona neoadjuvant radio-/chemotherapy cannot be predicted prethera-peutically. Nevertheless, a neoadjuvant treatment is recommended asthe standard therapy to induce a downstaging and/or a downsizing.The purpose of this investigation was to detect differences in genetictumor expression profiles between pretreated and untreated rectalcarcinomas by means of RNA-oligonucleotidarrays.Patients and methods: Within a phase I/II study of neoadjuvantradio-/chemotherapy of rectal carcinomas the asservation of tumortissue occurred before the beginning and after the end of a radio-/

chemotherapy. To define the gene expression patterns samples wereassorted (in each case untreated and neoadjuvant treated tumor tissueby six patients: complete remission n=2; good remission n=1 and noremission n=3) according to there response. The RNA isolation(RNeasy mini RNA-Isolation-Kit, Qiagen) , cleaning, amplificationand biotinylation was carried out according to the standard protocolof Affymetrix with next hybridisation on an Affymetrix HG U-133ARNA-oligonucleotidarray. Further data mining was carried out bycommercial mining software, Microarray Analysis Solution v1.0.3(SAS). In addition to the analysis of variances of the tumor spec-imens depending on the manner of remission, an hierarchical clusteranalysis was performed to detect differentially expressed genes.Results: Tissue specimens of rectal cancer with good compared topoor remission after radiochemotherapy showed a set of 502significant differentially expressed genes in the above describeanalysis. These genes mainly belong to apoptotic and antiapoptoticcascades and regulation of cell-cellinteraction, differentiation andproteinbiosynthesis. An upregulation in geneexpression occurred e.g.in amyloid beta precursor protein (APP), splicing factor arginine/serine-rich3 (SFRS3), insulin induced gene (INSIG1/2), TNF-Rezeptor subfamily 6 (TNFRSF6), while ribosomal protein L29/30/32, S10/27 (RPL), bone morphogenic protein 4 (BMP4),homebox A11 (HOXA11), BCL2-associated athanogene 3(BAG3), protocadherine gamma subfamily A11 (PCDHGA11), T-box 2 were downregulated.Conclusion: The present results show a clear modulation of thegenetic expression profiles of rectal carcinomas by a radio-/chemotherapy. The apoptotic cascade, cell-cell-interaction anddifferentiation demonstrate a marked increase in consecutivelyanalysed samples. Validation of the described gene patterns in alarge cohort of tumor specimen is mandatory.

32Load and surface motion for engineeringof functional cartilaginous tissueSibylle Grad1, Markus A. Wimmer2, Sylwester Gogolewski1,Mauro Alini11Biomaterials and Tissue Engineering Program, AO ResearchInstitute, Davos Platz, Switzerland2Department of Orthopedics, Rush University Medical Center,Chicago, IL, USABackground: Functional tissue engineering involves the applicationof controlled biomechanical loadings in an attempt to direct theformation of competent tissues in vitro. Our cartilage bioreactormimics the movements and contact pressures that are relevant duringnormal joint loading in vivo. The response of articular chondrocytesto different loading regimes was studied in a 3D culture system.Methods: Resorbable porous polyurethane scaffolds (8 mmdiameter; 4 mm thickness) were seeded with primary bovine articularchondrocytes suspended in fibrin gel. Cell-scaffold constructs wereheld in static culture for 5 days and then subjected to 1 hour ofmechanical conditioning twice a day over 5 consecutive days. Group1 was exposed to dynamic compression (10-20% sinusoidal strain;0.1 Hz) by pressing a ceramic hip ball against the construct; group 2was exposed to compression and simultaneous rotation around theaxis (±7.5°; 0.1 Hz), group 3 to compression and superimposedoscillation of the ball over the construct surface (±30°; 0.1 Hz), andgroup 4 to simultaneous compression, sample rotation and balloscillation (multidirectional articular motion). The mRNA expres-sion levels of different genes and the release of the boundarylubricant superficial zone protein (SZP/lubricin) and of hyaluronan(HA) into the culture media were assessed. Non-loaded samplesserved as controls.Results: Simultaneous compression and rotation around the axisenhanced the mRNA expression of aggrecan. Compression andsuperimposed ball oscillation increased the mRNA expression of

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aggrecan and type II collagen and markedly up-regulated the mRNAexpression of SZP/lubricin and of cartilage oligomeric matrix protein(COMP). No significant additional effect was observed by applica-tion of multidirectional articular motion (group 4). Type I collagengene expression was not affected by any loading regime. Condi-tioned media of constructs exposed to compression and balloscillation contained significantly elevated levels of SZP/lubricin(groups 3 and 4) and of HA (group 4).Conclusion: Application of joint specific biomechanical stimuliincreased the expression of important cartilage matrix proteins. Inparticular, articular motion generated by ball oscillation over theconstruct surface appears to be an efficient stimulus, which not onlyaffects the synthesis of molecules with key roles in joint lubrication.Thus, specific bioreactor conditioning may promote the properphenotype of articular chondrocytes, which is the precondition forthe development of a tissue with appropriate matrix and surfaceproperties.

33Massive dysregulation of the cellular transcriptome duringthe development of rectal adenocarcinomasMarian Grade1,2, Michael J. Difilippantonio2, Sudhir Varma3,Richard Simon3, Torsten Liersch1, Heinz Becker1, Thomas Ried2,B. Michael Ghadimi11Department of General Surgery, University Medical Center,Göttingen, Germany2Genetics Branch and 3Biometrics Research Branch, National CancerInstitute, National Institutes of Health, Bethesda, MD, USABackground: The goal of this analysis was to separate genes whichare responsible for the development of rectal adenocarcinomas and tounveil the genetic pathways underlying rectal carcinogenesis.Furthermore, we aimed to explore the relationship betweenchromosomal copy number changes and the expression level ofgenes residing on those chromosomes. This enabled us to evaluatethe extent to which genomic imbalances impact the cancer celltranscriptome.Patients and methods: Biopsies from 17 locally advanced rectaladenocarcinomas and 20 normal mucosa samples were analyzed forgene expression signatures using oligonucleotide microarrays (22231features). Furthermore, comparative genomic hybridization wasemployed to evaluate DNA copy number changes. Class comparisonand class prediction analysis (LOOCV) were performed to measurethe difference in gene expression between the two classes.Results: Expression profiles revealed the identity of 1722 genes withdifferences at a significance level of p<0.0001 between normal rectalmucosa and rectal carcinomas. We then applied stringent filteringcriteria to decrease this number to a reasonable size. The number ofgenes differentially regulated between the two groups withsignificance of p<0.0000001 was 351. Furthermore, 52 genes hadan average expression > 5-fold higher in the tumor group comparedto normal mucosa, while only about half as many genes (25) wereexpressed on average more than 5-fold higher in the mucosa samples.We also identified a group of 39 genes whose expression was alwaysincreased more than 2-fold in the tumors and 46 genes whoseexpression was always decreased more than 2-fold in the tumorsrelative to any of the normal mucosa. A total number of 12 genes metall three filtering criteria. Strikingly, we detected two groups ofnormal rectal mucosa that were clearly separated from the tumorsamples and from each other. Furthermore, recurrent DNA copynumber gains occurred on chromosome arms 7p, 8q, 13q, 20p and20q and losses were mapped to 8p, 15q, 16p, 17p, 17q, 18p, 18q, 19pand 22q. The effect of these tumor-specific aneuploidies was partiallyrevealed through the significant correlation of chromosome armaverage gene expression values with chromosome copy number.

Conclusion: We have specifically identified a number of geneswhose expression is significantly deregulated in rectal adenocarci-nomas. Expressed genes residing on chromosomes commonly foundto be aneuploid in this tumor type had a high likelihood of alteredexpression correlating with the chromosome copy number. Thus, weconclude that while rectal carcinogenesis requires the dysregulationof genes and particular genetic pathways, a more generalizeddisturbance of the transcriptional equilibrium in cancer cells occursthrough chromosome segregation errors leading to aneuploidy.

34Differentially expressed genes in colorectalcarcinoma: comparison between bulk and lasercapture microdissected materialJörn Gröne1, Esmeralda Heiden2, Klaus Hermann2, Jan Wiemer2,Bernd Hinzmann2, André Rosenthal2, Heinz J Buhr11Department of Surgery, Campus Benjamin Franklin, Charité Berlin,Berlin, Germany2Signature Diagnostics, Potsdam, GermanyBackground: For improvement of diagnostic tools, outcome anddrug response prediction, as well as effective drug discovery incolorectal cancer (CRC) identification of differentially expressedgenes (DEGs) and signatures are in focus of research. Combinationof gene expression analysis and Laser capture microdissection(LCM) enables the collection of separate cell populations such asnormal, premalignant, cancerous, and stromal tissue from the sametissue sample. It is not clear yet, wether bulk or laser capturemicrodissected material has an advantage for the identification ofDEGs. Our purpose was to compare specifity of LCM and bulk tissueconcerning identification of tumor-related genes in CRC.Patients and methods: In a pilot experiment, tumor and histologi-cally normal epithelial cells from five patients with CRC wereenriched using LCM. In parallel, bulk tumor and normal tissue fromthe same blocks were also analysed. RNA extraction, cDNAamplification, labeling, and hybridization to U133A Affymetrixchips were performed simultaneously in both types of samples.DEGs were determined by univariate statistical analysis (WelchTest). The degree of DEG list correspondency was determined bySpearman rank correlation of p-values. Differences between listswere quantified by statistical tests (binomial test, Wilcoxon Test).Results were additionally visualized by p-value scatterplots withkernel density estimation.The experiment resulted in two separatelists of DEGs.Results: Remarkably, our analysis shows that only about half of theDEGs identified in both lists are shared between LCM-derived andbulk material (Fig. 3). Thus, using only one of the two techniques,about half of the genes would not be identified. Therefore, in order togenerate more thorough DEG lists, both methods would need to beapplied. LCM resulted in a higher reproducibility. An overallstronger differential expression of genes was found when usingLCM, since p-values in the LCM list were significantly lower than p-values in the bulk list (Wilcoxon p-value: p=1,3 e-10, see Table 2 andFig. 4). Also, there were 10% more DEGs with p-values below 0.05in the LCM list than in the bulk list (Fig. 3). The higherreproducibility by using LCM improves the identification andquantification of DEGs, most likely due to more homogeneous cellpopulations.Conclusion: LCM provides higher specificity for the detection oftumor-related genes, and is therefore a useful tool for the discovery ofputative cell targets for new drugs. On the other hand, including thetumor microenvironment in the array-based analysis may yield DEGsimplicated in tumor progression which are needed to address otherimportant features like prognosis or therapy response of individualcancer patients. For this purpose a comprehensive analysis of thebulk of the tumor may be more informative.

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35Identification of differentially expressed genesin the stromal tissue of pancreatic ductal adenocarcinomaRobert Grützmann1, Jutta Lüttges2, Ole Ammerpohl3,Holger Kalthoff3, Günter Klöppel2, Hans Konrad Schackert4,Hans Detlev Saeger1, Christian Pilarsky11Department of Surgery, University Hospital Dresden, Dresden,Germany2Institute of Pathology, University of Schleswig-Holstein, Kiel,Germany3Department of Molecular Oncology, University of Schleswig-Holstein, Kiel, Germany4Department of Surgical Research, University Hospital Dresden,Dresden, GermanyBackground: Prognosis for patients with pancreatic carcinoma(PDAC) remains poor. Despite of increasing knowledge about themolecular basis of PDAC no specific marker for early diagnosis nor atarget protein for a new therapeutic approach have been identified sofar. Moreover, PDAC displays large desmoplasia indicating that theactivation of tumor surrounding stroma plays a key role duringPDAC development. We were therefore interested in the analysis ofdifferential gene expression between tumor surrounding stroma andthe stroma compartment of chronic pancreatitis, a benign disease.Methods: We microdissected the stromal tissue compartment of sixpancreatic cancer and six chronic pancreatitis tissues. The tissueswere obtained during surgery and freshly frozen. The mRNA wasextracted, amplified by repetitive in vitro transcription andhybridized to the Affymetrix U133 GeneChip set. The obtained datafrom the microarray were normalized and signal intensities werecalculated using dCHIP. Differentially expressed genes wereidentified using SAM (cut-off: fold change >3, q-value <5%).Results: We identified 195 differentially expressed genes fromwhich 39 were over- and 157 genes were under-expressed inpancreatic cancer stroma. Hierarchical clustering using the 195 genesand the gene expression profiles of microdissected pancreatic tumorepithelia identified a subset of pancreatic cancers epithelia displayinggene expression similar to the cancer stroma. Annotation of the 195genes resulted in the identification of soluble factors from the Wntand the Notch signaling pathways overexpressed in the stroma fromcancer tissue.Conclusion: In conclusion, gene expression analysis of the stromalcompartment could identify new markers and therapeutic targets forthe tumor associated stroma of pancreatic cancer.

36Ultrasound-associated cytoskeletal changesof cultivated osteoblast-like cell lines in vitroJoerg Hauser1, Manfred Hauser2, Manfred Koeller3, Gert Muhr3,Stefan A. Esenwein31Klinik fuer Plastische Chirurgie und Schwerbrandverletzte, BGKliniken Bergmannsheil, Universitaetsklinik, Bochum, Germany2Lehrstuhl für Zellmorphologie und Molekulare Neurobiologie,Ruhr-Universitaet, Bochum, Germany3Chirurgische Klinik mit Poliklinik, BG Kliniken Bergmannsheil,Universitaetsklinik, Bochum, GermanyBackground: In clinical and experimental studies an acceleration offracture healing and increased callus formation during additionaltreatment by low-intensity pulsed ultrasound has been proven.Although clinical results were promising, the molecular effectmechanism of ultrasonic treatment is almost unknown. Until now,less efforts have been made to find out whether morphologicalchanges of the cytoskeleton can be detected during ultrasonictreatment. In this study ultrasound transmitted cytoskeletal changesand changes of cell proliferation were examined.

Materials and methods: In vitro different osteoblast-like cell lines(MG-63 and SAOS-2) have been treated using low-intensity pulsedultrasound (frequency 1,5MHz, signal repetition frequency 1kHz,signal burst width 200 µs, intensity 30 mW/cm2). For visualizationof the cytoskeleton indirect immunofluorescence methods werechosen. After fixation of the cells anti-bodies against microtubuli,and rhodamine-phalloidin against actin filaments were added andmarked using fluorescence staining. Cytoskeletal changes were an-alyzed microscopically. To examine proliferation rates after ultra-sound treatment, cell counts were done.Results: By means of immunofluorescence analysis significantchanges in structure of the cytoskeleton were detected. Especiallythe amount of tyr-tubulin of the cytoskeleton has been significantlyincreased after ultrasound application. Moreover a detectablestimulation of cell proliferation was noticed.Conclusion: In our experiments we were able to demonstrate thatultrasound treatment stimulates cell proliferation in vitro and leads tosignificant changes of the cytoskeletal structure. Whether themolecular changes decribed play a role as an effect mechanisms ofthe clinical treatment with low-intensity, pulsed ultrasound, should befurther examined.

37Peripheral choline acetyl-transferase is expressedby monocytes and up-regulated during renalallograft rejection in ratsAndreas Hecker1, Katrin S. Lips2, Uwe Pfeil2, Wolfgang Kummer2,Winfried Padberg1, Veronika Grau11Laboratory of Experimental Surgery, Department of General andThoracic Surgery, and 2Institute of Anatomy and Cell Biology,Justus-Liebig-University Giessen, Giessen, GermanyBackground: Acetylcholine (ACh) has been shown to modulate thefunction of mononuclear leukocytes, both by muscarinic andnicotinic ACh receptors. Acute stimulation of lymphocytes withACh or muscarinic agonists enhances pro-inflammatory functionswhereas chronic application of the ACh agonist nicotine has an anti-inflammatory effect. In macrophages acute treatment with nicotinedown-modulates effector functions. ACh regulating leukocytesmight originate from the nervous system. However, once released,ACh is quickly degraded. Only in the direct vicinity of nerve endingsrelevant concentrations occur. Non-neuronal ACh acting in aparacrine or autocrine fashion is more likely to influence immunefunctions. Lymphocytes express all enzymes needed for AChsynthesis, including choline acetyl-transferase (ChAT). In the rat,alternative splicing generates common ChAT and peripheral ChAT(pChAT). Up to now, ChAT expression by monocytes has not beendemonstrated. We investigate pChAT in monocytes in an experi-mental model of acute renal allograft rejection. Inside the bloodvessels of the transplant, huge numbers of activated, cytotoxicmonocytes accumulate and probably contribute to graft destruction.Methods: Renal transplantation was performed in the fullyallogeneic DA to LEW rat strain combination. Intravascular graftleukocytes were harvested four days after renal transplantation byextensively perfusing the renal blood vessels. pChAT expression wasinvestigated by nested RT-PCR and immunoblotting. pChATexpressing cells were identified by immunohistochemical doublestaining with the pan monocyte/macrophage marker ED1.Results: After allogeneic renal transplantation, intravascular graftleukocytes express pChAT mRNA and protein. In isograftssignificantly lower mRNA and protein expression levels areobserved. Most pChAT-positive intravascular leukocytes are mono-cytes. They co-express the ED1-antigen, a CD68 like lysosomalmarker of monocytes/macrophages.Conclusion: We demonstrate for the first time that monocytes canexpress pChAT and up-regulate pChAT-expression during allograft

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rejection. It is likely that transplant monocytes can produce ACh. TheACh system of activated monocytes might represent a negativefeedback loop limiting immune reactions, as it has been recentlydemonstrated that ACh down-regulates proinflammatory responsesin monocytes/macrophages via the alpha-7 nicotinic receptor.Additionally, ACh of monocytic origin might influence T-lympho-cytes and the function of graft vessels.

38Role of adhesion molecules in the inductionof restenosis after angioplasty in the lower limbPeter Heider1, Moritz Wildgruber1, Oliver Wolf1, Marc Hanke1,Wolfgang Weiss2, Hermann Berger2, Hans-Henning Eckstein11Department of Vascular Surgery and 2Department of InterventionalRadiology, Rechts der Isar Medical Center, Technical UniversityMunich, Munich, GermanyBackground: Until now restenosis remains a major limitation to theclinical usefulness of percutaneous transluminal angioplasty (PTA).Despite a primary success rate up to 90%, the incidence of recurrentstenosis resulting mostly from vascular smooth muscle cell (VSMC)proliferation following PTA has been reported to be as high as 40%within 6 months. The pathophysiology of restenosis has beenextensively studied within coronary angioplasty, but the process ofperipheral intimal thickening and therapeutical conclusions is stillpoorly investigated. Endothelial surface connected adhesion mole-cules play a role in the beginning of the atherosclerotic process and inthe development of the restenosis. A variant of these adhesionmolecules, released from the cell surface of endothelial cells andplatelets, can be detected as circulating soluble molecules in theblood. We investigated the performance of the soluble (s) forms ofthe adhesion molecules p-Selectin, e-Selectin, VCAM, ICAM andMCP-1 in patients in the early course after angioplasty in the lowerlimb to focus on their role in the endothelial answer to angioplastyinduced injury and to get a better understanding of the delayed failureof angioplasty due to restenosis development.Patients and methods: Between July 2003 and December 2004 weinvestigated 44 patients (25 male, 19 female, age 67,7±8,5 years)with PAOD, all Fontaine stage IIb, and indication for interventionalprocedure. 12 patients (27,3%) underwent diagnostic angiography,32 (72,2%) received interventional treatment, 22 (68,8%) of themreceived balloon angioplasty, 10 (31,2%) required stent implantation.All patients were examined on the day before interventionalprocedure, on the day of discharge, two and four weeks afterintervention as well as 6 months post procedure. Follow-up includedclinical examination and ABI-measurement. Venous blood samplesfor ELISA testing (p-Selectin, e-Selectin, ICAM, VCAM and MCP-1) were drawn on the day before interventional procedure, 15 and 60minutes after the inflation of the angioplasty balloon or the placementof the stent, 24 hours after the intervention as well as 2 and 4 weeksafter therapy.Results: 10 patients (31,25%) developed restenosis in the first 6months following PTA. The group of patients that later developedrestenosis had significantly higher levels of e-Selectin over the entireobservation period (p=0,011). P-Selectin levels were higher inpatients treated with balloon angioplasty than in patients who onlyreceived diagnostic angiography and were highest in patients whorequired stent implantation, but without reaching a significantdifference between the different groups (p=0,62). Both in the groupwith and without restenosis ICAM serum levels were significantlyincreased at 24 hours and remained elevated at 2 and 4 weeks(p=0,002). Considering the intervention type the level increase at 24hours was significant in all three groups, the angiography group(p=0,029), balloon-angioplasty group (p=0,020) and stent angio-

plasty group (p=0,019). Also the serum levels of VCAM wereelevated over the whole time course in patients who later ondeveloped restenosis compared to those who did not, but only at 15minutes after intervention this difference reached borderlinesignificance (p=0,080). In MCP-1 we observed an inverse effectconsidering restenosis: the levels in patients with restenosis werelower than in patients without restenosis over the entire observationperiod (p=0,059).Conclusion: Our present study shows that: (a) circulating adhesionmolecules in the early course after PTA can be positively associatedwith restenosis development; (b) stent implantation leads to a moresevere irritation of the endothelium and activation of inflammationbut angiographic procedure itself leads to certain rise of pro-inflammatory pathways especially in the selectins. These resultsemphasise the important role of adhesion molecules in restenosisdevelopment but also show that marker levels in the period shortlyafter angioplasty have to be interpreted extremely carefully.Modulation of the early inflammatory response to angioplastyinduced vessel trauma, especially through interaction of adhesionmolecules, remains a promising opportunity for preventing restenosisformation after peripheral angioplasty procedures.

39Monocyte recruitment is a pre-requisitefor arteriogenesisMatthias Heil, Silvia Tribulova, Sawa Kostin,Thomas Schmitz-Rixen, Wolfgang SchaperMax-Planck-Institute for Heart and Lung Research, Bad Nauheim,GermanyDepartment of Vascular and Endovascular Surgery,J.W. Goethe-University, Frankfurt/Main, GermanyBackground: Mechanisms of arteriogenesis comprise complexinteractions including recruitment of leukocytes to the vascular wallby chemoattractive substances. Monocytes/macrophages, accumu-lated in the collateral vessel wall, are thought to release multiplearteriogenic substances. We hypothesized that fluid-shear-stressmediates the initial activation of the endothelium which inducesadhesion molecule expression and release of chemoattractivesubstances. Subsequently, monocytes may be attracted to thecollateral vessel. We investigated if macrophages, found aroundgrowing collaterals, may origin from blood monocytes and if acorrelation between blood monocyte concentration and the extent ofarteriogenesis was detectable.Methods and results: Monocytes were isolated from wild type ortransgenic GFP+ donors by cell sorting. After right femoral arteryligation mice received an i.v. injection of NaCl (controls), 2 or 3x105monocytes. Blood flow recovery as measured by laser Dopplerimaging was significantly increased in monocyte-injected groups.Comparison between groups which were treated with 2 and 3x105monocytes suggested that higher concentrations of monocytesimproved blood flow recovery. Compared to controls, hemoglobinoxygen saturation and active foot movement score of right hind limbswere improved in the monocyte-injection groups but did not differbetween both monocyte groups. Morphometric analysis showedincreased collateral vessel diameters in correlation with concentra-tion of injected monocytes. Histological analysis of a subgroupwhich had received injection of GFP± monocytes demonstratedrecruitment of GFP+ blood monocytes as well as co-expression ofmacrophage markers (F4/80) and growth factors (FGF-2).Conclusion: This study gives evidence that macrophages accumu-lating in growing collateral vessels origin from blood monocytes andare recruited to the vascular wall during early phases of arteriogen-esis. Tissue-derived macrophages seem to play a minor role.

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40Incidence and prognostic significance of neuroendocrinemarkers chromogranin A, synaptophysin and neuron-specific enolase in primary rectal cancer after curativeresectionMarcus Hilbert, Rainer Broll, Hans-Peter Bruch, Oliver SchwandnerDepartment of Surgery, University Hospital Schleswig-Holstein,Campus Lübeck, Lübeck, Germany, and Klinik für Chirurgie undChirurgisches Forschungslabor, Lübeck, GermanyBackground: It was the aim of the study, to determine theassociation between immunhistochemically proven neuroendocrinecells and clinical factors of rectal adenocarcinomas. The centralquestion was, if the “classical” diagnostic markers for neuroendo-crine tumors, Chromogranin A (CgA), Synaptophysin (Syn) andneuron-specific enolase (NSE) may be prognostic factors forsporadic rectal cancer.Patients and methods: Formalin-fixed and paraffin embedded tissuesections of curatively resected rectal carcinomas (n=94) wereexamined immunohistochemically for the expression of CgA, Synand NSE using a two-step-detection system and were correlated withclinical and histopathological parameters as well as the data of theprospective tumor registry (mean follow-up of 46 months). Only R0-resected rectal cancers (central ligation of the A. mesenterica inferior,TME, adjuvant radiation-chemotherapy at UICC stages II and III)have been included. End points of the prognostic analysis (91patients) were tumor progression (local recurrences, distant meta-chrone metastases) as well as survival according to Kaplan-Meier(disease-free, overall). Statistical significance regarding to theprognostic relevance were tested uni- and multivariate (p<0,05statistically significant).Results: 20% (19/94) of the carcinomas were CgA-positive, 7% (7/94) were Syn-positive and 3% (3/94) were NSE-positive. For amajority of 72% (68/94) of the carcinomas an expression ofneuroendocrine markers was not found, whereas in 3 cases (3%)the expression of two markers was proven. CgA was associatedsignificantly with the age of the patient (CgA positive: 28% youngerthan 70 years vs. 10% 70 years and older; p=0,03), but there was nocorrelation of the neuroendocrine markers with other clinical orhistopathological parameters (p>0,05). After a mean follow-up of 46months, a tumor progression was documented in 14 patients (15,4%):6 local recurrences (n=6; n=2 isolated, n=4 with distant metastases),12 distant metachronous metastases (n=12, from that n=8 withoutlocal recurrence) were found. With regard to the incidence of localrecurrence the univariate analysis did not show any significantcorrelation between CgA, Syn or NSE (p>0,05), whereas CgAexpression was correlated significantly with the occurrence ofmetachronous metastases (23,5% CgA positive vs. 5,9% CgAnegative, p=0,02). However, there was no association between theneuroendocrine markers and the 5-year survival rates (p>0,05).Additionally, there was no association between neuroendocrinemarkers and clinical parameters such as age, gender, type of resectionand lymph node status, whereas the prognosis of survival wascorrelated significantly with UICC stage and depth of invasion (pTstatus).Conclusion: The current results show a prognostic influence ofChromogranin A for the incidence of metachronous metastases aftercurative resection in patients with sporadic rectal carcinomas.

41Lung transplantation in the Fischer 344 to WistarKyoto rat strain combination is not suitableto study bronchiolitis obliteransMarkus Hirschburger1, Susanne Greschus2, Martin Obert2,Tim Kuchenbuch1, Winfried Padberg1, Horst Traupe2,Veronika Grau1

1Laboratory of Experimental Surgery, Department of General andThoracic Surgery, University of Giessen Lung Center,Justus-Liebig-University Giessen, Giessen, Germany2Department of Neuroradiology, Justus-Liebig-University Giessen,Giessen, GermanyBackground: In humans, chronic rejection of lung transplants ischaracterized by Bronchiolitis obliterans (BO), a fibroproliferativenarrowing and obliteration of the airways which develops in afunctioning lung. To elucidate the pathogenesis of BO a reliableanimal model is needed. According to the literature, lungtransplantation from Fischer 344 (F344) to Wistar Kyoto (WKY)rats is the only model that results in BO without a further stimulus.However, in other studies BO is not observed.Methods: We performed orthotopic left lung transplantation in theF344 to WKYas well as in the WKY to WKY rat strain combination.The time course of lung rejection was carefully documented bynoninvasive Flat-panel Computed tomography (fpVCT) using aprototype fpVCT scanner (GE Global Research, Schenectady, USA).Only grafts which were completely aerated one week aftertransplantation were included in this study. Graft histopathologywas analyzed three months posttransplantation on paraffin sectionsstained with H&E and acidic Orcein.Results: As revealed by fpVCT, about 50% of the allografts developperivascular and peribronchial infiltrates due to acute rejectionduring the first weeks after transplantation which mainly resolvethereafter. These grafts remain aerated throughout. Three monthsposttransplantation, the lungs exhibit transplant vasculopathy but noBO. The other half of the transplants develop almost complete andirreversible atelectasis during the first two weeks. Three monthsposttransplantation these allografts are shrunken. The lung parench-yma is almost entirely replaced by scar tissue. In graft blood vessels,the intima as well as the media are severely thickened. The airwaysincluding the bronchioles are difficult to discern, drasticallythickened, and the lumina are narrowed or obliterated.Conclusion: The outcome of F344 to WKY lung allografts isvariable. Initially nicely aerated transplants either remain functionalfor at least three months or are destroyed during the first two weeksposttransplantation. The clinical relevance of this experimentalmodel to study BO is doubtful: In aerated lungs BO does not occur atall and only vascular remodelling is evident. Remodelling of theairways comparable to BO only develops in atelectatic lungs withoutlife sustaining function.

42Beneficial effects of the protein heme oxygenase-1on the ischemia/reperfusion injury after isogeneicorthotopic renal transplantationJens Peter Hölzen1, Ralf Bahde1, Christian August2, Detlef Lang3,Karl-Heinz Dietl1, Stephan Heidenreich3, Hans Ullrich Spiegel11Surgical Research, Department of Surgery, University HospitalMünster, Münster, Germany2Gerhard Domagk Institute of Pathology, University HospitalMünster, Münster, Germany3Department of Nephrology, University Hospital Münster, Münster,GermanyBackground: Cytoprotective proteins are highly involved in cellularinjury and regeneration, apoptosis and inflammation in the setting ofsolid organ transplantation. In renal transplants, expression ofprotective proteins is demonstrable in the early ischemia/ reperfusionphase. The aim of the study was to investigate the effects of thecytoprotective protein heme oxygenase-1 (HO-1) induced by thesubstance hemin in the setting of isogeneic renal transplantation.Materials and methods: Thirty-five isogeneic male Lewis rats (200- 250 g) were divided into three grous. In group I and II we performedtotal nephrectomy following orthotopic kidney transplantation. But

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only in group I donor rats underwent a preconditioning treatmentwith the HO-1 inductor hemin. In group III only kidney mobilsation(sham-operation) was performed. After 24 h the renal function wasdetermined by examination of serum and urine. Overall renalfunction was assessed by assays of potassium, creatinine and urea.Urine-volume and creatinine-clearance was determined. After 24 hspecimens were taken from the transplant for investigating thehistological damage using light microscopy (tubular damage,microthrombosis, atrophy and necrosis). Renal microcirculationwas examined by in vivo microscopy of the renal surface 1 hour afterreperfusion. We investigated blood flow (FITC-marked erythrocytes)and capillary diameters.Results: Compared to transplantation without preconditioning, inhemin group the animals showed significantly lower creatinin values(group I: 0,97±0,14 mg/dl, group II: 1,30±0,37 mg/dl, p<0.05) andurea values (group I: 37,14±10,65 mg/dl, group II: 68,57±18,59 mg/dl, p<0.05). In hemin group, we observed a decreased histologicaldamage. In addition, we investigated an improvement of micro-circulation in hemin group with significant larger vessel diameters(group I: 5,86±0,38 µm, group II: 4,61±0,17 µm, p<0.05). In hemingroup the capillary flow was more than doubled (group I: 14680,8±2181,3 µm³/s, group II: 5745,4±448,2 µm³/s, p<0.05) in contrast totransplantation without preconditioning.Conclusion: Treatment with hemin improves microcirculation byinducing HO-I and reduces the ischemia/ reperfusion injury afterrenal transplantation. Hemin may be a promising approach forpreconditioning of kidney donors.

43Accelerated rejection of allogeneic and syngeneiccardiac grafts by intracoronary interferon-gammais prevented by tolerance inductionRuediger Hoerbelt1,2, Louis C. Benjamin2, Tsuyoshi Shojy2,Douglas R. Johnston2, Winfried Padberg1, David H. Sachs2,Joren C. Madsen21Department of General-, Thoracic- and Transplant Surgery, Justus-Liebig-University, Giessen, Germany2Transplantation Biology Research Center, Massachusetts GeneralHospital, Boston, MA, USABackground: The purpose of this study was to investigate the effectof intracoronary Interferon- (IFN-)gamma infusion on acute rejectionof cardiac allografts in models of delayed rejection, syngeneity andtolerance.Methods: Using MHC-inbred miniature swine, IFN- perfused heartgrafts (saline perfused control grafts) were transplanted into MHCclass I disparate (delayed rejection model) or histocompatiblerecipients (syngeneity model) that were treated with a 12-day courseof cyclosporine. To induce immunologic tolerance, recipients weresimultaneously transplanted with a MHC class I disparate heart andkidney from the same donor (tolerance model). Intracoronarycatheters were implanted continuously perfusing the heart graftswith IFN-� (100-200 ng/d) or normal saline (controls). All animalswere followed by cell mediated lympholysis (CML) assays and opengraft biopsies were performed at regular intervals. Production of anti-donor antibodies was assessed by indirect flowcytometry.Results: Intracoronary IFN-gamma perfusion accelerated rejectionof MHC class I mismatched heart grafts as compared to saline-perfused controls (mean survival time= 19±7.21 vs. 38±8.19, p=0.025). Anti-donor IgM antibodies were detectable by postoperativeday (POD) 5 in IFN-gamma infused animals, whereas no anti-donorantibodies were seen in controls. Likewise, intracoronary infusion ofIFN-gamma induced rejection of syngeneic heart grafts by POD 5, 17and 35, whereas non-treated syngeneic control grafts all survivedlong term without signs of acute rejection (survival time >300 days,

n=3). In contrast, IFN-gamma-perfused heart grafts in recipients ofMHC class I disparate heart/kidney transplants were all accepted(survival time >100 days, n=3). Like the controls, IFN-gammatreated heart/kidney recipients developed donor-specific unrespon-siveness on CML and no anti-donor antibodies were detectable.Conclusion: The fact that intracoronary perfusion with IFN-gammaaccelerated acute rejection of MHC class I mismatched as well assyngeneic heart grafts suggests a non-MHC related inflammatorymechanism. Our data indicate that induction of tolerance by heart-kidney transplantation sufficiently counteract the non-specific pro-inflammatory effects of IFN-gamma.

44Combinations of donor specific transfusionswith cyclosporine A or rapamycin have different effectson tolerance induction and the prevention of cardiacallograft vasculopathy in MHC inbred miniature swineRuediger Hoerbelt1,2, Tsuyoshi Shojy2, Douglas R. Johnston2,Winfried Padberg1, David H. Sachs2, Joren C. Madsen21Department of General-, Thoracic- and Transplant Surgery,Justus-Liebig-University, Giessen, Germany2Transplantation Biology Research Center, Massachusetts GeneralHospital, Boston, MA, USABackground: To evaluate the effect of pretransplant donor-specifictransfusion (DST) combined with cyclosporine A (CyA) andrapamycin (RPM) on tolerance induction to cardiac allografts andcardiac allograft vasculopathy (CAV) in a clinically relevant largeanimal model.Methods: MHC class I mismatched, heterotopic cardiac transplantswere performed in MHC-inbred miniature swine. Experimentalanimals were treated with two DSTs (1.4x108 PBMC), on 14 and 7days prior to transplantation. All animals received a 12-day course ofCyA (trough 300-800 ng/ml) or RPM (trough 10-20 ng/ml) startingon post-operative day (POD) 0. Control groups received CyA orRPM alone, DST alone or no treatment. CML assays were performedat regular intervals. T cell priming and activation induced cell death(AICD) were assessed by CFSE proliferation assays and Annexin Vstaining. Open graft biopsies were taken at regular intervals andhistological signs of acute cellular rejection and CAV were assessed.Results:Untreated (n=2) and DST-only (n=2) treated control animalsrejected between POD 6 and 8. Animals treated with CyA (n=3) orRPM (n=3) alone exhibited graft survival to 53, 52 and 59 days or 41,50 and 53 days, respectively. In contrast, DST-pretreatmentcombined with CyA (n=3) led to stable graft function for >200 daysin all animals. Grafts in recipients treated with pretransplant DST andRPM (n=5) survived 52, 45, 100 and >200 days. Like the controls,long-term acceptors in the DST+CyA group and DST+RPM groupmostly maintained peripheral CML response against donor and thirdparty antigen. Following DSTs, the donor-specific proliferativeresponse of CD8+ recipient T cells was significantly increased onCFSE assay (p=0.011), and a significant number of recipient CD8+ Tcells underwent apoptosis (10.1% on POD 0 versus 5.2% on POD 14;p=0.04). Tolerant animals in the DST+RPM group (n=2) developedsignificantly less epicardial lesions of CAV as compared to tolerantanimals in the group of DST+CyA (n=3; 2.4%±2.4 vs. 18.0%±8.1),while there was no difference of CAV prevalence in myocardialvessels (7.3%±0.9 bei DST+RPM vs. 6.9%±2.5 bei DST+CyA).Conclusion: The combination of pre-transplant DST and a shortcourse of CyA consistently prolonged the survival of MHC class Idisparate cardiac allografts in miniature swine. The sustainedperipheral anti-donor CML response and activation of alloreactiveT cells by the DST prior to transplantation suggest an activeregulatory mechanism rather than peripheral deletion. In contrast,rapamycin did not demonstrate with the tolerogenic effect of

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pretransplant DST potentially because it failed to achieve appropriateregulatory cells. Combination treatment with DST and RPM appearsto have some inhibitory effect on the development of CAV.

45Expression and secretion of Endostatin in thyroid cancerS. Hoffmann1,T. Musholt2, P. Musholt2, A. Wunderlich1, I. Celik1,A. Zielke11Department of Surgery, Philipps-University Marburg, Marburg,Germany2Department of Surgery, Gutenberg-University Mainz, Mainz,GermanyBackground: Endostatin has recently been identified as a powerfulnegative regulator of tumor angiogenesis, reducing tumor angiogen-esis and tumor growth in vivo. It is currently being evaluated for anti-tumor therapy in a variety of solid tumors in phase I clinical trials.The aim of this study was to evaluate the expression of Endostatin ina larger sample of thyroid cancers and its secretion followingstimulation with TSH and EGF in thyroid cancer cell lines.Methods: In a clinical series of differentiated (papillary-PTC, n=27,follicular-FTC, n=17) and anaplastic (n=7) thyroid cancer tumorspecimens Endostatin expression was determined by means ofimmunohistochemistry (DPC, BM 4098). In vitro, six differentiated(follicular-FTC 133, FTC 236, HTC, HTC-TSHr, Hürthle Cell-XTC,papillary-TPC 1) and three anaplastic (C 643, Hth 74, Kat 4) thyroidcancer cell lines were evaluated for basal and TSH (1-100 mU/ml)and EGF stimulated (1-100 ng/ml) Endostatin secretion (ELISA,Accucyte, PromoCell C-68306)Results: Endostatin immunoreactivity was detected in all of thethyroid tumor specimens and was more intense in differentiated ascompared to anaplastic thyroid cancers. However, no histiotype-specific pattern of expression was determined. In vitro, differences inbasal Endostatin secretion ranged from 33±5 pg/ml for FTC 236 to549±65 pg/ml in TPC 1 cells. In the FTC cell lines, basal Endostatinsecretion of the primary tumor (FTC133) was more than twice ashigh as compared to the metastatic FTC 236 clone. Some cell linesshowed a TSH induced up-regulation of Endostatin secretion (e.g.XTC: 60% at TSH 100 mU/ml). Other cell lines showed no increaseof Endostatin by TSH (HTC-TSHr) despite the documentedexpression of functional TSH receptor. An increase of Endostatinsecretion by EGF was detected only once (TPC1, 120% at EGF 100ng/ml).Conclusions: This study demonstrates for the first time thatEndostatin is expressed in a large sample of thyroid cancer tumorspecimens. In vitro, considerable individual differences of basal aswell as stimulated Endostatin levels were detected in various thyroidcancer cell lines. The stimulating effect of TSH and EGF wasinconsistent and deserves further functional studies. However, theresults already suggest the expression and regulation of Endostatin inthyroid cancer to be a tumor-specific rather than a histiotype-specificphenomenon.

46Development of a locking femur nail for miceJörg H. Holstein1,2, Michael D. Menger2, Tim Pohlemann11Department of Trauma-, Hand- and Reconstructive Surgery and2Institute for Clinical and Experimental Surgery, University ofSaarland, Homburg/Saar, GermanyTransgenic and knockout mouse models provide a wide spectrum ofopportunities to improve the understanding of the biology of fracturerepair. The aim of this study was to develop a novel locking in-tramedullary nail system in a murine closed femur fracture model.The nail system consists of a modified 24-gauge injection needleand a 0.1-mm-diameter tungsten guide wire. Rotation stability was

accomplished by flattening the proximal and distal end of theneedle. Torsional mechanical testing of the implants in osteoto-mized cadaveric femura revealed a superiority of the locking nail(3.9±1.0° rotation at a torque of 0.9 Nmm, n=10) compared to theunmodified injection needle (52.4±3.2°, n=10, p<0.05). None of theimplants, however, achieved the rotation stability of unfracturedfemura (0.3±0.5°, n=10). In a second step, we tested the feasibilityof the in vivo application of the locking nail to stabilize a closedfemoral midshaft fracture. Therefore, the tungsten guide wire wasinserted into the intramedullary canal of C57BL/6 mice (20-25 g,n=10). The femur was fractured by an impact device, which re-producibly resulted in a transverse fracture pattern. The needle wasthen placed over the introduced guide wire to stabilize the closedfracture. The procedure could be successfully performed in allanimals studied, and correct positioning of the nail was confirmedby X-ray analysis. With the advantage that closed fractures can befixed with rotation stability, the herein introduced model mayrepresent an ideal tool to study bone healing in transgenic and knock-out mice.

47Differential expression of apoptosis receptorsand activation markers on T- cells and monocytesafter severe tissue traumaArwed Hostmann, Markus Hellmuth, Sven K. Tschöke,Wolfgang Ertel, Andreas OberholzerDepartment of Trauma and Reconstructive Surgery, Charité –University Hospitals Berlin, Campus Benjamin Franklin, Berlin,GermanyBackground: Severe tissue trauma leads to acute activation of theinnate immune response with consecutive systemic inflammation andsubsequent immunosuppression. In the latter state of excessiveinflammatory mediator production and decreased immune functions,prolonged and uncorrected conditions often result in critical organdysfunction and finally multiple organ failure. Both, activation andsuppression of the innate immune defence are characterized by abalanced cellular environment and, in turn, primarily determinedthrough altered apoptosis and function of monocytes and T cells. Inthe present study, isolated monocytes and T cells from polytrauma-tized patients were functionally evaluated and analysed in regards totheir activation (CD25, CD69, HLA-DR) and expression ofapoptosis parameters (TNFR1, Fas, Bcl-2 and Bax).Patients and methods: Whole blood was obtained from multipletrauma patients (n=16) on days 0, 1, 3, and 5 after trauma. Monocytesand CD3+ T cells were isolated by negative selection (RosettSep®)and functionally characterized via flow-cytometry, TUNEL staining,caspase-assay, and Western Blot analysis.Results: Monocytes and T cells were significantly reduced inmultiple trauma patients on day 0 compared to healthy controlsubjects. Furthermore, severe tissue trauma induced an increasedexpression of the apoptosis markers Fas (CD95) predominantly on Tcells (29.2±10.6% patient (day 1) vs. 8.2±2.9% control) and TNFR(CD120a) on monocytes (61.5±10.2% patient (day 1) vs. 11.2±3.1%control), which correlated with an increase in apoptotic cells shownin TUNEL staining. Interestingly, the anti-apoptotic protein Bcl-2was significantly reduced in both T cells and monocytes, whereas thepro-apoptotic Bax protein remained relatively consistent on mono-cytes throughout the 5-day observation period. Further analysisrevealed a time-dependent decrease of the activation markers HLA-DR (monocytes) and CD25 (lymphocytes) after multiple trauma.Conclusions: The significant increase of Fas and TNFR1 expressionon T cells and monocytes, respectively, suggests the induction oftrauma-induced apoptosis through the caspase-dependent signalingpathway, which may be due to decreased presence of intracellularanti-apoptotic proteins.

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48Therapeutic potential of combined angiogenesis inhibitionand chemotherapy in experimental pancreatic cancerHubert G. Hotz, Sarah Bhargava, Birgit Hotz, Heinz J. BuhrDepartment of Surgery I, Charité Medical School, Campus BenjaminFranklin, Berlin, GermanyBackground: Monotherapy with antiangiogenic agents or che-motherapeutics has been shown to be of limited effect in pancreaticcancer. The aim of this study was to evaluate the therapeutic potentialof combined angiogenesis inhibition and chemotherapy in anorthotopic nude mouse model of human pancreatic cancer.Methods: 5x106 cells of 2 human pancreatic cancer cell lines(AsPC-1/poorly differentiated, HPAF-2/moderately differentiated)were injected subcutaneously into nude mice. 1 mm3 fragments ofthe resulting subcutaneous tumors were implanted into the pancreasof 72 other mice. Animals were randomized into control and 2treatment groups: combined application of the angiogenesis inhibitorIM862 (L-glutamyl-L-tryptophan; 100 mg/kg, daily ip.) andGemcitabine (125 mg/kg, weekly ip.) began either 3 days(prophylaxis) or 6 weeks (therapy) after tumor induction. Treatmentwas continued for 14 weeks or until death of the mice. Volume of theprimary tumor (TU-Vol), local infiltration and metastatic spread(dissemination score: D-Score) were determined at autopsy.Results: (p<0.05: * vs. control, # vs. therapy): HPAF-2 group:controls developed large primary tumors and aggressive tumordissemination (TU-Vol 4002±875 cmm, D-Score 16±2 pts., survival33%). Prophylactic combination therapy resulted in an almostcomplete suppression of tumor growth (TU-Vol 0 cmm*#, D-Score 4pts.*#, survival 100%*). The therapeutic setting was less effective(TU-Vol 357±102 cmm*, D-Score 7±2 pts.*, survival 66%). AsPC-1group: controls developed large primary tumors and aggressivetumor dissemination (TU-Vol 1550±131 cmm, D-Score 19±3 pts.,survival 17%). Prophylactic combination therapy resulted in asignificant reduction of TU-Vol (392±99 cmm*#), and improvedsurvival (50%). Tumor dissemination was not affected (D-Score 19±2 pts.), but only 50% of the animals developed metastasis. Thetherapeutic setting was less effective (TU-Vol 735±79 cmm*, D-Score 19±3 pts., survival 25%).Conclusion: Combined treatment with IM862 and Gemcitabineelicits therapeutic potential in a clinically relevant animal model ofpancreatic cancer. Effects were most striking in moderatelydifferentiated HPAF-2 tumors, where tumor growth and metastasiswas almost completely suppressed in the prophylactic setting.

49Blockade of mTOR inhibits lymphatic angiogenesisin vivo and in vitroStephan Huber, Gerald Schmid, Hanno Niess, Claudius Conrad,Karl-Walter Jauch, Christopher Heeschen, Christiane Bruns,Markus GubaDepartment of Surgery, University of Munich, KlinikumGrosshadern, Munich, GermanyBackground: Lymphangiogenesis is involved in many pathologicaldisorders. Importantly, tumor lymphangiogenesis promotes lympha-tic metastasis to the regional lymph nodes. Neutralization of thelymphangiogenetic key factor VEGF-C inhibits lymphatic metas-tasis. Since inhibition of the mammalian target of Rapamycin(mTOR) is known to significantly reduce VEGF-A inducedangiogenesis of blood vessels and inhibits tumor growth, weinvestigated the effect of mTOR inhibition on lymphangiogenesis.Methods: For in vitro experiments, lymphatic endothelial cells(LEC) were isolated from human dermal microvascular cells (PromoCell) via positive selection for VEGFR-3 (R&D Systems) using amagnetic beads sorting system (Milteny). In LECs treated with

VEGF-C (100 ng/ml) in the presence or absence of the mTORinhibitor rapamycin, phosphorylation of the p70S6 Kinase at theThr389 site, a specific target of mTOR, was quantified by WesternBlot analysis. For in vivo assessment of lymphangiogenesis, a skinflap was cut in the epigastric region of NMRI nude mice. Mice wererandomized for treatment with or without an oral solution of themTOR inhibitor Rapamycin (trough level 14.3±3,7 ng/ml). As anindicator of lymphangiogenesis, the number of crossing vessels andstained axillary lymph nodes was examined 20 minutes after theintracutaneous injection of 50 µl of Patent Blue in the center of theflap.Results:Western blot analysis for the phosphorylation of the P70S6-kinase, which is crucial for cell proliferation, demonstrated potentinhibition by pharmacological blockade of mTOR. In the skin flapmodel, treatment with Rapamycin significantly reduced both thenumber of lymphatic vessels crossing the flap (5.7±2.3 versus 1.4±0.9; P=0.004; n≥5) and the number of axillary sentinel lymph nodespositive for Patent Blue (1.7±0.5 versus 0.4±0.6; P=0.003; n≥5).Collateral lymphatic vessels bypassing the surgical incision tended tobe reduced in mice treated with Rapamycin (1.7±0.8 versus 1.0±1.0;n.s.).Conclusions: Inhibition of mTOR results in a marked inhibition oflymphangiogenesis. As mTOR inhibition also has a powerful inhi-bitory effect on angiogenesis, the combined inhibition of lymphaticand blood vessel growth may be of great importance for the treatmentof tumors that demonstrate strong lymphatic metastasis (e.g. breastcancer and melanoma).

50Changes of the C5a receptor on neutrophilsduring sepsis in humansM. Huber-Lang, H. Schreiber, U. Bruckner, M. Weiss*,P.A. Ward, F. GebhardDepartment of Trauma-, Hand- and Reconstructive Surgery and*Department of Anesthesiology, University of Ulm, Ulm, Germany§Department of Pathology, University of Michigan Medical School,USABackground: In animal models of sepsis, excessive generation of thecomplement activation product C5a and down-regulation of the C5areceptor (C5aR) on neutrophils have been described to be associatedwith an impaired innate immune response and lethal outcome. In atranslational research approach, a complement monitoring wasperformed in humans with sepsis and the C5aR expression onneutrophils as well as the clinical outcome was evaluated.Material and methods: 30 healthy volunteers and 60 patients withsevere sepsis or septic shock were enrolled for measurement of C5aRcontent on blood neutrophils by flow cytometry and westernblotting.In addition, hemolytic activity of complement (CH50), C3a, C5a, andmembrane attack complex (MAC) were determined in the serum.Approval from the Independent Ethics Committee of the Universityof Ulm: No. 82/2002.Results: Patients with sepsis exhibited significantly increased serumlevels of C3a, C5a and MAC (n=60) in comparison to healthyvolunteers. CH50 values of patients with sepsis were significantlyreduced. Patients who succumbed to sepsis within a 30 dayobservation period showed a significantly lower CH50 value incomparison to those who survived the lethal consequences of sepsis.Blood neutrophils isolated from patients with severe sepsis or septicshock demonstrated a significant decrease of C5aR expression incomparison to those found in healthy volunteers. In additional invitro studies, C5aR on neutrophils decreased with exposure toincreasing concentrations of C-reactive protein (1-300 mg/l). Basedon a ROC-analysis, a positive correlation between the C5aRexpression level on neutrophils and the survival of patients withsepsis was found.

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Conclusion: The data suggest, that in humans with severe sepsis orseptic shock there is an excessive generation of complementactivation products (C3a, C5a, MAC), impaired complementfunction (CH50) and reduction of C5aR on neutrophils. The lossof C5aR seems to play a crucial role in the pathophysiology andoutcome of sepsis in humans and may therefore be an interestingtarget for future therapies.

51Efficiency of Hirudin-Iloprost coated arteriovenousdialysis grafts in an animal modelInga Husmann1, Gerhard Schmidmaier2, Utz Settmacher1,Peter Neuhaus1, Michael Heise11Department of General Surgery, University Medicine Berlin, and2Department of Trauma and Reconstructive Surgery, Charité CampusVirchow Klinikum, Berlin, GermanyBackground: Thrombotic properties of alloplastic graft material andthe development of neointimal hyperplasia represent the mostcommon causes for dialysis graft failure. The purpose of this studywas to evaluate the efficiency of a hirudin-iloprost graft coating toprevent the formation of prosthetic pseudointima, and improvepatency rates. Hirudin-iloprost coating was applied to conventionalePTFE (expanded polytetrafluoroethylene) dialysis grafts and toarteriovenous patchprothesis (Venaflo-type) in an animal model.Methods and material: 32 arteriovenous ePTFE dialysis grafts wereimplanted in domestic pigs as unilateral loops, between the left iliacartery and vein (length 20 cm, diameter 7 mm). A biodegradablepoly-(D,L)-lactid acid (PLA) was used for graft coating. The PLAlayer served as carrier medium for hirudin and iloprost, providing along-term, local drug release from the graft surface. Standard ePTFEgrafts were implanted either native (group 1, n=8), or treated with thehirudin-iloprost coating (group 2, n=7). Analogously, Venaflo-typegrafts were tested uncoated (group 3, n=8) and coated (group 4, n=7).Individual grafts were assigned after randomization. Dialysis graftswere excised after a follow-up period of 6 weeks. Patency rates werecalculated and the development of pseudointima was noted.Results: Patency rate for uncoated conventional ePTFE dialysisgrafts was 25%, and increased to 62,5% when pretreated with ahirudin-iloprost coating. The patency rate of uncoated Venaflo-typeprothesis was 72%. A patency rate of 100% was achived in thehirudin-iloprost coated Venaflo-type group. Marked differencesbetween uncoated and coated ePTFE graft surfaces were noted atmacroscopic examination already. Hirudin-iloprost pretreated graftsshowed a smaller layer of pseudointima, compared to uncoatedePTFE dialysis grafts.Conclusion: The hirudin-iloprost coating effectively improvespatency rates of dialysis grafts. Arteriovenous patchprothesis(Venaflo-type) are superior to standard ePTFE-grafts. Best resultswere achieved by the implantation of hirudin-iloprost coatedVenaflo-type prothesis.

52Inhibition of Src tyrosine kinase by AZM475271enhances the efficacy of 5-Fluorouracil and Gemcitabinein human pancreatic carcinoma cellsIvan Ischenko, M. Yezhelyev, M. Guba, T. Green, M. Fennel,K-W. Jauch, C.J. BrunsDepartment of Surgery, University of Munich-Grosshadern LMU,Munich, Germany, and Cancer and Infection Research, AstraZeneca,Alderley Park, Macclesfeld, UKBackground: Src kinases are known to be over-expressed in humantumors, such as colon adenocarcinoma, breast cancer and pancreaticcarcinoma, and are active in numerous signaling pathways involved

in tumor proliferation, migration, adhesion and angiogenesis. Srcinvolvement has been implicated in signaling from many types ofreceptors including receptor tyrosine kinases, integrins and G-proteincoupled receptors. Recent studies demonstrated that Src transducesmany pathological signals to key modifiers of cancer cell survival,including MAPK, AKT and NF-kappa B. Src kinase over-expressionand increased activity has been reported in human pancreatic cancer.This study evaluated the efficacy of the novel selective Src kinaseinhibitor AZM475271 alone and in combination with 5-Fluorouracilor Gemcitabine on human pancreatic tumors growing orthotopicallyin nude mice.Methods: Cytotoxic effects of AZM475271, Gemcitabine and 5-Fluorouracil on L3.6pl human pancreatic carcinoma cells were testedassessed by MTT assays. In order to investigate the ability ofAZM475271 to inhibit Src kinase activity, an ELISA-based Srckinase inhibition test was used. Quantification of apoptosis wasperformed using propidium iodide staining for cell cycle analysis byFACS. To explore the in vitro effects of AZM475271 alone or incombination with conventional drugs, we injected 106 viable L3.6plcells into the pancreas of athymic nude mice. Seven days later,groups of mice received either: AZM475271 (50 mg/kg/day, orally),or 5-Fluorouracil (100 mg/kg/weekly, i.p. injection), or Gemcitabine(50 mg/kg/weekly, i.p. injection), or AZM475271 in combinationwith 5-Fluorouracil or in combination with Gemcitabine. Controlgroup was included. Mice were sacrificed on day 32 and the tumorvolume and weight was recorded, in addition to the incidence ofregional lymph node metastasis, and the number of liver metastases.Results: AZM475271 demonstrated strong selective dose-dependentinhibition of Src kinase, the IC50 concentration of AZM475271 toinhibit the phosphorylation of c-src was 0.1 µM, in comparison withIC50 of 0.7 µM to inhibit KDR. The in vitro concentrations ofGemcitabine or 5-Fluorouracil that produced no detectable cellgrowth were markedly reduced by combination with 5 µMAZM475271 compared with Gemcitabine or 5-Fluorouracil mono-therapy. In vitro FACS analysis detected a significantly elevatednumber of apoptotic cells after treatment with AZM475271 andGemcitabine. Consequently, combination therapy (AZM + Gemci-tabine or AZM + 5-Fluorouracil) was also associated with a growthdelay of primary pancreatic tumors after orthotopic tumor cellinjection. Our results indicate that the inhibition of Src kinase inhuman pancreatic cancer has antitumor efficacy that is markedlyenhanced by combination with Gemcitabine or 5-Fluorouracil.Additionally, a decreased incidence of lymph node metastases anda complete inhibition of the liver metastases were associated withadministration of AZM475271 monotherapy as well as combinationtherapy.Conclusion: Our preclinical study in pancreatic cancer hasdemonstrated that the antitumor efficacy of Gemcitabine or 5-Fluorouracil can be enhanced by additional treatment with inhibitorsof the tyrosine kinases (such as AZM475271). Further investigationswill be carried out in order to explain the ability of AZM475271 tosensitize tumor cells against the cytotoxic effects of Gemcitabine and5-Fluorouracil.

53In vivo analysis of host tissue response to scaffoldbiomaterials for tissue engineering: promotionof vascular ingrowth by PLGA but not hydrogelDominic Junker1, Matthias W. Laschke1, Martin Rücker2,Carlos Carvalho3, Michael D. Menger11Institute for Clinical and Experimental Surgery, University ofSaarland, Homburg/Saar, Germany2Department of Oral and Maxillofacial Surgery, Medical Universityof Hannover, Germany3FMF Freiburger Materialforschungszentrum, Freiburg, Germany

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Background: In tissue engineering, synthetically derived polymerscaffolds provide a matrix for cells to attach and proliferate that canbe implanted into a tissue defect site. For this purpose, the scaffoldsshould be biocompatible and should ensure an optimal interactionwith endothelial cells to promote angiogenesis. In fact, long-termsurvival and function of tissue constructs decisively depend on rapidand adequate vascularization after implantation into the hostorganism. However, the ideal scaffold, which sufficiently fulfillsthese material properties has not been determined yet. For a bettercharacterization of commonly used scaffold biomaterials, we used inthe present study the dorsal skinfold chamber model in order toquantitatively analyze in vivo the inflammatory host tissue responseand angiogenesis after scaffold implantation.Animals and methods: A dorsal skinfold chamber was prepared in22 balb/c mice for the implantation of two different scaffold typesconsisting of either poly-lactic-glycolic acid (PLGA) (n=8) or hydro-gel (n=7). Dorsal skinfold chambers without scaffolds served ascontrol (n=7). Using the technique of intravital fluorescence micro-scopy, we repetitively analyzed angiogenesis, microhemodynamics,microvascular permeability and venular leukocyte-endothelial cellinteraction over a time period of 14 days after scaffold implantation.Results: PLGA scaffolds induced an inflammatory response of thehost tissue with significantly increased values of adherent (day6, 210±32 cells/mm²) and rolling (day 6, 18±2 cells/min) leukocytesin postcapillary and collecting venules of the scaffold border zonewhen compared to control animals (day 6, 40±10 cells/mm² and 5±1cells/min; p<0.05). This cellular inflammatory response was accom-panied by an increase of macromolecular leakage (day 6, PLGA:0.68±0.01 vs. control: 0.45±0.03; p<0.05), indicating loss of in-tegrity of venular endothelial cells. Angiogenesis started at day 3after implantation by protrusion of capillary sprouts, originating fromthe host microvasculature. Until day 14, these sprouts interconnectedwith each other to form a new microvascular network. Histologyconfirmed the formation of granulation tissue with adequate incor-poration of the PLGA scaffolds within the host tissue. In contrast,hydrogel scaffolds failed to vascularize during the observationperiod. Moreover, their implantation into the dorsal skinfold cham-ber resulted in an even more pronounced leukocytic inflammatoryresponse when compared to PLGA scaffolds (day 6, 393±58 cells/mm² and 34±5 cells/min) and an increased endothelial damage(macromolecular leakage: 0.86±0.02; p<0.05).Conclusion: The dorsal skinfold chamber of balb/c mice is an usefulexperimental approach to study host tissue response and angiogen-esis of implanted scaffold biomaterials for tissue engineering. Inthe present study, we could demonstrate that PLGA scaffolds arecharacterized by a better biocompatibility when compared to hy-drogel scaffolds as indicated by a reduced inflammatory responseduring the first 14 days after implantation. Moreover, PLGA pro-motes vascular ingrowth from the surrounding host tissue, guar-anteeing an adequate incorporation of the biomaterial within thehost tissue.

54Growth inhibition of pancreatic cancerin vitro and in vivo by antihistaminesElias Karakas1, M. Soulaiman2 , O. Sürücü2, U. Schäfer3,Carsten Dietz1, Ilhan Celik21Department of Visceral-, Thoracic- und Vascular Surgery,Philipps-University, Marburg, Germany2Institute of Theoretical Surgery, Philipps-University Marburg,Marburg, Germany3Biochemical and Experimental Division, Faculty of Medicine,University of Cologne, Cologne, GermanyBackground: Pancreatic cancer has a very poor prognosis andresponse to therapeutic interventions. Clinical studies with H2receptor antagonists, particularly cimetidine, significantly improve

the survival rate of colon cancer patients. The aim of our study was toinvestigate the effect of cimetidine on human pancreatic cancerxenografts in SCID mice and the underlying mechanisms in vitro.Materials and methods: Two primary human pancreatic cancer celllines (BxPC-3 and ASPC-1) were implanted (5x106 cells/mouse) s.c.into the dorsa of SCID mice. After tumour volume reached 100 mm3,animals were randomised into three groups (n=8/group). Two groupswere injected (s.c.) with cimetidine 10 or 100 mg/kg/day and onegroup received placebo (saline). Tumours were measured every thirdday until day 14 or 20. The effect of Cimetidine on tumour cellproliferation, histamine release and endothelial cell (HUVEC)migration were analysed in vitro. Expression of tumour associatedantigens (sialyl Lexisa (sLea), sialyl Lewisx (sLex)) and histamine H2-receptors was examined in both cell lines (flow-cytometry).Results: Cimetidine (10 or 100 mg/kg/day) caused a significantinhibition of tumour growth (ASPC-1: 32% and 50%, p<0.05; BxPC-3: 56% and 65%; p<0.05) compared to placebo (no adverse sideeffects). Cimetidine dose-dependently inhibited tumour cell prolif-eration (1 mg/ml: ASPC-1: 80%, BxPC-3: 100%) and migration ofHUVECs (80% inhibition). Furthermore, incubation with tumourcells led to a dose-dependent increase in histamine release. Asignificant difference in sLea expression was detected comparingboth tumour cell lines (ASPC-1: 2.6% versus BxPC-3: 85%). Nodifferences were obtained with regard to sLex and histamine H2-receptors.Conclusion: Cimetidine potently inhibited tumour growth ofpancreatic cancer in vitro and in vivo. The different results with the2 cell lines may be due to different sLea expression and histaminereleasing effects. Given the safety of cimetidine, it is a promisingcandidate for adjuvant therapy of pancreatic cancer.

55Extracellular matrix specific responseof human mesenchymal stem cellsGrit Kasper1, Andrea Ode1, Jens Tuischer1, Georg Matziolis1,Simone Fuchs2, Georg N. Duda1, Carsten Perka11Center for Musculoskeletal Surgery, Charité, University MedicineBerlin, Berlin, Germany2Biotechnology Department, TU-Berlin, Berlin, GermanyBackground: The development of biomaterials for tissue engineer-ing applications has recently focused on the design of biomimeticmaterials that are capable of regulating cellular behaviour. To enablethe rational design of biomimetic materials, knowledge is neededabout the effect of the biological/biochemical microenvironment onprogenitor cells, which are crucial to regeneration processes. In thephysiological environment, cellular responses are elicited by avariety of biomolecules, among which components of the extra-cellular matrix (ECM) and growth factors play a pivotal role.Therefore this study aimed to investigate the influence of differentECM components on the migration, adhesion and proliferation ofhuman mesenchymal progenitor cells (MPCs) .Patients and methods: Human MPCs were obtained from theproximal femur during hip surgery and characterized by FACS fortheir marker protein expression (CD105+, CD106+, CD73+, CD44+,CD4-, CD14-, CD34-) as well as by their potential to differentiate intoosteogenic, adipogenic and chondrogenic lineages. To analysemigration, a Boyden chamber assay was established. Adhesion andproliferation were examined on coated 96-well plates.Results: When compared to the negative control, collagensstimulated both adhesion (+41% to +79%, p<0.001 to 0.005) andmigration (+46% to +78%, p=0.005 to 0.017) of MPCs while fibrinhad a positive effect on adhesion (+84%, p=0.004), but inhibitedmigration (-99%, p<0.001). In contrast laminin showed nosignificant influence on migration (p=0.731) but very poor adhesiveproperties (-31%, p=0.020). Fibronectin provided the maximalmigratory stimulus (+93%, p=0.015), whereas chondroitin sulfate

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A and fibrin maximally enhanced proliferation (+20%, p=0.024) andadhesion (+84%, p=0.004) respectively.Conclusions: Different ECM components elicit specific responses inhuman MPCs. This knowledge could be exploited in the design ofbiomimetic materials incorporating peptides mimicking thesemolecules.

56Beta-cell regeneration after subtotal pancreatectomyin a knock-out mouse modelStephan Kersting1,2, Hendrik Bergert1,2, Hassan Mziaut2,Klaus-Peter Knoch2, Michele Solimena2, Hans Detlev Saeger11Department of Visceral-, Thoracic and Vascular Surgery, and2Department of Experimental Diabetology, University ofTechnology, Dresden, GermanyBackground: In contrary to the general opinion pancreatic beta-cellmass is not static but can adapt to the demand of the metabolism, e.g.in pregnancy or obesity. As is known from clinical experience, a pre-existing diabetes in some cases disappears after pancreatic resections.This effect has been imitated in animal models studying beta-cellregeneration after partial pancreatectomy. Islet Cell Autoantigen 512(ICA 512) is a catalytically inactive receptor tyrosine phosphatasetargeted to the nucleus where it up-regulates insulin expression andgene transcription by enhancing the nuclear accumulation andtyrosine phosphorylation of STAT5b, a transcription factor activatedby growth hormones and presumably involved in proliferation ofbeta-cells. To investigate the role of the ICA 512 pathway inproliferation of pancreatic beta cells, the established animal model ofsubtotal pancreatectomy was further refined and applied to ICA-512knock-out mice.Material and methods: To establish the model, 30 wild-type c57/bl6mice were divided into two groups. One group was subtotallypancreatectomized, the other group was sham operated by removingonly the spleen. For the partial pancreatectomy the tail of thepancreas was mobilized from the transverse colon, the stomach andfrom the retroperitoneal adhesions up to the mesenteric root. Thepancreatic head was detached from the duodenum as far as the bileduct allowed. (The procedure will be demonstrated in a short film.)At the end of the operation all animals received a small osmotic pump(Alzet®) continuously releasing Bromodesoxyuridine (BrdU) forseven days. BrdU is a proliferation marker acting as a thymidineanalogon, which is incorporated into the DNA of dividing cells. Oneweek after the operation the animals were sacrificed and theremaining pancreas was removed. The tissue was paraffin embedded,serial sectioned and fluorescence-stained for insulin, DAPI andBrdU. The ratio of BrdU-positive (i.e. newly built) beta-cells wascalculated and compared statistically between the groups. Untilrelease of this abstract, one series of ICA-512 knock-out wasoperated likewise. One group of animals received partial pancrea-tectomy, the other was sham operated.Results: By our technique, a 80%(±7%) pancreatectomy wasachieved. As described before, even after the operation the animalswere able to maintain blood glucose control autonomously, albeitICA-512 -/- mice had significant higher levels of blood glucoseparticularly within the first days after the operation. Statisticallysignificant less BrdU-positive beta-cells were found in sham-operated animals compared to subtotally pancreatectomized miceindicating a successful stimulation of beta-cell regeneration by ourmodel. Differences in BrdU incorporation between pancreatecto-mized wild-type and knock-out mice could be demonstrated as well,even though they reached not statistically significant levels in thisvery first series. More data will be shown at the time of presentation.Conclusion: We successfully established a model for stimulation ofbeta-cell regeneration by partial pancreatectomy. BrdU administra-tion by continuous release of an intraperitoneal pump allowspermanent labelling of dividing cells, thus reaching –different fromsingle daily injections- all proliferating cells in the observation

period. Further experiments are necessary to demonstrate differencesin the regeneration of beta-cells of ICA 512 knock-out mice.

57Secretion of N terminal-pro brain natriuretic peptidein patients with severe traumatic brain injuryC. Kirchhoff1, T. Mussack1, V. Bogner1, J. Stegmaier1,U. Kreimeier2, W. Mutschler1, P. Biberthaler11Chirurgische Klinik und Poliklinik and 2Klinik für Anästhesiologie,Klinikum Innenstadt, Ludwig-Maximilians-Universität, München,GermanyBackground: The pathophysiological consequence of TBI isinfluenced by primary and secondary brain damage; in this contextsecondary damage might be worsened by dysregulation of cerebralwater - sodium homeostasis in terms of cerebral salt wasting (CSW).Therefore, recent studies gave evidence to the important role of BNPin CSW in patients suffering from aneurysmatic subarachnoidalhaemorrhage (SAH). Moreover it has been shown, that in patientswith SAH serum BNP levels correlate with intracranial pressure(ICP) and clinical outcome. Therefore, the aim of this study was toassess the influence of the systemic and intrathecal excretion of NT-proBNP on the dysfunction of the blood brain barrier (BBB) and thecorrelation of NT-proBNP levels to increased ICP in patients withsevere TBI.Patients and methods: 14 patients suffering from TBI (GCS <8)were enrolled in this study. Immediately after the placement of anextraventricular drainage (EVD), as well as 12, 24, 48 and 72 hourspost trauma, 3 ml cerebrospinal fluid (CSF) samples as well as 5mlperipheral blood samples were drawn. Pro-BNP levels weredetermined in CSF and blood, using ®NT-proBNP-test systems(Roche Diagnostics). ICP was monitored on a permanent basis viathe EVD (TraumaCath®, IntegraNeurosciences, Plainsboro, USA)and recorded at each drawing time point. Patients were divided intotwo sub-collectives according to ICP; group I (n=8) exhibited anICP<15 over the complete observation period, whereas group II(n=6) was at least once above that level. Due to its reliability as aparameter for the assessment of BBB function, the ratio of CSF/serum albumin (Qa) was daily calculated in all patients (normalvalue<0.007). 90 days post trauma the clinical outcome wasevaluated using GOS. Statistical analysis was done using t-test(group I vs. group II) and spearman rank correlation for assessingrelationships between NT-proBNP and ICP levels (p<0.05).Results: Patients in group I had a mean ICP of 11.1±3.5 mmHg(mean±SD,), group II a mean of 27.6±9.2. GOS of patients in groupII accounted for 2±2 vs. 4±1 in group I. Serum (800±150 pg/ml) aswell as CSF levels (120±15 pg/ml) of NT-proBNP rose in group II incorrelation to ICP-values and were therefore significantly elevated incomparison to the measured levels of group I (serum: 40±8.8 pg/ml),respectively in CSG 110±13 pg/ml as well as to admission andcontrol group values. However, there was no significant disruption ofBBB as the Qa accounted for 0.006±0.0008.Conclusions: In this study we showed significant increases of serumand intrathecal NT-proBNP levels, directly correlating to the increaseof the ICP in patients with severe TBI. We could demonstrate that thisincrease seems not to be influenced by integrity of the blood brainbarrier. A possible explanation could be the intrathecal synthesis ofproBNP. However, the physiologic role of NT-proBNP in TBI needsto be elucidated in further studies.

58Immunohistochemistry - from simple countingto quality controlled quantificationR. Kleinert, O. Dirsch*, F. Madrahimova, H. Qing, H. Chi,N. Madrahimov, C.E. Broelsch, U. DahmenDepartment of General, Visceral and Transplantation Surgery,

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University Hospital Essen, Essen, Germany*Institute of Pathology, University Hospital Cologne, Cologne,GermanyBackground: In order to decrease the “human factor” and the timerequired for the evaluation in the analysis of immunohistochemicalstains (IHC) computer based image analysis can be employed. Theuse of an image analysis system requires the exact mathematicaldefinition of the target objects whereas the quantification by thehuman observer is mainly done by the subjective judgement of targetobjects. Aim of this study was the adaptation of a custom designed,cost-efficient image analysis system for the quantitative analysis of adefined IHC assay (nuclear staining of a defined target cell type, inthis case determination of BrdU-proliferation index in regeneratingrat livers) and the validation of both, the image analysis and theimmunohistochemical procedure in terms of precision, accuracy,sensitivity and specificity.Material and methods: Samples from BrdU-labeled regeneratingrat livers were subjected to immunohistochemical staining using analkaline phosphatase based detection system. Up to 20 images/section from zone 1 (periportal) and zone 3 (pericentral) according toRappaport were acquired using a digital camera (40x magnification,image resolution 2048x1024 pixel). Images were analyzed repeat-edly with the conventional method and with the specifically adaptedImage analysis program (Sigma Scan Pro 5). Validation of the systemwas done on the level of the image, the region of interest and theassay itself.Results: An image analysis system was custom designed usingspecifically selected standard hard- and software. Extraction of targetobjects was achieved by color based segmentation. Size (300 to 2500pixel) and roundness (greater 0,7) were selected as key parameters toidentify and classify hepatocyte nuclei (HC). Validation of the imageanalysis system revealed a high precision and accuracy with asensitivity >96% and a rate of true HC >95%. Observer basedselection of regions of interest (ROI) was the most critical part of thesystem and required a precise definition of criteria for choosing theROI. The immunohistochemical assay itself showed a high intra- andinterassay reproducibility.Conclusion: Computer assisted analysis of IHC images can be usedas a precise, accurate, sensitive and specific diagnostic tool for thedetermination of nuclear objects under defined conditions.

59Long-time intensive care therapy in anhepatic pigsKarolin Knubben, Martin Schenk, Christian Thiel, Klaus Dietrich,Matthias H. Morgalla*, Ruth Ladurner, Horst D. Becker,Alfred KönigsrainerDepartment of General, Visceral and Transplantation Surgery, and*Department of Neurosurgery, University Hospital, Tuebingen,GermanyBackground: Establishment of new intensive care therapy options inanhepatic animal model is required to realize new long-time survivalstudies of liver failure.Methods: Hepatectomy was performed in 15 female pigs (30-41 kg)by reconstructing the vena cava and portal vein through a three-wayvascular prosthesis with end to side anastomosis and afterwardspostoperatively attended in the intensive care unit. Postoperativelyanimals stayed under deep narcosis and pressure controlled ventila-tion. The following parameters were recorded continuously: electro-cardiogram, mean arterial pressure, SO2 oximetry, core bodytemperature, intracranial pressure, urinary output. Serum electrolytes,acid-base balance, blood gases, blood glucose levels and haemoglo-bin were hourly monitored and immediately corrected as required.Pigs received sodiumchloride 0,9%, hydroxyethylstrach 6% andfresh-frozen-plasma units. Erythrocytes units were given to coverblood loss. All pigs received furosemid to keep on dieresis as long aspossible, when renal failure occurred they were treated by dialysis.

Results: After hepatectomy a continuous worsening of the otherorgan systems appeared. To maintain adequate mean arterial pressureNoradrenalin was given in heightening dosage up to 30 µg/min.Blood lactate concentration stayed stable during sufficient circulationand raised when decompensation occurred. Pulmonal function im-paired progressively and therefore it was necessary to increase PEEPand airway pressure to maintain sufficient ventilation and oxygena-tion. All animals suffered from renal failure, which was treated bydialyses. Though this regime blood concentration of ammonia couldbe kept constantly stabile (500 µg/dl) for a long time. Maximumsurvival time was 88.5 hours (mean survival time 52 hours).Conclusion: We established a new intensive care therapy treatmentwhich can be used for long-time survival studies examining anhepaticsituations and also other questions about liver failure.

60Activated leukocytes promote osteogenic differentiationof human mesenchymal stem cellsManfred Köller, Vanessa Kroll, Thomas A. Schildhauer, Gert MuhrChirurgische Klinik und Poliklinik, Chirurgische Forschung, BGKliniken Bergmannsheil – Universitätsklinik, Bochum, GermanyBackground: Autologous transplantation of mesenchymal stemcells (MSCs) from bone marrow may offer new therapeuticalapproaches for the therapy of bone defects. Currently, first clinicalstudies -mainly feasibility trials- are performed. In these studiesbiomimetic porous calcium phosphates are commonly used as cellcarrier materials for bone repair applications. After implantation ofcell-loaded biomaterial leukocytes belong to the first cells which willimmediately get into close contact with the implanted material. It isknown that especially polymorphonuclear neutrophil granulocytes(PMN) were activated at calcium phosphates after adherence to thesurface. Thus, it was the purpose of this study to analyse theinfluence of PMN-conditioned cell culture media on the proliferationand differentiation of human mesenchymal stem cells (hMSC).Methods: PMN were isolated by Ficoll-separation from whole bloodof healthy volunteers. Subsequently the PMN (1x106/ml supple-mented RPMI1640) were stimulated with lipopolysaccharide (1 or 5µg LPS, E. coli O55:B5) for 24 h and the cell culture supernatants(conditioned media) were transferred to human mesenchymal stemcells (Cambrex Bio Science Walkersville) grown in 6-well-plates.After an additional cell culture period up to 35d a histochemicalexamination (Alizarin red-S stains) followed to detect osteogenic celldifferentiation and bone nodule formation. Respective cell culturesupernatants were analysed for cytokine content by ELISA.Results: In contrast to non-conditioned cell culture medium or LPS-conditioned medium the PMN/LPS-conditioned media decreasedhMSC proliferation and induced osteogenic cell differentiationindicated by Alizarin-positive bone nodule formation. In the cellculture supernatants an increase in IL-8 was observed.Conclusions: These data indicate the potential role of activatedleukocytes in the regulation of progenitor cell differentiation. Themolecular nature of the PMN-derived osteogenic factor or factors hasto be defined.

61Endothelial cell compatibility of porous Hi-PorLeila Kolios, Ronald Unger, C. James KirkpatrickInstitute of Pathology, Johannes Gutenberg University,Mainz, GermanyBackground: Hi-Por represents a new ceramic-biomaterial withorthopedic applications as a long-term implant. Spongiosa-likeporosity for use in osteogenic biomaterials has been found useful inthat it permits tissue ingrowth and apposition. Furthermore Hi-Por

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appears to offer long-term fixation, biofunctionality and biocompat-ibility. It has been shown to be an excellent substrate for osteogeniccell integration and demonstrated to be a non-sensitizing, non-irritating and non-toxic biomaterial. For a porous biomaterial tosucceed vascularisation of the implant must occur. Endothelial cellsare the major cell-type of the microvasculature. How these cellsrespond to biomaterial is of profound importance to the success of thebiomaterial. Neovascularisation is an important step that willdetermine the capacity for bone ingrowth in a biomaterial andeventual osseointegration. This study examines the in vitro interactionof human endothelial cells with Hi-Por.Material and methods: Biomaterials: Hi-Por discs, consisting ofhydroxyapatite (HA) or hydroxyapatite-tricalciumphosphat (HA-TCP), approximately 10 mm in diameter and 2 mm thick weresterilized by 70% ethanol. The pore size ranged from 50 µm over 200µm to 500 µm. Prior to cell addition, the discs were incubated withfibronectin (100 µg/ml) for 1 hr and placed into culture medium.Cells:Human microvascular endothelial cells from the skin (HDMEC) andHuman macrovascular primary endothelial cells (ISO-HAS) werecultured and used as previously described [1]. Detection of cellgrowth: Hi-POR discs were stained with Calcein-AM and examinedby confocal microscopy. Gene regulation of endothelial cells: RNAwas isolated from cells growing on Hi-Por and equal amounts ofcDNA were subjected to PCR with primers specific for VCAM-1,ICAM-1 and E-Selectin [2]. In certain experiments cells werestimulated with 1µg/ml LPS for 4 hr prior to the extraction of RNA. Inaddition, immunofluorescence with antibodies specific to thesemarkers and to PECAM-1 was also carried out to examine theexpression of genes at the single-cell level.Results: Both endothelial cell types readily adhered and spread overthe entire threedimensional Hi-Por surface with time. Fibronectincoating increased the initial adhesion of cells, but did not influence theeventual coverage of Hi-Por. Cells grew on all surfaces, into pores,and exhibited normal cell-cell contacts and endothelial cell structures.The cells remained in contact with Hi-Por and did not span acrossopen pore areas. The cells growing on the Hi-Por exhibited a normalpattern of adhesion molecule expression. In some cases only a lowlevel of expression of VCAM-1, ICAM-1 and E–Selectin wasobserved in the analysis of RNA from these cells. After stimulation ofthese cells with LPS a pattern of induction of these molecules wasobserved comparable to cells growing on normal cell culture plastic.Immunofluorescent staining also confirmed that the strong inductionof adhesion molecule RNA expression in the presence of LPSobserved in the PCR was due to a global upregulation on all of thecells. PECAM-1 staining with typical expression at cell-cell contactswas observed. Cells growing on Hi-POR formed microvessel-likestructures under conditions that have been shown to induceangiogenic behavior in cultured cells in vitro. Formation ofcapillary-like structures was observed after 3-5 days.Discussion and conclusions: Hi-Por is an excellent substrate for thegrowth of human endothelial cells required for tissue vascularization.Cells maintain the most essential of endothelial cell-specificphenotypes and functions while growing on this material. Mostimportant is the ability to form microvessel-like structures underangiogenesis-stimulating conditions, one of the first steps observed inosseointegration. It will be important to determine whether thismaterial supports the culture/coculture of osteoblasts and endothelialcells and whether cell-specific phenotypes and functions arepreserved under these conditions.

62Gastrointestinal field effect and dendritic cellsArne Koscielny, Thomas Börner, Andreas Hirner, Jörg C. KalffKlinik und Poliklinik für Allgemein-, Viszeral-, Thorax- undGefäßchirurgie, Universität Bonn, Bonn, GermanyBackground: Intestinal manipulation leads to bowel wall inflamma-tion not only at the traumatized site but also of the entire small bowel,

stomach and colon. Previously, this gastrointestinal field effect wasdemonstrated in a rodent model. Previous investigations did notshow a humoral or neurally mediated propagation of the inflamma-tion and we postulate a reaction mediated by immunologically activecells. The aim of this study was to isolate and characterize dendriticcells (DC) out of the intestinal smooth muscle and to study theirreceptor expression for maturation and migration following surgicaltrauma and LPS challenge.Material and methods: Mice underwent standardized intestinalmanipulation or i.p. LPS administration (5 μg/kg KG) and severaltissues (intestinal muscularis, Peyer’s Plaques, mesenteric lymphnodes and spleen) were obtained at different times after themanipulation. DC were isolated by tissue digestion with collagenaseIVand separated by CD11c-iMAG. The harvested DC were analysedby FACS or brought into culture. The activation pattern of DC wasanalysed by RT-PCR for CCR 2, CCR 5, CCR 7, CCL-19, IL-12a.Results: We found a statistical increase in DC within the intestinalmuscularis, the Peyer’s Plaques and the mesenteric lymph nodes at 6and 12 hours following intestinal manipulation and injection of LPS.There was an upregulation of the costimulatory molecules MHC II,CD 40, CD 80, CD 86 and of the phagocytosis marker CD 205 in theDC after intestinal manipulation. CCR 2, CCR 5, CCR 7, CCL-19and IL-12a were upregulated in a time- and tissue-depending mannerafter intestinal manipulation.Conclusion: Intestinal manipulation or LPS challenge induces arecruitment of DC into the muscularis externa and mesenterial lymphnodes combined with an upregulation of costimulatory immuno-competent molecules and migratory surface markers in DCs. Thesefindings demonstrate a precondition for an immunological responsewithin the gastrointestinal tract and a possible immunologicallytransmitted field effect.

63Quantification of nondirected tumor cellmotility using time-lapse microscopyJ. Krasnyanska, K. Fisch, P. Gaßmann, J. HaierDepartment of General Surgery, University Hospital Münster,Münster, GermanyBackground: Tumor cell migration and chemotactic cell invasioninto host organs appear to be important steps during formation oforgan specific distant metastasis. These processes require variousmorphological and structural alterations of the tumor cell. We usedtime lapse microscopy and single cell tracking to establish atechnique allowing the quantification of these alterations and distinctsteps of cell movement.Methods: MDA-MB468 breast cancer cells were plated on differentextracellular matrix (ECM) proteins for 20 min. Using an invertedfluorescence microscope time lapse recording was performed for 10min. Direction of cell movement, distance and velocity wereanalyzed at defined time intervalls. Cell motility was comparedbetween different ECM components (type I and IV collagen, laminin,fibronectin) and using various FCS concentrations for stimulation.Statistical analyses were performed using Students t-test.Results: Non-directed cell motility of single cells differed over time(range 0-14,00 µm/min) and was only partially dependent from FCS(1-5%) concentrations used which was found for all ECMcomponents (mean: 3,26-4,10 µm/min). However, significantdifferences of movement velocities were observed between theECM components and cells showed less motility on laminin (mean:1,11-2,50 µm/min). Cell surface areas remained comparable overtime for each cell, but was heterogenous between different cells.Conclusions: Single cell tracking over time enabled the quantifica-tion of nondirected motility of tumor cells on ECM components.Variable velocities of cell movement were found suggesting shortterm dynamics of intracellular signaling processes. Furthermore, thisvariability can result in misinterpretation of summative techniques,such as Transwell chambers. Additionally, the heterogeneity between

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the cells may be a determinant for host organ colonization duringmetastasis formation.

64Reticular tissue generation by bioreactor-expanded humanbone marrow cells after long term cell cultureVanessa Kroll, Thomas A.Schildhauer, Gert Muhr, Manfred KöllerChirurgische Klinik und Poliklinik, Chirurgische Forschung, BGKliniken Bergmannsheil – Universitätsklinik, Bochum, GermanyBackground: The expanding potential of mesenchymal stem cells(MSCs) for tissue repair and regeneration using autologues cellsstimulates clinical research to develop applicable cell culture systemsfor clinical use. The maintenance and propagation of progenitor cellpopulations are crucially dependent on the conditions of cultureincluding supplements in the culture medium. It was the purpose ofthis study to analyse harvest cells expanded with a closed and sterilecell production bioreactor during a long term culture.Methods:Density-gradient isolated mononuclear cell (MC) fractionsof bone marrow (BM) taken from the iliac crest from healthyvolunteers were inoculated into single-use disposable cell cassettewhich contained the bioreactor-chamber (AastromReplicell™ CellProduction System) for 12 days using supplemented medium(IMDM containing FCS, HS, EPO, PIXY321, Flt-3-ligand, L-glutamine, gentamicin, and vancomycin). Harvested cells weretransferred to conventional cell culture and after two passages cellculture flasks were left for long term culture (up to 312 days) withoutfurther supplements or stimulations. Soluble mediators in cell culturesupernatants were analyzed by ELISA and the nature of generatedtissue was determined by a histochemical silver impregnationmethod (Bio-Optica, Milano, Italy).Results: During long term culture a converging of randomly seededcells was observed. Reticular tissue structures then emerged whichextended up to 7 cm. The histochemical analysis revealed theoccurrence of agyrophilic reticular fibers (reticulin) and positivecollagen staining. Together with morphologic criteria the tissueshowed elements of a reticular connective tissue. In the respectivecell culture supernatants an increase in IL-6, IL-8 and IL-11 wasobserved.Conclusions: These results clearly demonstrate the particularlongevity of human bone marrow derived progenitor cells and theintrinsic capacity for tissue generation. The molecular signals for thistissue generation are obviously provided by different cell types sincethe reticular tissue formation was not observed with highly enrichedhumans mesenchymal stem cells.

65The hemodynamics at a venous anastomosisof artificial grafts used as hemodialysis accessUlf Krueger, Juergen Zanow, Hans ScholzDepartment for Vascular Surgery, Queen Elisabeth Hospital,Berlin, GermanyBackground: An arteriovenous (AV) graft causes unphysiologicalhemodynamics. The physiological reaction of the body may lead tointimal hyperplasia and subsequent occlusion or stenosis. Thestenosis is responsible for the late failure of AVaccess grafts, and it ismainly observed at the venous anastomosis. The conventional end-to-side anastomosis, a hooded Venaflo graft (Bard/IMPRA, Tempe,AZ, USA), and a correct trimmed Venaflo anastomosis werecompared.Materials and methods: By using Computational Fluid Dynamics(software CFX 5.6, ANSYS Ltd. USA), the flow patterns, the pres-sure and the wall shear stresses were calculated. A time-dependedmass flow profile with flow rates of 1500 ml/min (systolic) and 120

ml/min (diastolic, mean 700 ml/min) was specified as the inletboundary condition. The use of a non-Newtonian incompressiblefluid considered the effect of shear-thinning. The results werecompared with in vitro investigations performed on a pulsatile flowsystem.Results: The velocity profile of the correct trimmed Venaflo isobviously better than the one for the conventional form. In thehooded Venaflo anastomosis, the velocity levels near the floor arehigher compared to the correct Venaflo anastomosis. A large regionof boundary layer separation is observed at the inner wall, theretrograde flow movement formed a vortex in the correct Venaflo. Inthe diastole, very slow velocities were seen in the hood region of thehooded Venaflo anastomosis. At the vein floor, the exposed areas aswell as the duration of high wall shear stress are higher in both theconventional anastomosis and the hooded Venaflo.Conclusion: The correct Venaflo graft performs significantly better,whereas the impact of the blood stream at the vein floor caused astagnation point at both the conventional and hooded anastomosis.The hooded anastomosis leads to a combination of a conventionalanastomosis with an additional dead water region.

66Beta-catenin interacts with the COP9 signalosome (CSN)Corinna Langelotz, Bettina Hetfeld, Wolfgang Schwenk, WolfgangDubielDepartment of General-, Visceral-, Vascular- and Thoracic Surgery,Charité - Universitätsmedizin Berlin, Berlin, GermanyBackground: The COP9 signalosome (CSN) is a conservedmultiprotein complex consisting of eight subunits. It is involved ina wide variety of regulatory processes including cell cycle control,signal transduction and transcriptional activation. One of the majorplayers in the wnt signaling pathway is beta-catenin, being the focusof a number of regulatory systems. Its interactions with adhesion,transcription and destruction complexes ensure proper tissuearchitecture and cell-fate decisions during normal development.Disturbed beta-catenin turnover can be oncogenic, as the failure todestroy cytoplasmic beta-catenin plays a role in most colon cancers.Its proteolysis is mediated by an interaction with the adenomatouspolyposis coli (APC) leading to phosphorylation and subsequentubiquitination and degradation via the ubiquitin-proteasome system.Beta-catenin is ubiquitinated by a cullin-RING ubiquitin ligase(CRL) complex. Because the CSN is a regulator of CRLs, we studiedits impact on beta-catenin degradation.Methods: A PCR for beta-catenin was performed, the full lengthbeta-catenin constructed, cloned into the vectors pCMVTag3C,pQE32 and pcDNA3.1 for transfection and recombinant proteinproduction, respectively. The expression of endogenous beta-cateninin colon cancer cells (HT29, Caco-2, SW480), MCF7 breast cancercells and Hela cervical cancer cells was analyzed in western blots anddetected with a monoclonal antibody against beta-catenin (R&Dsystems). Glycerol density gradient ultracentrifugation with recom-binant beta-catenin and purified CSN as well as with cell lysates wasperformed. Immunoprecipitations with recombinant beta-catenin andcell lysates using the anti-CSN7 antibody or the anti-beta-cateninantibody were also established.Results: Glycerol density gradients with lysate from HT29 andMCF7 cells revealed that beta-catenin and the CSN co-sedimentedinto the same fractions. These fractions also contains CRLs. To studywhether there is a direct interaction between beta-catenin and theCSN, glycerol gradients with recombinant beta-catenin and purifiedCSN were performed. Beta-catenin appeared to shift into the samefractions as the CSN, whereas beta-catenin alone was detected inmuch lighter fractions. Moreover, immunoprecipitations showed aco-precipitation of beta-catenin and the CSN.Conclusion: We could demonstrate an interaction between beta-catenin and the CSN, possibly as a degradation complex in the

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ubiquitin-proteasome system. Further experiments are needed toclarify the role of the CSN in the wnt-pathway and beta-cateninregulation and are currently under way in our laboratory.

67Development of a vascularized bioartificialendocrine pancreasNicolas Lembert1,2, Marc Waidmann3, Michael Doser4,Alfred Königsrainer11Department of General-, Visceral and Transplantation Surgery,2Department of Pharmacology, and 3Department of TransfusionMedicine, University of Tübingen, Germany4Institute of Textile and Process Engineering (ITV), Denkendorf,GermanyBackground: The transplantation of macroencapsulated islets ofLangerhans is one approach to treat type 1 diabetes without the needof a lifelong immunosuppressive therapy. For this purpose abiomaterial for encapsulation is needed combining immunoisolationand biocompatibility with optimized diffusion properties for glucoseand insulin. This study was performed to analyse the function ofislets after encapsulation in differently modified polysulfonecapillaries.Methods: Isolated rat islets were macroencapsulated and insulinsecretion and VEGF release were monitored immediately afterisolation and after cell culture of three days. Expression of VEGF,MCP-1 and IL8 in pig islets was examined during isolation andpurification. Encapsulated islets from rats or pigs were transplantedinto streptozotocin-diabetic rats. The islet function was followed bymeasuring blood glucose concentrations and the host reactiontowards the biomaterial was histologically examined.Results: Out of 4 different poylsulfone capillaries (PSU) only highlyhydroxymethylated PSU was suited since glucose induced insulinsecretion of freshly isolated rat islets was not affected afterencapsulation. AUC (ng/mlx90 min) of free islets was 126.0±21.6compared with 105.0±24.9 for encapsulated islets. However, glucoseinduced insulin secretion of encapsulated islets was almost lost after3 days of cell culture whereas free floating islets remained active. Theloss of glucose responsiveness after encapsulation was paralleled byVEGF release indicating hypoxia in the capillary lumen. Transplan-tation of encapsulated rat islets (4 transplantations) or highly purifiedporcine islets (2 transplantations) lowered blood glucose concentra-tions in diabetic rats over a period of 4 weeks. The outer surface ofthe explanted capillaries filled with islets was covered with bloodvessels whereas transplanted empty capillaries remained free of cellgrowth. By contrast, partly purified pig islet preparations (4 trans-plantations) lowered blood glucose concentrations only transientlyand eventually lost cell function due to a fibrotic capsule formation. Inconclusion, modified PSU capillaries allow prolonged islet functionafter encapsulation. The function of transplanted encapsulated isletsmay depend on the graft specific release of cytokines. With highlypurified islets, a functional model of a vascularized bioartificial en-docrine pancreas can be generated in a diabetic rat model.

68Defined microdisplacement at a bone-implantsurface leads skeletal progenitor cellsinto the chondrogenic lineagePhilipp Leucht1,2, Jae-Beom Kim1, Jennifer Currey3,John B. Brunski3, Jill A. Helms11Department of Plastic and Reconstructive Surgery, StanfordUniversity School of Medicine, Stanford, USA2Department of Trauma, Hand and Reconstructive Surgery, Goethe-University of Frankfurt/Main, Germany

3Department of Biomedical Engineering, Rensselaer PolytechnicInstitute, Troy, USABackground: During healing of both fractures and bone-implantinterfaces, little is known about the cellular/molecular mechanismsregulating cell fate decisions and tissue differentiation in response tosignals from the mechanical environment. Therefore, we developed amurine model to study the interactions between mechanobiology andcell fate during the osseointegration of a surface-characterizedpolymer implant.Methods and results: In a “stable” environment (i.e., in the absenceof an applied mechanical load to the tip of the implant)osseointegration was initiated after five days. We observed bothendochondral and intramembranous ossification in the gap betweenthe cortical drill edge and the implant surface. In contrast, onlyintramembranous ossification was detected between the implantsurface and the bone marrow. Ossification was initiated in both sitesby 7 days post-surgery, and was completed by 28 days post-surgery.By applying a defined motion (i.e., 1 Hz frequency, with a 60 secduration for a total of 7 days that created a displacement of 150 µm)we were able to show a change in the histological pattern of theosseointegration. The process of bone formation was interruptedsuch that the cells interposed between the implant surface and thebone marrow differentiated into fibrocartilage rather than intoosteoblasts. This region is correlated with a region of high-concentrated strain. Immunohistochemical localization of endothelialcells, using a PECAM-1 antibody, showed that in contrast to thestable implant situation, the applied motion resulted in a reduction ofvascular invasion which corresponded to the domain of fibrocarti-lage. By using micro-CT system and image analysis, strain fieldswere also detected. Therefore, we were able to directly compare theconsequences of motion on the molecular and cellular responses ofcells in the implant interface.Conclusions: This model offers the opportunity to direct skeletalprogenitor cells into a chondrogenic lineage by applying controlledmechanical stress. Preliminary data raise hope that by introducingWnt-protein into the differentiated fibrocartilage, transdifferentiationinto bone tissue might take place, which would provide a promisingapproach towards the therapy of implant loosening.

69Src-induced uPAR gene expression is also mediatedvia an AP-1 motif and is associated with increasedinvasive capacity in vivoJ.H. Leupold1, E. Lengyel2, S. Post1, H. Allgayer11Department of Experimental Surgery/Molecular OncologyMannheim, University of Heidelberg, Mannheim, Germany2Department of Gynecology, University of Chicago, Chicago,IL, USABackground: The urokinase receptor (u-PAR) promotes invasionand metastasis and is associated with a poor survival in differentcarcinomas. It is regulated transcriptionally via diverse cis- elements,among them being an AP-1-element at region -190/-171 and acombined AP-2, Sp-1, Sp-3-motif at region -152/-135. Previously wedemonstrated that Src, being overly active in about 80% of colorectalcancers, induces u-PAR gene expression via Sp1 bound to u-PARpromoter region -152/-135. The present study was conducted toexamine as to whether other transcription factors bound to the uPARpromoter act as downstream mediators of Src and to investigate as towhether Src-induced u-PAR gene expression is leading to increasedinvasion in vivo.Experimental design: uPAR promoter wildtype and deletionconstructs were compared for their ability to be regulated by Src inreportergene assays, and the specific binding of transcription factorsto promotor elements was tested by Electromobility shift. Decreaseof invasive capacity by a specific Src tyrosine kinase inhibitor was

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tested by Matrigel and CAM Assays using SW480 colon cancer cellsstably expressing constitutively active Src (Y-c-src527F).Results: A CAT-reporter plasmid driven by 3 tandem repeats of anAP-2-consensus motif was not induced by a constitutively active Srcin SW 480 colon cancer cells. Co-transfection of a dominant negativeAP-2 construct (AP-2αB) did not inhibit Src-inducible u-PARpromoter activity. To elucidate the role of AP-1 (-190/-171), SW480cells were either co-transfected with a CAT-reporter driven by thewildtype u-PAR promoter or a construct deleted for AP-1 binding atregion -190/-171, and Y-c-src527F. Src-stimulation of the AP-1-mutated u-PAR promoter was significantly reduced as compared tothe wildtype. In gelshift analysis, SW480 cells stably transfected withconstitutively active Src demonstrated an increase of AP-1transcription factors (c-Jun, JunD, c-Fos, Fra-1) bound to region –190/-171. SW480 clones stably expressing Y-c-src527F showed anincreased invasive capacity in Matrigel assays and a chorionallan-tois-membrane (CAM)-intravasion model (chicken embryo). Treat-ment of these cells with a Src-specific kinase-inhibitor stronglyreduced their ability to invade in vivo.Conclusion: These data suggest that uPAR gene expression, inaddition to Sp1 bound to promoter region -152/-135, is also mediatedvia AP-1-transcription factors bound to region -190/-171, but not viaAP-2. Furthermore, Src increases invasion of cultured colon cancerin vivo. This extends the hypothesis of Src inhibition as an anti-invasive therapy in colon cancer.

70Construction of tissue-engineered skinby genetically modified human keratinocytesand fibroblasts in a collagen-GAG matrixF. Liu1,2, T. Egana1,3, T. Krengel4, S. Krüger5, W. Lindenmaier6,H.G. Machens11Department of Plastic Surgery and Hand Surgery, Burn Care Center,UKSH, Campus Lübeck, Lübeck, Germany2Institute of Burns, Wuhan Hospital No.3, Wuhan University,PR China3Institute for Pharmaceutical Chemistry University of Santiago deChile, Chile4Department of Dermatology and 5Institute for Pathology, UKSH,Campus Lübeck, Lübeck, Germany6German Research Center for Biotechnology, Braunschweig,GermanyBackground: To observe the biological character of PDGF-BB genemodified human keratinocytes in tissue-engineered skin templatesincluding fibroblasts and collagen-GAG matrix for full-thicknessskin defects.Methods: We isolated human keratinocytes from human foreskin,cultured them in vitro and introduced a gene encoding human PDGF-BB into human keratinocytes using a adenovirus-derived vector(pAdcos bicistronic). Similarily, human fibroblasts were modified toexpress a combination of VEGF165 and bFGF as angiogenic stimuli.Growth curve, cloning efficiency and target gene expression weremeasured in vitro. The gene modified keratinocytes and fibroblastswere seeded into the collagen-GAG matrix to reconstruct the tissues-engineered skin and to observe the growth of seeded cells in vitro fortwo weeks. Two types of skin substitutes were prepared withkeratinocytes and fibroblasts: group A, containing both modifiedkeratinocytes and fibroblasts and group B, containing bothunmodified keratinocytes and fibroblasts. Matrices were transplantedinto full-thickness wounds on athymic nu/nu mice. Wound healingwas evaluated, tissue samples were harvested and examined bymeans of histology, immunohistochemistry and microangiography.The results were compared among the groups.Results: The in vitro results showed successful gene transfection ofboth keratinocytes and fibroblasts with the adenoviral vector by

fluorescent microscope inspection of the cells and ELISA measure-ment of the culture medium. Protein expression lasted at least 1 weekwith a peak at about 3 to 7 days after transfection. Keratinocytes andfibroblasts of group A maintained significantly higher proliferativecapacity in vitro after seeding in collagen-GAG matrix both in vitroand in vivo, compared to group B (P<0.001). Wound healing andangiogenesis was also significantly improved in grafts of group Acompared to the control grafts in group B (P<0.05).Conclusion: PDGF-BB gene transfection can promote the prolifera-tion of human keratinocytes in our model. Our model may become afeasible tool for constructing bioengineered skin substitutes usingcell-based gene therapy.

71Influence of neoadjuvant chemotherapyon liver integrity and ischemic toleranceS. Manekeller1, T. Minor21Klinik und Poliklinik für Allgemein-, Viszeral, Thorax- undGefäßchirurgie and 2Chirurgische Forschung, Universität Bonn,Bonn, GermanyBackground: The increasing interest in neoadjuvant chemotherapyof liver metastasis after colorectal carcinoma prior to resection hasfocused surgical concerns to the influence of oncologic chemother-apy on hepatic integrity and parenchymal tolerance to intraoperativeischemia as produced by Pringle’s maneuver. The present study wasthus undertaken in order to produce first experimental data on liverfunction and morphology after neoadjuvant chemotherapy andsubsequent ischemic challenge in a rat model.Methods:MaleWistar rats (250-300 g) were randomized in 2 groups.One group (CH) received an intraperitoneal chemotherapy compris-ing 5FU (1000 mg/m2), FA (200 mg/m2) und Oxaliplatin (85 mg/m2)on day 1,3 and 5, while the placebo group (PL) was given only salineaccording to the same protocol. On day 6, all animals were subjectedto 30min of total hepatic ischemia induced by Pringle’s maneuver andsubsequent reperfusion for 1h (n=5, resp.) or 24h (n=5, resp.). Duringthe 1h reperfusion experiments, total bile flow was measured as wasmicrocirculatory tissue perfusion after 15 min by means of laserDoppler flowmetry. At the end of the respective observation periods,the animals were sacrificed and serum activities of ALT, AST andGLDHwere determined. Tissue samples were used for standard histo-logical evaluation and quantification of cellular apoptosis by TUNEL.Results: Serum activities of hepatic enzymes rose significantly afterischemia with highest values after 24h, but no differences were seenbetween CH and PL. Moreover, histological examination (H&E stain)or quantification of cellular apoptosis by TUNEL did not reveal anyabnormalities in either group. Bile flow, however, was foundsignificantly reduced in CH prior and after ischemia (0,09± 0,04 vs.0,40±0,14 ml/h, p<0,05) compared with PL, and hepatic tissueperfusion during early reperfusion was also significantly impaired bypreceding Chemotherapy (81±19,49 vs. 133±42,51, p<0,05).Conclusions: Despite induction of altered macroscopic liver appear-ance after chemotherapy, no histological or enzymatical evidence wasfound for an altered ischemic tolerance of these livers with theexception of an only transient suppression of post ischemic tissueperfusion and a, however notable, reduction in bile flow.

72First time assessment of whole blood SOCS1mRNA expression in multiple trauma patientsin respect of clinical outcomeM. Matz, J. Stegmaier, J. Landes, C. Kirchhoff, V. Bogner,W. Mutschler, P. Biberthaler

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Chirurgische Klinik und Poliklinik Innenstadt,Ludwig-Maximilians-University, Munich, GermanyBackground: Inflammation in response to trauma might induceMultiple Organ Failure and death. In this context, Suppressors OfCytokine Signalling (SOCS) act as negative regulators of inflamma-tory cytokine release. Especially, SOCS1 seems to play a critical rolein immune regulation after burn or LPS-induced sepsis in animalmodels. However, in humans the role of SOCS1 remains incom-pletely characterized in multiple injured patients, so far. Hence, theaim of this pilot study was to investigate the dynamic of SOCS1expression in the early posttraumatic period after severe trauma.Patients and methods: 21 multiple injured (ISS>16 points) patientswere included into this study. Whole blood samples were drawnwithin 90 minutes after injury, as well as consecutively 6h, 12h, 24h,48h and 72 hours after trauma. mRNAwas stabilised and isolated bythe PAXgene RNA-System (PreAnalytix), selective SOCS1 mRNAexpression was quantified using RT-PCR (Light Cycler, Roche),respectively. All data are given in [copies/1 ng analysed RNA] andpresented as mean±SEM. Statistic analysis was performed byANOVA on ranks followed by SNK-test.Results: The mean ISS was 42±3 points. The mean initial SOCS1mRNA expression on admission (251±27) was significantlyincreased (p<0.05) as compared to the following time points of ourmeasurements (6h: 196±28, 12h: 152±24, 24h: 182±37, 48h: 157±39, 72h: 79±12; p<0.05). 15 patients survived, 6 died within theposttraumatic period. SOCS1 mRNA expression in deceased patientswas significantly reduced 12h (79±17 vs.182±29; p<0.05), as well as72h (32±20 vs.101±12; p<0.05) after the traumatic event ascompared to the survivors group.Conclusion:Within this pilot study, we demonstrate for the first timequantitative SOCS1 mRNA expression analysis in whole blood sam-ples of multiple injured patients. Interestingly, the suppressor SOCS1expression was decreased in deceased patients as compared to sur-vivors although it is well recognized, that cytokine production is alsolower in patients facing an unfavourable outcome. Further investiga-tions are currently on the way to further illuminate this phenomenon.

73Induction of urokinase-receptor (u-PAR) gene expressionvia Src and two different u-PAR promoter elements:first analysis of in vivo and prognostic relevancein resected colorectal carcinomasG.D. Maurer1, J.H. Leupold1, D.M. Schewe1, T. Biller1,H.M. Hornung2, K.U. Grützner2, U. Lau-Werner2, S. Post1and H. Allgayer11Department of Experimental Surgery/Molecular OncologyMannheim, University of Heidelberg, Mannheim, Germany2Klinikum Grosshadern, LMU, Munich, GermanyBackground: The u-PAR promotes the invasive phenotype and isassociated with a poor prognosis in human cancers. In previousstudies we demonstrated that u-PAR gene expression in culturedcolon cancer is induced by Src, this being mediated by Sp1 bound toan AP-2/Sp1 promoter region (-152/-135). Furthermore, an AP-1-consensus promoter motif (-190/-171) conveys an induction of u-PAR gene expression initiated by mutation-activated K-Ras and alsoSrc.Experimental design: The present study was performed toinvestigate these regulatory mechanisms in vivo by applying kinaseassays, Western blotting, mutation-specific PCR and gel shift assays.In addition, first evidence for a potential clinical-prognosticrelevance should be obtained. In a series of 92 resected patientswith colorectal cancer, endogenous Src activity and expression aswell as K-ras mutations (codons 12/13), and transcription factorbinding to the AP-2/Sp1 and the AP-1-motif of the u-PAR promoterwere analyzed in both tumors and corresponding normal tissues.

Results: High Src protein amounts in tumors correlated significantlywith high binding of Sp1 to the AP-2/Sp1 promoter element(p<0.01). Src-activity was positively associated with transcriptionfactor binding to the AP-1-motif (p<0.01). Interestingly, Src-activityin tumors was significantly lower in the presence of K-ras mutations(p<0.05). Preliminary Kaplan-Meier-analysis (Log Rank, medianfollow-up 16.6 months) showed a trend for a tumor-specific bindingof Sp1, Sp3 or AP-1 transcription factors correlating with poorrecurrence-free survival (p=0.10). The most significant associationwith a poor disease-free survival was found for the combination of ahigh Src protein amount and a strong Sp1-binding to u-PARpromoter motif -152/-135 (p=0.01).Conclusion: This is the first study to demonstrate an in vivorelevance of different regulation pathways of the u-PAR gene in aseries of resected colorectal tumors and corresponding normaltissues. It suggests that clinical prognosis in colorectal cancer isinfluenced by an activation of these molecular regulators. Detailedanalysis of suchlike regulators will help to define more preciselyprognostic subgroups which can be appropriate candidates for anindividualized molecular targeting.

74The application of norepinephrine reduces 6-hourmortality at the critical hemoglobin concentrationJens Meier, Andreas Pape, Daria Loniewska, Patrick Lauscher,Harry Kertscho, Bernhard Zwißler, Oliver HablerClinic of Anesthesiology, Intensive Care Medicine, and Pain Control,Johann Wolfgang Goethe–University, Frankfurt am Main, GermanyBackground: Life-threatening normovolemic anemia is character-ized by a disparity of oxygen demand and oxygen delivery, and bycirculatory failure due to distinct hypotension. The aim of the presentstudy was to investigate, whether the application of norepinephrineas sole treatment could restore tissue oxygenation and prevent deathof otherwise lethal normovolemic anemia by stabilizing coronaryperfusion pressure.Material and methods: In 14 anesthetized domestic pigs (28.9±3.1kg) extreme anemia was induced by simultaneous exchange of wholeblood with hydroxyethyl starch (Voluven, 6%-HES, MW 130.000/0.4) until the individual critical hemoglobin concentration (Hbcrit) ofeach animal was reached. Hbcrit was defined as a significant decreaseof total body VO2 as compared to the baseline value. At Hbcrit theanimals were randomized to two experimental groups: seven animalswere observed during a 6-hour observation period without anyfurther intervention (control group: CG), while in the other 7 animalsdiastolic aortic pressure was raised to baseline values by continuousinfusion of norepinephrine during the 6-hour observation period(verum group: NOR).Results:All animals of CG died within the 6-hour observation period(i.e. 6 hrs-mortality 100%). In contrast 6 of the 7 animals of NORsurvived (p<0.05; CG cs. NOR). Parameters of macrohemodynamics(mean arterial pressure, cardiac index) and oxygen transport (oxygendelivery, oxygen consumption) improved significantly after initiationof the norepinephrine infusion, whereas parameters of peripheraltissue oxygenation (tissue oxygen partial pressure of skeletal muscle,arterial lactate concentration, arterial base excess) only slightlyimproved after initiation of the norepinephrine infusion. However, allfavourable effects of norepinephrine infusion dimished throughoutthe 6 hour-observation period.Conclusion: The application of norepinephrine as the soletherapeutic modality enables survival during otherwise lethal, acuteanemia. However, this increase of survival is accompanied by aredistribution of nutritive organ blood flow, limiting the benefit ofnorepinephrine to organs of central circulation for a short period oftime. Long term treatment of extreme anemia must therefore includefurther measures aiming at the increase of arterial oxygen content andperipheral oxygen delivery.

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75Cerebrospinal fluid interleukin-1 receptor antagonistfollowing severe traumatic brain injuryT. Mueller, C. Kirchhoff, J. Stegmaier, V. Bogner, K.G. Kanz,W. Mutschler, P. BiberthalerChirurgische Klinik und Poliklinik-Innenstadt,Ludwig-Maximilians-Universität, München, GermanyBackground: Severe traumatic brain injury (TBI) induces inflam-matory reactions with consequent secondary brain damage. Asrecently shown, a major issue of this inflammation is astrocytic andmicroglial activation and production of the cytokine interleukin-1(IL-1). The physiologic inhibitor of IL-1 and its proinflammatoryleukocyte attracting impact is the IL-1 receptor antagonist (IL-1ra),mainly synthesized by CD14+monocytes (MØ). Recently a sig-nificant correlation between IL1-ra levels and clinical outcome ofpatients suffering from subarachnoid haemorrhage (SAH) wasreported. However the role of IL1-ra in patients suffering from TBIstill remains uncharacterized. Therefore, the aim of our study was toanalyze the intrathecal dynamics of IL-1ra in respect to CD14+ MØin the early phase after TBI.Patients and methods:We enrolled 8 patients, suffering from severeTBI (initial GCS<8pts) and an intracranial lesion. After placement ofan external ventricular drainage (45±30 min after trauma) as well as12, 24, 48 and 72hrs post trauma CSF samples were drawn. ICP wasmonitored at every sampling point. CSF of 8 healthy patientsconceiving spinal anaesthesia, served as control group. For analysisof CD14+ MØ, CSF was stained using anti-CD14-TC (Caltag,Hamburg) and quantified using flow cytometry. Data are given aspercentage value relating to the total CSF cell population. For detec-tion of IL-1ra we used an ELISA technique (R&D Systems, Minne-apolis, USA). Statistics were performed using the ANOVA followedby SNK-test vs. Baseline, and Mann-Whitney-U vs. control.Results: The values of IL-1ra in the CSF were significantly increasedat every sampling point 569±151.1 pg/ml (Mean±SEM) in respect tothe control group 44.5±6.9 pg/ml (Mean±SEM). No significantincrease of IL-1ra over time was observed within the first 72hrs posttrauma. CD14+ MØ were continuously elevated over 48hrs with asignificant increase of 3.2±0.8 (Mean±SEM)72hrs post traumaaccording to values at admission1.1±0.4 (Mean±SEM) as well asto the control group values 1±0.8 (Mean±SEM).Conclusion: In this study we were able to demonstrate significantlyincreased levels of Interleukin-1 receptor antagonist (IL1-ra) in CSFof patients suffering from TBI directly after admission. In contrast, anincrease of IL1-ra in patients with SAH is described starting thefourth day after haemorrhage. However, we also showed a significantrise of CD14+ MØ, the main source of IL1-ra 72hrs post trauma.Further studies need to be done to elucidate possible intrathecalsources of IL1-ra.

76Cancer pathway analysis: new tools for explorativeanalysis of c-DNA microarraysJ. Neff1, M. Niedergethmann1, F. Alves2, B. Heidrich1,F. Willeke1, S. Post1, N. Gretz31Chirurgische Universitätsklinik Mannheim, UniversitätsklinikumMannheim, Mannheim, Germany2Hämatologie und Onkologie, Universitätsklinikum Göttingen,Göttingen, Germany3Zentrum für Medizinische Forschung, UniversitätsklinikumMannheim, Mannheim, GermanyBackground: Using cDNA - microarrays it is possible to identifyderegulated genes in the primary tumor and its metastases. Rightnow, the extend of data is too complex and there is no possibility forfast analysis which can estimate the gained data. The new establishedGene Set Enrichment analysis and Ingenuity analysis should afford

to assign differential expressed genes of cDNA-arrays to specificpathways.Materials and methods: In our orthotopic model, human MiaPaca-cells are implanted in SCID (Severe-combined immune deficien-cy)-mice. RNA is gained of the dissected primary tumor, thetumorinvasion to the duodenum and liver metastases. After con-verting RNA into cDNA and the hybridization on HG-U133A arraythe data is analyzed with ANOVA and differentially expressed genesare validated with external data bases (NCBI: PubMed, LokuslLink,Unigene, Swissprot, Geneontology). Finally, the pathway analysisis performed by Ingenuity and Gene Set Enrichment. The Ingenuityanalysis is based on focus genes, according to genes on the array,whereas the Gene Set Enrichment analysis calculates the sig-nificance of specific pathways with Fisher’s Exact Test by com-paring the genes of the pathways with the significant genes of thechip.Results: The Gene Set Enrichment Analysis revealed 11 significantpathways for tumor invasion and metastases (for example TGF-β).The Ingenuity-Analysis identified 6 significant regulated pathwayswhich consist of cell adhesion and tumor specific migration (forexample Integrin). All data could be validated by current externaldata bases.Conclusion: Pathway analyses using Ingenuity and Gene SetEnrichment provide the facility to gain fast and statistically specificanalysis of the microarray data. These techniques may detectrelevant, pathway-related groups of genes concerning invasion andmetastases and may relief the search of candidate genes.

77Prediction of metastases and local tumor invasionin pancreatic cancer using an orthotopic SCID mousemodelM. Niedergethmann1, F. Alves2, B. Heidrich1, J. Neff1, C. Pilarsky3,R. Grützmann3, F. Willeke1, S. Post1, N. Gretz41Chirurgische Universitätsklinik, Universitätsklinikum Mannheim,Mannheim, Germany2Hämatologie und Onkologie, Universitätsklinikum Göttingen,Göttingen, Germany3Klinik für Viszeral-, Thorax- und Gefäßchirurgie,Universitätsklinikum Dresden, Dresden, Germany4Zentrum für Medizinische Forschung, UniversitätsklinikumMannheim, Mannheim, GermanyBackground: Genexpression profiling in pancreatic cancer iscomplicated by the high amount of RNAses in human tissue andsuitable models. In order to reflect early metastasizing, modelsshould be constructed with respect to the anatomical environment.Using the orthotopic pancreatic tumor SCID mouse model theseinteractions are taken into account. In order to identify genes asso-ciated with local tumor invasion and metastases in ductal pancreaticcancer we investigated pancreatic tumor cell lines derived from anorthotopic pancreatic tumor model in SCID mice. Differential geneexpression was performed based on cDNA microarray technique.Material and methods: Human MiaPaca cell lines were orthotopi-cally implanted in SCID mice. Transcriptional profiling (Affymetrix,HGU133) was performed with tissue derived from the primarytumor, duodenal invasion, and liver metastases. Differentiallyexpressed genes were identified after statistical analysis (ANOVA)and validated with external data bases (NCBI: PubMed, LokuslLink,Unigene, Swissprot, Geneontology).Results: Of 12024 genes investigated, 51 (0,424%) derived fromliver metastases, and 15 (0,125%) derived from the duodenal tumorinvasion were deregulated with respect to the primary tumor. Com-paring current data bases we validated 18 genes being differentiallyexpressed. Up-regulation was archieved of e.g. RAB17, a member ofthe RAS oncogene family, derived from metastatic/invasion lesions,

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whereas e.g. tumor suppressor genes such tumor protein p73-like, orserine proteinase inhibitor were down-regulated.Conclusion: Using transcriptional profiling in the SCID mousemodel marker genes for local invasion and liver metastases can beidentified. These marker genes may predict early metastasizing inductal pancreatic cancer.

78Therapy of ductal pancreatic cancer in an immuno-competent rat model: suramin inhibits tumorgrowth and metastasis almost completelySusanne D. Otto, Birgit Hotz, Heinz J. Buhr, Hubert G. HotzDepartment of Surgery I, Charité Medical School,Campus Benjamin Franklin, Berlin, GermanyBackground: A previous study demonstrated that Suramin, a naph-thyl urea derivative, reduces the proliferation of human pancreascancer cells in vitro. This was accompanied by reduced secretion ofthe proangiogenic key factor VEGF. The aim of the present study wasto systematically evaluate the effect of Suramin on tumor growth,metastasis and angiogenesis in vivo using a clinically relevantorthotopic immuno-competent rat model of ductal pancreatic cancer.Methods: 107 cells of the rat ductal pancreatic cancer cell line DSL-6A/C1 were subcutaneously injected into 2 Lewis rats. 1 cmmfragments of the resulting subcutaneous donor tumors wereorthotopically implanted into the pancreas of 16 other Lewis rats.The animals were randomized into 2 groups: the therapy group(Suramin 60 mg/kg, weekly ip.) and the control-group (vehicle ip.).Intravital microscopy was performed 4 weeks after tumor induction.The volume of the primary tumor and local infiltration, as well assystemic metastasis (score) and microvascular density were deter-mined at autopsy.Results: The animals of the control group developed large primarytumors (1978,5±231,2 cmm) and metastasis (9,0±1,1 pts.). Incontrast, half of the animals in the therapy group displayed notumor growth, the other half showed only small tumors (4,1±1,9cmm; p<0,05) without distant spread. Control animals showedtypical tumor vessels with a vessel density of 421±39 cm/scm,whereas vessel density of primary tumors in treated animals wasreduced to 266±31 cm/scm (p<0,05). The weight of the animals wassimilar in both groups (controls: 265±12 g; therapy: 259±16 g).Conclusion: Sumarin reduces primary tumor growth, dissemination,and angiogenesis in the early stage of tumor development in animmuno-competent rat model of ductal pancreatic cancer.

79Role of Foxp3 in pancreatic adenocarcinoma cell lines: animmuno-suppressive mimickry of regulatory T-cells?Laia Pagerols Raluy1, Sebastian Hinz1, Ole Ammerpohl1,Christian Röder1, Heiner Oberg2, Daniela Wesch2, Dieter Kabelitz2,Robert Grützmann3, Christian Pilarsky3, Hans Konrad Schackert4,Hans Detlev Saeger3 , Günter Klöppel5, Bence Sipos5,Hendrik Ungefroren1, Bernd Kremer1, Holger Kalthoff11Clinic of General Surgery and Thoracic Surgery, UniversityHospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany2Institute of Immunology, University Hospital of Schleswig-Holstein, Campus Kiel, Kiel, Germany3Department of Visceral, Thoracic, and Vascular Surgery, UniversityHospital Carl Gustav Carus, Technical University of Dresden,Dresden, Germany4Department of Surgical Research, University Hospital Carl GustavCarus, Technical University of Dresden, Dresden, Germany5Department of Pathology, University Hospitalof Schleswig-Holstein, Campus Kiel, Kiel, Germany

Background: Pancreatic cancer, despite all efforts in developingnew adjuvant therapies and improving surgical techniques, remainsone of the most aggressive malignancies with a five year survival ratebetween 3-8%. For the development of more effective therapies,particularly immunotherapies, the local and systemic immune escapemechanisms in pancreatic cancer need to be further explored. Recentreports suggested an important role for regulatory T-cells (Treg) inthe immune regulation in different malignant tumours. A highernumber of Treg was detected in the peripheral blood of pancreaticand breast cancer patients. Furthermore, it could be shown in ovariancancer patients, that Treg accumulate in the vicinity of the tumourand the amount of Treg negatively correlated with patient survival.The key proteins that confer a regulatory phenotype to Treg are stillpoorly defined. Foxp3, a member of the forkhead family oftranscription factors is highly expressed in CD4+ CD25+ T-cellsand linked to the regulatory function of CD4+ CD25+ T-cells, but thetarget genes that are (negatively) regulated by Foxp3 remain elusive.Patients and methods: Cryosections from tissues (n=10 for normal;n=20 for malignant samples) were tested by immunohistochemistry.FoxP3 mRNA expression was also analyzed in tissue samples. RNAwas isolated from Panc1, PancTUI, Panc89, Capan1, Colo357, Kif5fibroblasts, TCD4+CD25-,TCD4+CD25- and anti CD3 and anti CD28stimulated TCD4+CD25-. Real Time PCR was carried for FoxP3 anddifferent cytokines and FoxP3 mRNA levels were downregulated byspecific siRNAs. FoxP3 protein levels were also analyzed byWesternBlot in cell lines stimulated with TNF α as well as TGF β1 and TGF β2with or without their corresponding neutralising antibodies. Further-more, we carried out co-culture experiments of different pancreatictumour cell lines with naïve Tcells and measured their proliferation rate.Results: Real Time PCR analysis revealed a significant increase ofFoxP3 mRNA in nearly 60% of the tumour specimens, whereas theremaining 40% showed no or little FoxP3-specific transcripts. Foxp3RNA expression was positive in all five pancreatic cell lines, butlower than in the three T cells populations. Kif5 fibroblasts werenearly negative. Remarkably, at the protein level we observed a strongand comparable positivity between pancreatic tumour cell lines and T-cells, against with fibroblasts being only marginally positive. In orderto elucidate the role of FoxP3 in pancreatic tumour cells we appliedspecific siRNAs resulting in a clear Knock-down of FoxP3 mRNAafter 48h followed by a dramatic decrease of FoxP3 protein after 72h.The specific knock-down of FoxP3 in Panc89 cells resulted in anincrease of some specific Interleukines and modulation of othercytokines. Incubation of cell lines with TNF α , TGF β1 and TGF β2resulted in an increase in FoxP3 protein expression . Whereas TNF αmediates a discrete and early (10 min) increase of FoxP3 proteinexpression, both TGF β1 and TGF β2 exhibited a delayed and morerobust effect. Co-culture of tumour cells with naïve T cells showed aninhibition on the proliferation of the Tcells following stimulation withanti CD3 and anti CD28.Conclusions: Our striking finding of the expression of FoxP3 inmalignant epithelial cells support the novel concept that pancreaticcancer cells mimic the effects of Tregs and are able to regulate thefunction of the immune system. This adoption of specific regulatoryfunctions of Tregs by pancreatic tumour cells may prevent tumourcells from being targeted by CTL.

80Hyperoxic ventilation increases the tolerance of acutenormovolemic anemia in anesthetised pigsAndreas Pape, Jens Meier, Harry Kertscho, Max Steche,Mohammed Laout, Frank Schwerdel, Malte Wedel,Bernhard Zwissler, Oliver HablerIntensive Care Medicine and Pain Management,Clinic of Anesthesiology, Johann Wolfgang Goethe-University,Frankfurt/Main, Germany

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Background: Hyperoxic ventilation (HV, ventilation with supra-normal FiO2) decreases the mortality of otherwise lethal normovo-lemic anemia when established as a “rescue therapy” at criticalhemoglobin concentrations. Up to date, it has not investigatedwhether (1) “prophylactic”HV (i.e. HV during the induction of acutenormovolemic anemia) has an impact on the maximally tolerableextent of anemia and (2) whether this effect can already be achievedwith FiO2 < 1.0.Methods: 14 anesthetized pigs were randomly ventilated with eitherFiO2 0.21 (G 0.21, n=7) or FiO2 0.6 (G 0.6, n=7). Acutenormovolemic anemia was induced by exchange of blood for 6%hydroxyethyl starch 130/0.4. The maximally tolerable extent of acuteanemia (primary endpoint) was defined as the onset of O2-supply-dependency of total body O2-consumption VO2 with the correspond-ing hemoglobin-concentration (Hb) being defined as “critical”(Hbcrit). Secondary endpoints were changes in myocardial function,central hemodynamics, O2-transport and tissue-oxygenation.Results: HV with FiO2 0.6 enabled a higher fractional exchange ofcirculating blood volume (138.8±15.9% vs. 87.3±12.9%, p<0.05),resulting in a significantly lower Hbcrit (1.5±0.4 vs. 2.4±0.4 g/dL). AtHb 2.4 g/dL (i.e. Hbcrit in G 0.21), animals of G 0.6 covered 39.8±8.5% of their O2-demand by utilisation of O2 physically dissolved inplasma (vs. G 0.21: 14.7±5.0%) and their condition was still superiorregarding (1) coronary perfusion pressure, (2) left ventricular systolicand diastolic function (dp/dtmax, dp/dtmin) and (3) O2-transport andtissue-oxygenation (VO2, mixed-venous pO2, lactate concentration).Compared with G 0.21, the condition of animals in G 0.6 was de-teriorated more severely at their individual Hbcrit (i.e. Hb 1.5 g/dL).Conclusion: During cell-free volume-replacement, HV with FiO20.6 generates a readily utilizable plasmatic O2-reserve and therebyincreases the tolerance of acute normovolemic anemia.

81Functional high throughput characterizationof differentially expressed genes of pancreaticductal adenocarcinomaChristian Pilarsky1, Kerstin Korn2, Ole Ammerpohl3,Holger Kalthoff3, Günter Klöppel4, Hans Konrad Schackert5,Eberhard Krauß2, Marino Zerial2, Hans Detlev Saeger1,Robert Grützmann11Department of Surgery, University Hospital Dresden,Dresden, Germany2Max-Planck Institute for Cell Biology and Genetics,Dresden, Germany3Department of Molecular Oncology, University ofSchleswig-Holstein, Kiel, Germany4Institute of Pathology, University of Schleswig-Holstein,Kiel, Germany5Department of Surgical Research, University HospitalDresden, Dresden, GermanyBackground: Prognosis for patients with pancreatic carcinoma(PDAC) remains poor. Despite of increasing knowledge about themolecular basis of PDAC neither a specific marker for earlydiagnosis nor a target protein for a new therapeutic approach havebeen identified so far. In recent years gene expression profilingexperiments have identified a large number of candidate genes fordiagnosis and therapy. However, given the large number ofdifferentially expressed genes in PDAC the functional characteriza-tion of those genes in a one by one approach is time consuming andbiases further analysis to genes with at least some existent knowl-edge. With the development of the siRNA technology new ap-proaches for functional characterization in large scale have emerged.Methods: In the last years we were able to characterize the geneexpression of PDAC by different methods. All identified candidategenes were assembled in one file and we randomly selected 140 genes

for functional high throughput characterization. For these genescDNA clones were obtained from the RZPD and sequenced. We wereable to confirm the sequence of 125 genes. From 103 of these wesuccessfully generated dsRNA by in vitro transcription using the T7Megascript kit from Ambion. The dsRNA was digested using therecombinant Dicer enzyme from Stratagene. After purification ofthe digested RNA by a combination of gel- and ultrafiltration theresulting dsiRNA was analyzed using PAGE. The amount was cal-culated based on the ethidium bromide staining intensity and com-pared to a chemical synthesized siRNA with known concentration.The dsiRNAwas transfected into 4.000 cells of either Panc89 (30 ngdsiRNA/well) or MiaPaca-2 (50 ng dsiRNA/well) cells in 96 wellformat using oligofectamine. Growth inhibition of the investigateddsiRNA was analyzed via nuclear staining using DraqV and induc-tion of apoptosis was identified using an Annexin Vassay. All assayswere performed in triplicate.Results: Of the transfection of 103 generated dsiRNA molecules 20resulted in a growth inhibition in both assays and in both of the celllines analyzed, whereas in 30 no growth inhibition could be observedin any assay or cell line. Within these 20 genes are three (15%) geneswithout a known functional characterization. These three genesmight have been un-analyzed by conventional techniques and couldlead to new insights in the development of this dismal disease.Conclusion: High throughput functional characterization is a newand useful tool for the further investigation of gene lists resultingfrom gene expression profiling experiments. It will lead to a betterunderstanding of the development of cancer and it will provide newtargets for new therapeutic approaches.

82Functional analysis of in vivo formed heterotopic liverneo-tissue after matrix based hepatocyte transplantationusing confocal laser-scanning microscopyJörg-Matthias Pollok, Marc Luetgehetmann, Sándor Paku,Éva Török, Peter X. Ma, Maura Dandri, Jörg Petersen,Péter Nagy, Xavier RogiersDepartment of Hepatobiliary Surgery and Visceral Transplantation,University Medical Center Hamburg-Eppendorf, GermanyDepartment of Biologic and Materials Sciences, University ofMichigan, Ann Arbor, MI, USADepartment of Internal Medicine I, University Medical CenterHamburg-Eppendorf, Hamburg, GermanyFirst Department of Pathology and Experimental Cancer Research,Semmelweis University, Budapest, HungaryJoint Research Organisation Hungarian Academy of Science andSemmelweis University, Budapest, HungaryBackground: In previous studies we could demonstrate that matrixbased hepatocyte transplantation leads to heterotopic liver neo-tissue,which shows strong proliferative activity and increases dramaticallyin size over the course of 24 weeks in vivo. The liver cells wereglycogen, CK18 and HepPar 1 positive and displayed liver like Actindistribution with formation of a bile canaliculi network. In this studywe were especially interested in cell polarity and function of theheterotopic liver neo-tissue.Methods: Primary rat hepatocytes were isolated using a two stepcollagenase digestion. PLLA polymers were seeded with 4x106hepatocytes, pre-cultured in a flow-bioreactor and transplanted ontothe small bowel mesentery and explanted after 1, 4, 12 and 24 weeks.The samples were marked for the following primary antibodies forfurther analysis using confocal laser-scanning microscopy: Pan-Cytokeratin, Actin, Laminin, HNF-4α, α1-Integrin, CD26, Connexin32, Claudin, and β-Catenin.Results: Via proliferation of the hepatocytes during the time course,stretched areas of liver neo-tissue were formed already after 4 weeksin vivo. Laminin-staining showed multiple new blood vessels within

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and around the heterotopic liver tissue. Among them no sinusoidalendothelial cells could be identified, as demonstrated by negative α1-Integrin staining. The heterotopic hepatocytes displayed a liver likecytoskeleton, as shown by Pan-Cytokeratin and Actin staining. Thecell areas were surrounded by a newly formed thin Laminin mem-brane. The hepatocyte polarity markers CD26 und β-Catenin showeda liver typical distribution signal. The hepatocytes were negative forthe transcription factor HNF-4α, for Connexin 32 and Claudin.Conclusions: Rat hepatocytes, heterotopically transplanted on PLLApolymer matrix, showed an impressive proliferative activity. Theliver neo-tissue increases in size and is nourished by multiple newlyformed small blood vessels. The cells forming the heterotopic liverneo-tissue display, next to the previously found specific markers,positive signals for a liver typical cytoskeleton by Cytokeratin andActin staining. The cells display intact liver specific polarity, asdemonstrated by the markers CD26 and β-Catenin. The negativity forClaudin though may be a sign for immature formation of tightjunctions. The negativity for HNF-4α and for Connexin 32 could beattributed to the fact, that the transplanted liver cells can not gainfully differentiated function in their heterotopic location. Possibleexplanations are the maintained high proliferative activity, or aprocess of retro-differentiation, since the heterotopic hepatocytes,like their progenitor cells (oval cells) are surrounded by a newlyformed Laminin membrane.

83Evaluation of the physical properties of a new fullydegradable suture material with a shapememory effect for visceral surgeryC. Reißfelder1, S. Kelch2, J.P. Ritz1, K. Kratz2, A. Lendlein2,H.-J. Buhr11Department of Surgery I, Charité - Universitätsmedizin Berlin,Campus Benjamin Franklin, Berlin, Germany2GKSS Research Center Geesthacht GmbH, Institute of Chemistry,Teltow, GermanyIntroduction: The insufficiency rate of colorectal anastomosesdecisively influences the results and prognosis of colonic operations.The suturing and knotting technique is a major risk factor due to thepossible induction of microcirculatory disturbances and/or inade-quate adaptation Novel suture materials with a shape memory effectare potentially able to offset such risk factors by readaptation (self-knotting). Suitable new polymer-based suture materials wereevaluated for their mechanical and shape memory properties andcompared with conventional suture materials.Materials and methods: Linear multiblock copolymers were chosenfor the shape memory material. Apart from the crystallizing oligo(p-dioxanone)diol for the hard segment, oligo(ς-caprolactone)diol wasused as the precursor for the switch segments. The shape memorymaterial was programmed for 40% shortening. The physicalproperties (shape memory effect with temperature elevation) of theshape memory polymers were examined in various solutions (0.9%NaCl, blood, air) at 38° and 45° C and compared with conventionalsuture material. To measure the bursting strength, an end-to-endanastomosis was created with interrupted sutures in fresh pig intes-tine. Three groups were formed: colonic anastomosis with 3-0 Vicyl®,

3-0 PDS® and 3-0 shape memory material (5 anastomoses each). Thebursting strength of the anastomosis was recorded by a Millar-Tip®catheter before and after heating the anastomosis and defined as thevalue measured on appearance of the first drop.Results:Compared with room temperature, rising temperatures led toshortening of the shape memory material in all solutions. Addition ofphysiological saline or heparinized rat blood increased the sutureshortening. The two solutions did not differ. There was no reduction inthe length of conventional suture material (Vicryl®, PDS®). The threedifferent suture material did not differ in their bursting strength. –aftera temperature rise in the anastomosis region, the bursting strength ofthe shape memory material could be increased to 46.8 mmHg. Nochanges were found in the bursting strength with Vicryl®, PDS®.Conclusion: 1. The newly developed shapememorymaterial shows asignificant length reduction under physiological conditions and aftertemperature elevation. 2. The bursting strength of the anastomoticregion was highest when shape memory suture material was used. 3.This novel suture material is potentially able to ensure temporarilyjuxtapositioned self-knotting in the interval (tightening effect) and tothus possibly improve the suture insufficiency rate.

84Role of the chemokine receptor CXCR4in prostate tumor malignancyBorna Relja, Wolf-Dietrich Beecken, Tobias Engl,Dietger Jonas, Elsie Oppermann, Roman BlahetaZentrum der Chirurgie, Klinik für Urologie und Kinderurologie,J.W. Goethe-Universitätsklinik, Frankfurt/Main, GermanyBackground: The CXC chemokines constitute a subgroup of thechemokine superfamily and are involved in proinflammatory,chemotactic and tumor growth regulating processes. There is agrowing evidence that the chemokine stromal-cell-derived factor-1α(SDF-1α) and its receptor CXCR4 play an important role in a varietyof cancers. However, the relevance of this receptor in prostate canceris still not fully understood. In this study, we investigated the role ofCXCR4 receptors in prostate cancer cell lines DU145 and LNCaP.Methods: Flow cytometry and confocal laser scanning microscopyhas been used to evaluate CXCR4 surface expression level andcytosolic CXCR4 accumulation. Protein and mRNA expression weredetermined by Western Blot technique and RT-PCR, respectively.Furthermore, chemotaxis and adhesion studies were carried out usingtumor cells pretreated with siRNA against CXCR4 receptors or withan anti CXCR4 blocking antibody. This was necessary to investigatethe functional relevance of CXCR4 in prostate cancer malignancy.Results: Both DU145 and LNCaP cell lines expressed low levels ofCXCR4 receptors on the cell surface, but high amounts of CXCR4proteins were detected intracellularly. Using RNA interference(RNAi) we demonstrated the functional relevance of CXCR4 inSDF-1α-chemotaxis and adhesion assays. Further experimentsshowed that CXCR4 might act as a triggering element which isinvolved in integrin mediated tumor cell attachment. p38 proteinphosphorylation became downregulated in presence of SDF-1α.Conclusion: The data provide a critical role of CXCR4/SDF-1αinteraction in the progression of prostate cancer. We postulate thatbinding of SDF-1α to CXCR4 might activate integrin molecules

Shortening at Shortening at Shortening at Shortening at Shortening at Shortening at Bursting strength38°C + air (%) 45°C + air (%) 38°C + 0.9% 45°C + 0.9% 38°C + 45°C + (mmHg) at 20°C

NaCl (%) NaCl (%) blood (%) blood (%)

Shape 13.7±1.92 33,3±2.72 26.7±2,72 37.7±0.15 26.5±0.25 38.1±1.33 45.7±3.1memoryPDS® 0 0 0 0 0 0 45.8±2,8Vicryl® 0 0 0 0 0 0 46.1±2,7

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responsible for tumor cell-endothelial cell and/or tumor cell-extracellular matrix interaction. Presumably, p38 could be involvedin this process. From a clinical point of view, CXCR4 might providea novel candidate for future therapeutic regimen.

85The function of VEGF (vascular endothelial growth factor)and the importance of S100-Staining for realneovascularization as a cause of recurrentvaricose veins in the groinS. Rewerk1, T. Riester1, M. Winkler2, H. Nüllen31Department of Vascular Surgery, University Hospital of Mannheim,Mannheim, Germany2Practice of Vascular Surgery, Nürnberg, Germany3Practice of Vascular Surgery, Mönchengladbach, GermanyBackground: 1:VEGF is known as a potent mitogen inducingangiogenesis. On the other hand it is still unclear wether VEGF isinvolved in the development of recurrent varicose veins aftercrossectomie. 2: S100 is a protein for staining nerve fibres. There isonly one investigation (1) describing the absence of S100-staining inthe wall of veins as a proof of real neovascularization. This trial isused worldwide as “golden-standard”. That is not very convincingbecause many factors are known to be involved in regeneration ofmany different types of tissues. Why should nerve fibres notregenerate in real neovascularizationes? The aim of this study wasto investigate the potential function of VEGF in the course ofdeveloping recurrent varicose veins. Furthermore this study wantsto reexamine the importance of S100-staining in vein-walls.Patients, material, methods: After approving this trial by the ethicscommission (University Hospital of Mannheim) two groups werebuilt with consecutive patients. Group1: Incompetent great saphe-nous vein, n=91. Group2: Recurrent varicose veins in the groin,n=15. Both groups were operated on and venous specimen weretaken from the sapheno-femoral-junction (group1) or from venousvessels as near as possible to their junction with the deep vein(group2). Subsequently these specimen were stained histochemicallyfor semiquantitative evaluation of intimal VEGF and S100.Results:VEGF: group1: 1,88±0,77; group2: 3,26±0,88 p<0,005S100: group1: 1,77±0,56; group2: 1,82±0,6 n.s.Conclusion: 1. S100-staining appears in all types of varicose veins,even in real venous neovascularization. Therefore S100-stainingcannot distinguish between real venous neovascularization and var-icose veins which were not removed in former surgical procedures. 2.VEGF is involved in real neovascularization perhaps as a main reasonof recurrent varicose veins developing after correct performed sur-gical procedures. Further investigations to clear up the function of theVEGF-receptor and NGF (nerve growth factor) have already begun.

86Time-lapse digital-based intravital microscopy:an improved technology to assess leukocyte migrationin the intra- and extravascular spaceE. Ryschich, P. Lizdenis, V. Kerkadze, H.P. Knaebel,W. Gross1, M.M. Gebhard1, M.W. Büchler, J. SchmidtDepartment of Surgery and 1Experimental Surgery, University ofHeidelberg, Heidelberg, GermanyBackground: The ability of active movement is an important featureof leukocytes. Here, we evaluated the technique which combinesintravital microscopy and time-lapse video documentation toinvestigate the kinetics of leukocyte movement in vivo.

Methods: Intravital microscopy of mesenterial tissue was performedin 30 male Lewis-rats using digital imaging and time-lapse videocompression. Leukocyte transmigration, migration velocity and thenumber of migrating leukocytes were measured over 60 min. Thelocal effect of LPS and TNF-α on intravascular, transendothelial andextravascular leukocyte movement was investigated.Results: Only few leukocytes transmigrated through the endothelialwall under unstimulated conditions. The leukocyte transmigrationwas significantly increased after stimulation with LPS and TNF-αThe mean time of transmigration was 128±9 sec (stimulation withLPS) and 115±32 sec (stimulation with TNF-α). Baseline measure-ment showed a mean velocity of the extravascular leukocytes of9.9±1.1 µm/min and a mean percentage of migrating cells of 83.7±3.3%. Local concentration of LPS up to 20 µg/ml and con-centration of TNF-α up to 50 ng/ml significantly activated theleukocyte locomotion in a dose-dependent manner, whereas higherconcentrations significantly decreased the migratory activity ofextravascular leukocytes. Leukocyte labelling with rhodamine 6Gand light caused a strong phototoxic reaction resulted in anirreversible blockage of leukocyte locomotion.Conclusions: The method of time-lapse digitally-based intravitalmicroscopy represents a promising technology for the investigationof intravascular, transendothelial and extravascular migration ofleukocytes. The activating or inhibiting action of LPS and TNF-αon intravascular, transendothelial and extravascular leukocytemigration strongly depends on the concentration of the substance.The inhibition of leukocyte locomotion with high concentrations ofLPS and TNF-α can represent a relevant immunosuppressive factorin sepsis and infections.

87Do adherent leukocytes move in vivo?E. Ryschich, V. Kerkadze, H.P. Knaebel, W. Gross1,M. M. Gebhard1, M. W. Büchler, J. SchmidtDepartment of Surgery and 1Experimental Surgery, Universityof Heidelberg, Heidelberg, GermanyIntroduction and aim: Leukocyte-endothelial-interactions are aprerequisite for the leukocytes recruitment during acute inflamma-tion. We utilized time-lapse intravital microscopy and digital-basedvideo processing to investigate leukocyte adhesion and migration.Methods: Intravital microscopy of mesenteric venules wasperformed in 11 male Wistar-rats using digital video recording andtime-lapse image compression. Leukocyte-endothelial-interactionsand extravasation of leukocytes were recorded over 90 min. Adherentleukocytes were divided into rollers (adhesion for less than 1 sec),transient stickers (adhesion for 1 to 30 sec), and permanent stickers(adhesion for more than 30 sec).Results: Most permanent stickers (84±13%) moved (crawled)actively on the intraluminal site of venules. Baseline measurementof leukocyte crawling velocity yielded an average 9.0±4.5 µm/min(mean±SD) which was not significantly different from crawlingvelocity of extravascular leukocytes (8.9±4.5 µm/min). Intraluminalcrawlers traveled over a mean distance of 35±17 µm with the aver-age duration of 4.5±2.3 min. The maximum distance of leukocytecrawling observed was 150 µm. The maximum time of crawlingwas 15 min. Under unstimulated conditions, crawling leukocytesdetached from the endothelium and did not migrate through thevascular wall. All investigated parameters did not significantlychange over a period of 90 min.Conclusions: Leukocyte-endothelial-interactions are an active anddynamic process under unstimulated conditions. This processinvolves long-time (several minutes) interactions of leukocytes withthe endothelium, intraluminal leukocyte migration and, finally,detachment from the endothelium. The exact mechanisms andbiological relevance of this finding should be clarified in furtherstudies.

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88Hepatocytes in polyurethane non-woven fleece as earlystage of a biohybrid liver support systemMartin Schenk1, Annette Zipfel1, Carsten Linti2, Michael Dauner2,Heinrich Planck2, Richard Viebahn1,3, Alfred Königsrainer11Department of General, Visceral- and Transplantation Surgery,University Hospital Tuebingen, Tuebingen, Germany2Institute of Textile and Process Engineering, Denkendorf, Germany3General Surgery, Knappschafts Hospital Bochum-Langendreer,Bochum, GermanyBackground: Due to the shortage of donor organs, many patientssuffering from end-stage liver disease cannot be transplanted withinreasonable time. In cases of acute liver failure sometimes noappropriate liver graft can be made available. The long-time purposeof this study is the development of a bioartificial liver device, by themeans of which a patient’s blood could temporarily be cleared fromtoxins until the liver is regenerated or until a liver graft fortransplantation is available.Methods: A circuit consisting of a device bearing a three-dimensional cell culture system with a polyurethane non-wovenfleece and measuring possibilities for pressure, pH, temperature,ammonia, and oxygen was developed. Hepatocytes were isolatedfrom rats or pigs by collagenase perfusion and infused into themedium-perfused circuit. For quantification of cell viability lactatedehydrogenase (LDH) activities of the medium was determined. Forevalution of the cell function the MEGX test was performed: To thehepatocytes lidocaine was added and cytochrome P-450-dependentrelease of monoethylglycinexylidide (MEGX) was measured byFluorescence Polarization Immunoassay (FPIA) technology. Glu-cose, albumine and ammonia concentrations as well as urea synthesiswere monitored as further metabolites. Distribution and density ofthe hepatocytes within the non-woven fleece were judged by electronmicroscopy.Results: LDH measurements showed that apart from a short peakwhen perfusion started LDH concentrations remained stable for morethan 15 hours, which means that the cells were vital. After addition oflidocaine the MEGX concentration increased continuously duringthe next hour indicating that the hepatocytes were metabolicallyactive. The hepatocytes released albumin and glucose. Ammonia wasdepleted from the circuit by the hepatocytes, and urea concentrationsincreased. During a run without hepatocytes ammonia levelsremained high. The pressure within the circuit remained constantduring the whole perfusion period. Hepatocytes filled the gaps of thenon-woven fleece evenly and had a high oxygen consumption. Theseperfusion conditions remained stable for up to one week. When thehepatocyte-bearing device was perfused by human plasma, cellsperformed hepatocyte-specific functions for the next few hours.Conclusion: The hepatocytes adhere evenly to the non-wovenfleece, are metabolically active, consume oxygen and performhepatocyte-specific functions. Galactose and sorbitol eliminationtests will be established soon. The next step in this study will be theexpansion of the perfusion circuit to a larger scale to facilitate animalstudies with anhepatic pigs.

89Comparison of the osteogenic capacityof osteogenic differentiating mediaAgmal Scherzed, Robyn Tewksbury, Caroline Seebach,Dirk Henrich, Kerstin Wilhelm, Ingo MarziDepartment of Trauma Surgery, University Hospital,Frankfurt/Main, GermanyIntroduction/aim: Mesenchymal stem cells (MSC) could provide apowerful therapeutic option for the treatment of e.g. large bonedefects, and meanwhile, MSC combined with an appropriate scaffoldhave been shown to support bone repair in different animal models.

But it still requires high efforts, such as approval of the ethicscommittee, to obtain human mesenchymal stem cells for researchpurposes. Moreover, the commercially available osteogenic differ-entiating media are costly. For the purpose to establish the techniquesthat will be needed for a succsessful research in tissue engineering itis reasonable to save valuable MSC by the use of cell lines like SaOS-2 as surrogate and to save costs by economically priced alternativesto induce the osteogenic differentiation. Thus, in this study wecompared a standard medium supplemented with commercialosteogenic substances with a specific osteogenic medium on theireffects on MSC and SaOS-2 cells.Materials and methods: Cell line Saos-2 (ACC243) was obtainedfrom the DSMZ. The cells were cultured in DMEM-F12 supple-mented with 10% FCS. MSC were obtained intraoperatively from theilliac crest of 3 volunteers, with permission of the local ethicscommittee. SaOS-2 and MSC, respectively, were seeded in a densityof 103cells/cm2 in 6-Well plates. For osteogenic differentiation ofMSC and SaOS-2 cells MesenCult + osteogenic Stem cell Kit (Cell-System) was compared with DMEM-F12 + 10% FCS that wassupplemented with commercially available ascorbic acid [AA, 5*10-5 M] and beta-glycerolphosphate [BGP, 1*10-2 M]. The basicmedia without osteogenic supplements were used as control. Thecells were cultured for 14 days, the medium was changed threetimes a week. Osteogenic differentiation was assessed by van Kossastaining and alkaline phosphatase (AP) staining.Results: Both media could induce a significant, comparableosteogenic differentiation of MSC and SaOS-2 cells as assessedby van Kossa and AP staining.

Table (+++ = strong staining, ++ = normal staining, + = weakstaining, - = no staining)

Cells MesenCult + DMEM+ MesenCult DMEMOsteogenic AA+BGPStem cell Kit

van MSC ++ + - -Kossavan SaOS-2 ++ +++ - -KossaAP MSC + ++ - -AP SaOS-2 + ++ - -

Conclusion: Our data show that the immortal cell line Saos-2 couldbe a suitable tool to establish the necessary methods for the basicresearch in tissue engineering. Thus, the use of these inexpensivecells will help to prevent the waste of valuable MSC. Moreover, weshow that a standard medium is sufficient to induce the osteogenicdifferentiation effectively.

90Identification and differentiation of highly proliferatingsingle human mesenchymal stem cells for tissueengineering applicationsMatthias Schieker, Matthias Wierer, Florian Haasters, Christina Ern,M. Shakibaei*, Denitsa Docheva, W. MutschlerExperimental Surgery and Regenerative Medicine,Department of Surgery, and *Department of Anatomy,University of Munich (LMU), München, GermanyBackground: Due to their high proliferative capacity, humanmesenchymal stem cells (hMSC) are increasingly used in tissueengineering research applications. Cell culture of hMSC in vitroshow heterogeneous morphologies with three main subtypes: a)large, flat cells with very low proliferation capacity (FC cells), b)long, spindle shaped cells with medium doubling rates (SS cells) andc) small, round cells that rapidly self renew (RS cells). In this study,

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we aimed to characterise these 3 morphological groups of hMSC byelectron microscopy and immunocytochemical labelling. Further-more, we showed the differentiation potential of human mesench-ymal stem cells starting from one single cell.Material and methods: HMSCs (Cambrex, USA) were culturedaccording to providers’ recommendations. Single cells were isolatedby single cell picking and clonally expanded. For electronmicroscopy cells of cell clones (2 for each type of cells – RS, FCand SS) were fixed in Karnowsky fixative solution. For standarddifferentiation assays, cell clones derived from one single RS cellwere split into three subclones and cultured in media enriched withadipogenic, osteogenic or chondrogenic supplements respectively.Histological analysis was carried out by staining for oil red-o(adipogenic cells), von Kossa (osteogenic cells) and toluidin blue(chondrogenic cells). In addition, the chondrogenic pellets wereimmunohistochemically labelled for collagen type I, collagen type IIand aggrecan.Results: The EM data suggest that the RS and SS cells are verymetabolically active. They are rich in organelles and their nuclei havethe appearance of dividing cells. In contrast, the FC cells show signsof slight degeneration such as dilated rER, some areas of condensedchromatin in their nuclei and vacuoles. Moreover, some features ofapoptotic cell death such as apoptotic bodies and annular chromatincondensation at their nuclear membrane were found and TUNELstaining confirmed apoptosis. Clonal expansion from one RS cellrevealed approximaly one million cells and the adipogenic,osteogenic and chondrogenic differentiation was verified byrespective (immuno-)histological methods.Conclusion: We present for the first time ultrastructural analysis ofsubtypes of human mesenchymal stem cells and show the potential ofone single mesenchymal stem cell to differentiate into three differentlineages. Thus, additionaly to their high proliferation capacity invitro, these single cells fulfill the stem cell definition and are veryinteresting for tissue engineering applications.

91Clinicopathologic and prognostic significanceof matrix-metalloproteinases in rectal cancerAlexander Schlamp, Rainer Broll, Hans-Peter Bruch,Oliver SchwandnerDepartment of Surgery, University Hospital Schleswig-Holstein,Campus Lübeck, Lübeck, GermanyBackground: The aim of this study was to determine the prognosticrole of matrix-metalloproteinases in human rectal cancer.Methods: Formalin-fixed and paraffin-embedded tissue sections of94 rectal carcinomas resected curatively within 6 years (1996-2002)were used for immunohistochemical analysis, and results werecorrelated with clinical and histopathologic data and prognosis. Theprimary antibodies used in this study were monoclonal mouseantibodies for MMP-2 (eMMP-2: epithelium; sMMP-2: stroma)(clone 75-7F7), MMP-7 (clone ID2), MT1-MMP (clone 114-6G6)und TIMP-2 (clone T2-N IC3). Sections with immunostainingsignals in more than 10% of carcinoma and stromal cells were jugdedas being positive. The expression of the MMPs were assessed by twoobservers blinded to the clinical data. A standardized oncologicaltechnique with total mesorectal excision was used in all patients.Clinical, surgical, histopathologic and follow-up data were prospec-tively recorded in a computerized registry. End points of theprognostic analysis (91 patients) were local recurrence, metachro-nous distant metastasis and 5-year survival (disease-free and overall).To assess prognostic significance statistics included univariate andmultivariate analysis (p<0,05 statistically significant).Results: Of the 94 rectal carcinomas 35% (33/94) were eMMP-2-positive, 77% (72/94) were sMMP-2-positive, 54% (51/94) wereMMP-7-positive, each 47% (46/94) showed a positive MT1-MMP-

respectively TIMP-2-status. The stromal MMP-2-stainig pattern wascorrelated with depth of invasion (pT-status, p=0,006) and withMMP-7 (p=0,016) und TIMP-2 (p=0,036), positive expression ofMMP-2 in tumour epithelium correlated with MMP-7 (p=0,027),MT1-MMP (p=0,036) and TIMP-2 (p<0,0001). Positive expressionof MMP-7 was significantly correlated with depth of invasion (pT-status) and with TIMP-2 (p<0,01). The expression of MT1-MMPcorrelated with eMMP-2 (p=0,036), MMP-7 (p=0,004) und TIMP-2(p=0,002). TIMP-2 immunoreactivity correlated with depth ofinvasion (p=0,013), eMMP-2 (p<0,0001), sMMP-2 (p=0,036),MMP-7 (p<0,0001) and MT1-MMP (p=0,002). Neither patterncorrelated with age, gender, tumor stage (UICC), grading, pre-operative serum CEA level or nodal status (p>0,05). Within a meanfollow-up of 46 months, tumor progression, caused by either localrecurrence and distant metastasis, occured in 14 patients (15,4%).There was no association between the MMP-expression and theappearance of a local recurrence and/or distant metastasis. In terms ofsurvival, preoperative CEA level (disease-free 5-year-survival 46%with increased CEA vs. 70% with normal CEA, p=0,01; overall 5-year-survival 43% vs. 74%, p<0,01) and UICC stage were the onlyfactors to be significantly related to 5-year-survival by univariateanalysis, whereas the others failed to show a significant association.However, the expression of MMPs were not significantly associatedwith survival (p>0,05).In multivariate analysis CEA and UICC stagewere not identified as independent factors predictive of survival.Conclusion: MMP-2, MMP-7, MT1-MMP and TIMP-2 do notappear to be significant indicators of prognosis in a collective ofcuratively resected rectal carcinomas.

92Minimal-invasive vein harvest for peripheral arterialrevascularizationThomas C. Schmandra, Farzin Adili, Ralf Ritter,Matthias Tenholt, Thomas Schmitz-RixenDepartment of Vascular and Endovascular Surgery, JohannWolfgangGoethe-University, Frankfurt/Main, GermanyBackground: Conventional open harvesting of the long saphenousvein for peripheral bypass surgery is often associated with significantwound pain and increased morbidity in some patients. Endoscopicvein harvest attempts to reduce this morbidity and improve patientsatisfaction with no compromise in outcome. We report our firstresults using a new vein harvest system.Patients and methods: For peripheral arterial revascularization weperformed saphenous vein harvesting in 17 patients using a new,reusable endoscopic vein harvest system (Richard Wolf GmbH,Knittlingen). Long saphenous vein preparation was entirelyperformed under visual control with permanent CO2-insufflation.For dissection special instruments were used. Small feedingtributaries were divided between clips.Results: In all patients endoscopic vein harvesting was successfuland arterial reconstruction was performed using the dissected longsaphenous vein. Bypass function was satisfying in all cases. Thepatency rate is 100% (surveillance 1-20 months/mean 12). Meanpreparation time was 48 (±17) min. Total operation time was clearlyprolonged to open vein harvest-bypass surgery. In 4 patientsadditional incisions were performed for tributary dissection. In 3patients a low grade postoperative hematoma developed. No surgicalintervention was necessary. Wound healing was uneventful in allpatients.Conclusion: Endoscopic saphenous vein harvesting is a safe andwell practicable procedure with a low rate of impaired woundhealing. Dissection should be performed under CO2-insufflation toimprove visual control. The data support a prospective randomizedtrial with endpoints including impaired wound healing, operative andharvest time, vein quality, outcome and postoperative pain.

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93FTY720 Dose-dependently inhibits angiogenesis in vitroand in vivo and decreases tumor growthGerald Schmid, Markus Guba, Armine Papyan, Ivan Ischenko,Christiane J. Bruns, Karl-Walter Jauch, Christopher Heeschen,Christian GraebDepartment of Surgery, Grosshadern,Ludwig-Maximilians-University Munich, Munich, GermanyBackground: The immunosuppressant FTY720 induces apoptosis inactivated lymphocytes and reduces their concentration in peripheralblood by inhibiting the egress of activated lymphocytes via efferentlymph vessels. This effect is achieved through sphingosine 1phosphate receptor agonist blockade. Recently, FTY720 was shownto reduce tumor growth and metastasis. In this study we investigatedwhether the anti-tumor effect of FTY720 is mediated throughinhibition of angiogenesis.Patients and methods: To evaluate the proposed anti-angiogenicproperties of FTY720 we used a human umbilical vein endothelialcell (HUVEC) spheroid model. The matrigel plug assay was used toassess the effect of FTY720 (10 mg/kg/d) on angiogenesis in vivo. Tosubstantiate our findings the effect of FTY720 on tumor growth wasillustrated by subcutaneous injection of Lewis Lung Carcinoma(LLC1) cells into the back of syngeneic C57BL/6 mice. Afterestablishment of palpable tumors animals were randomized fortreatment with FTY720, or the mTOR inhibitor everolimus(RAD001).Results: FTY720 showed a strong anti-angiogenic effect in vitro.The vascular sprouting of VEGF-stimulated HUVEC spheroids wassignificantly inhibited even at nanomolar FTY720 concentrations(10nM: –73.4±12.7% relative to control; P<0.001). Mechanistically,a neutralizing CXCR4 antibody was capable of abrogating the anti-angiogenic properties of FTY720 (isotype antibody: –39.5±22.1%relative to control; CXCR4 antibody: +38.8±26.9%; P<0.001 versusisotype). Utilizing an in vivo matrigel plug assay we were able toconsistently demonstrate inhibition of angiogenesis following treat-ment with 10 mg/kg/d FTY720 (– 87.7 ± 8.4%). Consequently,FTY720 also inhibited subcutaneous tumor growth in a dose-de-pendent manner. Maximal inhibition of tumor growth was achievedwith a FTY720 concentration of 10 mg/kg/d (–53.3±28.1% comparedto control). However, the observed reduction was significantly small-er as compared to the inhibition achieved with 1.5 mg/kg/d RAD001(–79.4±9.9% compared to control; P<0.001 versus FTY720).Conclusion: FTY720 demonstrates a pronounced anti-angiogenicactivity resulting in a substantial anti-tumor effect in vivo. The anti-angiogenic activity of FTY720 is mediated through activation ofCXCR4, a specific receptor for the stromal cell-derived factor-1.Therefore, the treatment with FTY720, preferably in combinationwith other immunosuppressants with anti-angiogenic properties suchas mTOR inhibitors, may proof to be useful in preventing tumordevelopment and progression in high-risk patients following organtransplantation.

94Acute rejection of experimental lung transplants -time course of graft infiltration by macrophagesand T-lymphocytesAndree Schmidt, Renate Plaß, Ute Nau, Winfried Padberg,Veronika GrauLaboratory of Experimental Surgery, Department of Generaland Thoracic Surgery, University of Giessen Lung Center,Justus-Liebig-University, Giessen, GermanyBackground: An early hallmark of lung allograft rejection istransplant infiltration by leukocytes. There is evidence that inaddition to T lymphocytes, monocytes/macrophages play a pivotalrole in graft rejection as antigen presenting as well as effector cells. In

the lung, at least three different populations of monocytes/macrophages can be differentiated: 1) intravascular monocytes, 2)interstitial macrophages, and 3) alveolar macrophages. All of themare likely to be involved in graft rejection, but their role is not clearlydefined yet. In this study we investigate the time course of lungtransplant infiltration by macrophages and compare it to infiltrationby T- and B-lymphocytes.Methods: Orthotopic transplantation of the left lung was performedin the isogeneic LEW to LEW and in the allogeneic DA to LEW ratstrain combination. Groups of animals were sacrificed at intervalls of24 h until day 6 posttransplantation. The lungs were fixed in 4%buffered paraformaldehyde and embedded for paraffin histology. Todetect macrophages we used mAbs ED1 and ED2. T- lymphocyteswere stained with mAb R73 directed against the alpha/beta-T-cellreceptor and B-lymphocytes with mAb OX33. In the intact lungalveolar macrophages are positive for ED1 and negative for ED2,whereas interstitial macrophages are only positive for ED2. Graftinfiltration was semiquantitatively analyzed by computer assisteddensitometry.Results: During the first 2 days after lung transplantation, interstitialand intraalveolar edema develops in allografts. Thereafter, leuko-cytes infiltrate perivascular, peribronchial, interstitial, and intraal-veolar regions of allografts. On days 5-6 the parenchyma of theallografts is essentially destroyed. Starting on day 2 posttransplanta-tion, the number of ED1-positive alveolar macrophages increases. 5days after transplantation the proportion of the ED1 postive areaamounts to about 16%. At this point of time both alveolarmacrophages as well as interstitial macrophages are ED1-positive.Initially the number of ED2-postive cells remains about constant.Late during allograft rejection on days 5-6 about 14% of the tissue areED-2 positive. Again, interstitial and alveolar macrophages expressthis antigen. The increase in the R73-positive area (T-lymphocytes)starts on day 3 and reaches its maximum of about 3% on day 5. Inisografts there is a slight increase in the number of ED1-positivealveolar macrophages during the first two days after surgery, whichdeclines thereafter. The number of T-lymphocytes and ED2-positivemacrophages does not augment significantly in isografts. In bothisografts and allografts only minor changes in the frequency of B-lymphocytes are seen.Conclusion: After allogeneic lung transplantation, the grafts areheavily infiltrated by ED1-positive macrophages. Macrophagesoutnumber T-lymphocytes during all phases of allograft rejectionand emerge earlier. Further studies are needed to clarify the role ofmacrophages during rejection of lung transplants and to evaluatetherapies targeting these cells.

95Intravital microscopy of the coronary microcirculationin heterotopically transplanted mouse heartsRené Schramm, Michael D. Menger, Frank Langer, Yves Harder,Jürg Hamacher, Hans-Joachim SchäfersDepartment of Thoracic and Cardiovascular Surgery, Institute forClinical and Experimental Surgery, University of Saarland,Homburg/Saar, GermanyBackground: The aim of this study was to establish an experimentalmodel for the intravital microscopic analysis of the coronarymicrocirculation in mice during myocardial ischemia/reperfusioninjury.Methods: Murine cardiac syngrafts were exposed to 60 or 240 minof cold ischemia before subsequent heterotopic transplantation to therecipient neck vessels. High resolution intravital fluorescencemicroscopy (IVM) was used to visualize the subepicardial coronarymicrocirculation of the right ventricle in cardiac syngrafts.Results: IVM allowed for detailed visualization of the rightventricular subepicardial coronary microcirculation, including feed-ing coronary arterioles, nutritive capillaries, and postcapillaryvenules. In addition to blood vessels, dilated lymphatic ducts were

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illuminated in the deep myocardium due to FITC-dextran lymphatictransport. NADH-dependent autofluorescence, indicating the mito-chondrial redox state, showed limited recovery during reperfusion inthe 240 min cold ischemic syngrafts (P<0.05 vs. 60 min coldischemic syngrafts), indicating a persistently higher mismatchbetween oxygen supply and demand after exposure to prolongedischemia times. Quantitative analysis of the microscopic imagesrevealed that the exposure to prolonged ischemia of 240 minaggravates the postischemic nutritive capillary perfusion failure, asindicated by significant reduction of the functional capillary densityby 63%, the capillary blood flow velocitiy by 45%, and the red bloodcell flow velocities in draining venules by 34% (P<0.05 vs. 60 mincold ischemic syngrafts). Moreover, markedly elevated numbers ofrecipient leukocytes were found to interact with the coronarymicrovascular endothelium during reperfusion in 240 min coldischemic syngrafts. Both leukocyte rolling and firm adhesion weresignificantly elevated by approximately 6- and 11-fold in 240 mincold ischemic syngrafts (P<0.05 vs. 60 min cold ischemic syngrafts).Conclusion: This study introduces a novel approach to visualize indetail the murine coronary microcirculation by intravital fluorescencemicroscopy. Our data indicate that prolonged ischemia times provokeboth pronounced nutritive coronary perfusion failure and enhancedleukocyte responses during myocardial reperfusion.

96Simvastatin inhibits lymphocyte homingto peripheral lymph nodesRené Schramm, Michael D. Menger, Gabriele Weitz-Schmidt,Yves Harder, Rudolf Schmits, Hans-Joachim SchäfersDepartment of Thoracic and Cardiovascular Surgery,Institute for Clinical and Experimental Surgery, Department ofInternal Medicine I, University of Saarland, Homburg/Saar,GermanyNovartis Pharma AG, Preclinical Research, Basel, SwitzerlandBackground: Lymphocyte homing to peripheral lymph nodes isgoverned by the coordinated expression and function of certainadhesion molecules, including lymphocyte function-associatedantigen (LFA)-1. Statins are HMG-CoA reductase inhibitors andare well recognized to exert anti-inflammatory effects throughinhibition of LFA-1 function. It remained elusive, however, whetherstatin compounds are capable of inhibiting lymphocyte homing toperipheral lymph nodes in vivo.Methods: We utilized the cervical lymph node preparation to studythe effects of simvastatin on lymphocyte adhesion to high endothelialvenules (HEVs) by means of intravital fluorescence microscopy(IVM).Results: IVM revealed that lymphocyte firm adhesion to theendothelium of the cervical lymph node HEVs was criticallydependent on the adhesive function of LFA-1. The over-all numbersof firmly attached lymphocytes was reduced by 58% in LFA-1-deficient mice (P<0.05 vs. wild type controls). Similarly to thefindings in mutant mice, a two-hour pretreatment (i.p.) withsimvastatin inhibited the firm adhesion of lymphocytes to cervicallymph node HEV endothelium by 63% (P<0.05 vs. vehicle-treatedwild type controls). Histological analysis of lymph node crosssections revealed that a 10-day treatment with simvastatin reducedthe cellularity of cervical lymph nodes, i.e. the relative area ofhematoxylin-stained cell nuclei was reduced from 94±0% in vehicle-treated controls to 77±3% in simvastatin-treated mice (P<0.05). Inaddition, we demonstrate that acute treatment with the syntheticstatin-derivate LFA878, but not LFA703, reduced lymphocyte firmadhesion in peripheral lymph node HEVs by 63% (P>0.05 vs.placebo-treated controls).Conclusion: This study confirms a key role for LFA-1 in lymphocytehoming to peripheral lymph nodes, mediating the firm adhesion of

lymphocytes to HEVendothelium. We demonstrate that the clinicallyrelevant HMG-CoA reductase inhibitor simvastatin as well as thesynthetic statin analogue LFA878 are capable of attenuating LFA-1-dependent firm adhesion of lymphocytes to HEV endothelium incervical lymph nodes in vivo. Moreover, our novel data indicate thatthe LFA-1-dependent firm adhesion of lymphocytes is a criticalprerequisite for effective transendothelial migration into the nodalmatrix. Thus, we conclude from our data that statin compounds arecapable of inhibiting lymphocyte homing to murine peripheral lymphnodes in vivo. Our findings may therefore have implications for theclincical treatment of adaptive immune responses, e.g. transplantrejection.

97Blunt chest trauma reduces the number of alveolartype 2 epithelial cells. Role of alveolar macrophagesin apoptosis inductionDaniel H. Seitz, Mario Perl, Max G. Bachem*,Sonja T. Braumüller, Markus S. Huber-Lang, Markus W. KnöferlDepartment of Trauma, Hand- and Reconstructive Surgery,University of Ulm, Ulm, Germany*Department of Clinical Chemistry, University of Ulm, Ulm,GermanyBackground: Blunt chest trauma is a very common injury. It isknown to induce severe complications like acute respiratory distresssyndrome or multiple organ failure. In addition to a well definedsystemic inflammatory response local alterations in the contusedlungs might contribute to the development of these complications.An important cell population of the lung are alveolar type 2 epithelial(AT2) cells, which produce surfactant, maintain the alveolarepithelium and contribute to host defense. Apoptosis of AT2 cellsis an essential mechanism regulating these processes. Previousstudies have shown that alveolar macrophages are activated by bluntchest trauma to release inflammatory mediators. Due to theirlocalization alveolar macrophages have the potential to interact withAT2 cells. The present study was performed to elucidate whetherblunt chest trauma induces apoptotic processes in AT2 cells and itsdependency on alveolar macrophages.Methods: To study this, male CD rats were subjected to either shamprocedure or blunt chest trauma induced by a single blast wavecentered on the chest. At various time points after injury (0.5 hours to7 days) lungs were analyzed immunohistochemically with an anti-cytokeratin-antibody (clone MNF-116), which allows to detect andquantify AT2 cells. To identify apoptotic cells, a stain for activatedcaspase 3 was used. In a subsequent study, AT2 cells were isolatedfrom healthy, untreated rats and cultured for 48 hours without anystimulation. Alveolar macrophages were isolated from eithertraumatized or sham operated animals at 4, 24 or 48 hours after theinsult and cultured for 24 hours without any stimulation. AT2 cellswere then cultured for another 6 hours in the presence of the alveolarmacrophage supernatants. In some cultures, oxidative stress wasinduced by added H2O2. Both, TUNEL staining of AT2 cells andWestern-blot analysis of AT2 cell lysates for active caspase 3 wereused to detect apoptotic events.Results: MNF-116 staining of lung tissue revealed a significantdecrease in AT2 cell number 48 hours after trauma. In addition, anincreased count of caspase 3 positive AT2 cells versus sham operatedlittermates was detected at the same timepoint after blunt chesttrauma. TUNEL stain of AT2 cells, cultured in presence of alveolarmacrophage supernatants, obtained at 4, 24 or 48 hours after chesttrauma revealed increased numbers of apoptotic cells whenadditional oxidative stress was induced by H2O2. The concentrationof active caspase 3 was elevated in cultured AT2 cells stimulated withsupernatants of alveolar macrophages isolated after chest traumacompared to those from sham animals.

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Conclusion: The decrease of AT2 cell number in lungs observedafter blunt chest trauma is most likely due to apoptosis, sinceactivated caspase 3 was increased in the remaining AT2 cells at thesame timepoint. The results of the second study indicate that solublefactors released by alveolar macrophages contribute to the inductionof apoptosis. Oxidative stress increased the susceptibility of AT2cells to programmed cell death. Since polymorphonuclear granulo-cytes transmigrate into lung tissue and induce oxidative stress afterblunt chest trauma, further studies should focus on their contributionto the induction of AT2 cell apoptosis. (DFG KN 475/3-1)

98hMSC-human mesenchymal stem and progenitor cells:characterization and chemotactic activation of migrationand homing by blood plasma and wound-secreted fluidOliver Seitz, Brigitte Rüster, Cornelius Klein, Robert Sader,Erhard Seifried, Reinhard HenschlerInstitute for Transfusion Medicine and Immune Hematology,University of Frankfurt, Frankfurt, GermanyClinic for Maxillofacial Plastic Surgery, Surgical Center,University of Frankfurt, Frankfurt, GermanyBackground: Cells which can give rise to a multitude ofmesenchymal lineages (MSC) have been widely characterized, andcould be successfully re-infused into patients. However, their homingand migration behaviour is nearly unknown, and its regulation iswidely unclear.Methods: Bone marrow samples were seeded onto plastic dishes invarious serum-containing culture media. Immunohistochemistry andRT-PCR analyses were established to characterize differentiationmarkers. A modified Boyden Chamber assay was developed toanalyze the potential for chemotactic migration.Results: We found reproducible and efficient expansion of MSCwith multilineage differentiation capability over 3-4 log withinculture periods of 3-4 weeks. Preselected fetal calf serum provedsuperior to commercially available expansion supplement for MSC,or serum-free medium to promote optimal expansion rates. MSCcould be differentiated into osteoblasts (assayed by expression ofbone sialoprotein, osteocalcin, or alkaline phosphatase), chondro-cytes (by staining with toluidine blue), myocytes (by expression ofmyogenin) or fat cells (assayed by oil red O staining) at different timepoints of the expansion culture. Nevertheless, activation of MSCmigration proved difficult. Platelet Derived Growth Factor (PDGF)which was shown to induce chemotaxis of mature fibroblasts andosteocytes, stimulated MSC migration only to limited degrees. Incontrast, blood plasma efficiently induced the transmigration of MSCthrough 8 µm micropores within 4 h. Furthermore wound-secretedfluid 24 h after surgery proved as a further potent stimulus ofmigration. When immunodeficient NOD/SCID mice were infusedi.v. with 1x106 MSC, human cells were still contained in the lungsafter 24h, but very little signal was found in many other organs.Conclusion: This study describes conditions for efficient isolation,expansion and differentiation of MSC from human bone marrowsamples. We show, that activation of MSC migration may followvery different mechanisms compared with those known for he-matopoietic stem cells. It may underly a multicomponent regulationand require special signals from regenerating tissues.

99A new animal model for studies in the peripheralnervous system of rats with a gap distance of 4 cmusing a median nerve cross-chest transferNektarios Sinis1, Hans-Eberhard Schaller1,Caterina Schulte-Eversum2, Burkhard Schlosshauer3,

Michael Doser4, Klaus Dietz6, Harald Rösner5,Hans-Werner Müller2, Max Haerle11Klinik für Hand-, Plastische-, Rekonstruktive- undVerbrennungschirurgie, Universität Tübingen, BG-Unfallklinik,Tübingen, Germany2Labor für Molekulare Neurobiologie, Neurologische Klinik derUniversität Düsseldorf, Düsseldorf, Germany3NMI Naturwissenschaftliches und Medizinisches Institut an derUniversität Tübingen, Reutlingen, Germany4Deutsches Zentrum für Biomaterialien und Organersatz,Denkendorf, Germany5Institut für Zoologie, Zell- und Entwicklungsneurobiologie,Universität Hohenheim, Stuttgart, Germany6Institut für Medizinische Biometrie, Universität Tübingen,Tübingen, GermanyBackground: In former studies we could demonstrate a successfulregeneration across a 2 cm defect in a rat median nerve model with abioartificial tube (trimethylenecarbonate-co-epsilon-caprolactone)seeded with autologous Schwann Cells (SC) [1]. We compared theregenerative potential of this biohybride conduit with an autologousgraft across this 2 cm defect. Our results could demonstrate acomparable regeneration regarding force (grasping-test), immuno-histochemistry (S-100, PAM), weight of the end-organ (flexordigitorum sublimis muscle), electrophysiology, and electronmicro-scopy after nine postoperative months. In this study an augmentationof the nerve gap should clarify the regenerative potential of suchtissue-engineered products in a rat model, since it is known, that ratsdisplay a high capability of spontaneous regeneration in theperipheral nerve. If any differences between autologous grafts andconduits filled with SC were evident the large distances should makethem obvious.Materials and methods: Female inbred Lewis rats weighingbetween 220 and 250 g were anesthetized and the median nervesof both sides were exposed. On the left, the median nerve wassectioned far distally in the cubital fossa were the nerve branches intothe forearm muscles. Then the right median nerve was mobilized anddissected far proximal in the axilla. A 4 cm conduit filled with SCwas sutured on the left distal median nerve using an 11-0 micro-surgical suture. The same was done on the right proximal stump ofthe median nerve. The purpose was to use the sprouting axons fromthe left median nerve to find the distal stump on the right and re-innervate the end-organ (flexor digitorum sublimis muscle). Theconduits were put subcutaneous from the left across the chest tothe right. To imitate the autologous nerve graft, which is normallyused to bridge nerve gaps, both ulnar nerves of the forearms weresectioned and sutured together. After that the ulnar nerves weresutured between the left distal median nerve stump and the proximalmedian nerve stump on the right. In both groups 12 animals wereoperated.Results: No infections, autotomies or ulcerations were observedafter six months post operation. All animals but two of themsurvived the operative intervention where the mean operative timewas about 60 to 70 minutes. No signs of regeneration were foundsix months post operation in any of the both groups. A progressiveatrophy was found in the flexor digitorum sublimis muscle of allanimals (group I: - 73%±21, group II: - 67%±15). No functionalrecovery was found after six months, nor were any electrophysio-logical recordings possible. The histological analysis of the distalcoaptation side could not reveal any tissue demonstrating neuralorigin in group I. In animals of group II at the distal coaptation pointsigns of a Wallerian degeneration and neuroma formation werefound.Discussion: To augment a gap distance of 2 cm in the median nerve ofrats in order to reconstruct this gap by means of tubulisationtechniques one has to take the anatomy of the animals’ peripheralnerves into further consideration. After the median nerve passesacross the cubital fossa the first branches are leaving into the forearmmuscles. Therefore a larger gap than 2 cm reaching these branches

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seems impossible. To augment the distance in the way to change thetarget nerve and use another nerve, e.g. the sciatic nerve, leaves thesame problem since the nerve branches into the peroneal and tibialnerves. Bertelli described in 1999 a model in that he used a radialnerve cross-chest transfer for neurotisation of an injured brachialplexus in rats [2]. To use the median nerve, especially with the ulnarnerve as an autograft between the nerve stumps, was never reportedbefore, to our knowledge. Due to the excellent spontaneous regen-eration found in rats, we recommend to use larger distances then 1cm or less. A few studies disregard this important point leading intoresults which cannot be transferred into higher species. Even sixmonths after observation no signs of regeneration were found inboth groups. A longer period of observation might be necessary,since 4 cm seem very large in comparison to the size of one upperextremity in rats.

100Preformed bioartificial bone substitute: differentiationof osteoblasts in a processed bovine cancellousbone matrix in vitroLars Stangenberg1, Dirk J. Schaefer1,2, Jens Stern-Straeter1,Olaf Buettner1, Jan Ohnolz1,3, Raymund E. Horch3, G. Björn Stark1Ulrich Kneser1,31Department of Plastic and Hand Surgery, University of FreiburgMedical Centre, Freiburg, Germany2Department of Plastic Surgery, Kantonsspital Basel, Basel,Switzerland3Department of Plastic and Hand Surgery, University of ErlangenMedical Centre, Erlangen, GermanyBackground: Bone defects, e.g. caused by tumour or trauma, arestill a major therapeutic challenge. The golden standard in treatmentis the use of autologous bone grafts. However, shortcomings aremainly the creation of a significant donor site morbidity as well asthe limited availability. Processed bovine cancellous bone (PBCB)might be a suitable matrix for a tissue engineering based approach.It is biocompatible, osteoconductive, non-immunogenic, porous andits biomechanic properties are comparable to native bone. In thisstudy, cell specific differentiation of primary rat osteoblasts (rOB)seeded into PBCB was investigated in vitro.Methods: rOB were isolated from syngenic rats and expanded invitro. rOB were seeded into PBCB discs, immobilized by fibrin andeither cultivated in basal medium (BM) or differentiation medium(DM) containing ascorbic acid, dexamethason and beta-glycerolphosphate. Marker genes alkaline phosphatase (ALP), bonesialoprotein (BSP), collagen I (COL), osteocalcin (OC) andosteopontin (OSP) were examined by real-time RT-PCR over a14 day period. ALP was also assayed by chemiluminiscence.Standard histology was performed after 14 days.Results: ALP activity as well as expression increased over theobserved period independent of cultivation conditions. BSP wassignificantly higher expressed in DM and peaked at day 8. COLshowed a significantly higher expression in BM and a moderateincrease. OC again was significantly higher expressed in BM with atime course similar to BSP, whereas OSP was significantly higherexpressed in DM. Matrix calcification was only detectable in theDM group by von Kossa stain.Conclusion: In this study we investigated the differentiation ofrOB in a three-dimensional environment in a PBCB matrix. Theobserved expression patterns suggest a physiological response ofrOB to the differentiation stimuli of media and PBCB. In DM therOB were able to synthesize calcified extracellular matrix and thusexhibit full functional capacity. So PBCB is a efficient and powerfulscaffold for in vitro differentiation of rOB. Cell-seeded PBCB is apotential osteogeneic construct for in vivo application.

101Dynamic of STAT3 and SOCS3 in neutrophils of multipleinjured patients in the early posttraumatic periodJ. Stegmaier1, H. Vester1, C. Kirchhoff1, V. Bogner1,K. Ruckdeschel2, J. Heesemann2, W. Mutschler1, P. Biberthaler11Chirurgische Klinik und Poliklinik,Ludwig-Maximilians-Universität, München, Germany2Max-von-Pettenkofer-Institut, Ludwig-Maximilians-Universität,München, GermanyBackground: Destabilisation of the human immune system follow-ing severe trauma induces Systemic Inflammatory ResponseSyndrome, SIRS and Multiple Organ Failure, MOF. In this context,Suppressors of Cytokine Signaling, (SOCS) seem to play asubstantial role for modulation of cytokine expression after trauma,which are controlled by Signal Transducers and Activators ofTranscription, STAT3. Although it was demonstrated in animalmodels that i.e. SOCS3 mediates part of the posttraumatic immunemodulation its role within the human neutrophils obtained frommultiple injured patients remains unclear, so far.Patients and methods: Peripheral blood neutrophils (PMN) wereisolated from whole blood samples of 24 multiple injured patients(Injury Severity Score, ISS≥16) within 90 minutes after trauma, aswell as 6, 12, 24, 48 and 72h standardized after the traumatic event.STAT3 translocation was analysed by Electrophoretic Mobility ShiftAssay, EMSA in the nuclear protein fraction, mRNA expression ofSTAT3 and SOCS3 was analysed by RT-PCR (Light Cycler, Roche)in the cytosolic compartment. Statistical analyses were performedby ANOVA on ranks and SNK-test. Data [arbitrary units] given asmean±SEM for nuclear translocation, in copies/µg RNA as mean±SEM.Results: The mean ISS of the enrolled patients was 35±11. 17survived, 7 died in the posttraumatic period. Transcriptional activityof STAT3 in trauma patients was significantly increased at 6h and12h after trauma as compared to healthy controls (p<0.01). In moreseverely traumatized patients (ISS>41) STAT3-translocation wassignificantly decreased compared to patients of lesser injury severity(ISS 16-41) at 6 hours (926±230 vs. 2534±387; p<0.01), 12h (663±186 vs. 2503±396; p<0.05) and 24 hours after trauma (858±188 vs.2481±532; p<0.05). SOCS3 mRNA expression in neutrophils wassignificantly elevated in deceased patients on admission (1412±127vs. 958±237). In contrast, the SOCS3 mRNA expression was signif-icantly reduced in the deceased group 24 hours (633±33 vs. 1325±374; p<0.05), as well as 48 hours after trauma (873±196 vs. 1208±581; p<0.05) as compared to the survivors group.Conclusion: Our results demonstrate for the first time that STAT3 iselevated in multiple trauma patients. Patients of adverse outcomeshow minor activity of STAT3 than those of good outcome. Thisreduced activation is accompanied by reduced mRNA expression ofSOCS3 reporter gene. As preliminary data, further investigation willbe performed in order to increase the number of enrolled patients aswell as to correlate the demonstrated results with peripheral cytokineconcentrations.

102Proliferation of alveolar macrophagesafter lung transplantation in the ratJochen Sucke, Gabriele Fuchs-Moll, Petra Freitag,Winfried Padberg, Veronika GrauLaboratory of Experimental Surgery, Department of Generaland Thoracic Surgery, University of Giessen Lung Center,Justus-Liebig-University, Giessen, GermanyBackground: Alveolar macrophages play a pivotal role in the innateand adaptive immune system of the lung. Their number stronglyincreases after lung transplantation and they are probably involved in

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allograft rejection. However, the knowledge on this topic is scarce.Increased numbers of alveolar macrophages are generally explainedby an increased recruitment of blood monocytes or by slowing downtheir elimination from the airways. Additionally, there are some hintsthat alveolar macrophages can proliferate in situ. However it is notknown, if proliferation in situ can significantly contribute to theregeneration or enlargement of the pool of alveolar macrophages. Inthis study we investigate the proliferation of alveolar macrophagesafter isogeneic and allogeneic lung transplantation in the rat.Methods: Orthotopic transplantation of the left lung was performedin the isogeneic LEW to LEW and in the allogeneic DA to LEW ratstrain combination. To label cell nuclei in the S-phase of the cellcycle 5-Bromo-2’-deoxyuridin (BrdU) was injected on day 2posttransplantation 30 min before sacrificing the animals. We choosethis point of time because we observed the strongest increase in thenumber of alveolar macrophages between day 2 and day 3. Alveolarmacrophages in the S-phase were detected on paraffin sections indouble staining experiments using monoclonal antibody ED1 andantibodies to BrdU.Results: On the second day after experimental lung transplantationup to 21% of the alveolar macrophages display cell nuclei positivefor BrdU, indicating that they are in the process of DNA synthesis.No differences in the frequency of BrdU positive macrophages wereobvious between isofgrafts and allografts. However, in the nativeright lung of the graft recipients the proportion of proliferatingalveolar macrophages was significantly lower.Conclusion: We demonstrate that lung transplantation stronglyinduces proliferation of alveolar macrophages in the alveoli of thegraft. Comparable proliferation rates have never been describedbefore in any model of experimental lung damage. When cells arecontinuously proliferating, the S-phase takes about one third of thetime needed to complete one cell cycle. We thus conclude that almostall alveolar macrophages are proliferating in lung transplants on thesecond day after surgery.We clearly demonstrate that alveolarmacrophages still possess a good proliferative potential. Mitoticproliferation considerably contributes to the increase in the numberof alveolar macrophages observed after isogeneic or allogeneic lungtransplantation.

103Hemorrhage/resuscitation induced liver injury in the ratcan be reduced by a peptide inhibitor of c-JUN N-terminalkinaseVeronika Sun-Young Lee, Mark Lehnert, Dirk Henrich,Christoph Czerny, Tiziana Borsello*, Ingo MarziDepartment of Trauma Surgery, J.W. Goethe University, Frankfurt,Germany*Département de Biologie Cellulaire et de Morphologie, Universityof Lausanne, Lausanne, SwitzerlandBackground: Hemorrhagic shock/resuscitation (H/R) result in aninflammatory response that leads to subsequent organ failure. Inprevious studies, we and others demonstrated that H/R leads tophosphorylation of mitogen-activated stress kinases (MAPKs),specifically c-JUN and ERK, an event that was associated with liverdamage. Recently, a specific, cell-penetrating, protease resistantpeptide inhibitor peptide of c-JUN N-terminal kinase was developed(D-JNKI-1). Here, using this peptide, we tested if inhibition of JNKprotects against organ damage after H/R.Methods: Male Sprague-Dawley rats were treated intraperitoneallywith D-JNKI-1 (11 mg/kg, Alexis Corp.) or vehicle. 30 min laterhemorrhage was induced to a mean arterial blood pressure of 30-35 mmHg for 60 mins. Resuscitation was induced by reinjecting 60%of the shed blood and twice the shed blood volume as Ringers Lactateover a 20 min period. The rats were sacrificed 2h after the end ofresuscitation. Serum Alanine aminotransferase (ALT), Creatineki-

nase (CK), Lipase and lactate dehydrogenase (LDH) activity weremeasured using standard laboratory assays. Pathological evaluationwas performed by an expert pathologist in a blinded manner.Polymorphnuclear leukocytes were identified by chloroacetateesterase staining and counted in a blinded fashion. Serum Il-6concentration was measured via using CBA-Flex-Set and aFacscalibur flowcytometer. Results were evaluated using thesoftware FCAP-Array 1.0 (all BD-Biosciences). Statistics: ANOVAwith Tukey post hoc analysis or Kruskal-Wallis One Way Analysis ofVariance on Ranks, p<0.05 was considered significant.Results: ALT as a marker of liver injury increased to 2963±670 IU/L. In rats treated with d-JNKI-1, ALT level was blunted to 897±151IU/L (p<0.05). Serum CK levels were also attenuated in rats thatreceived d-JNKI-1 treatment to 5647±1037 IU/L compared to 17203±3989 IU/L in the vehicle group (p<0.05). d-JNKI-1 treatmentshowed a tendency to reduce pancreatic injury as indicated by serumlipase measurements (785±267 IU/L vs. 219±59 IU/L, p=0.089).Serum LDH levels rose to 10638±1343 IU/L after vehicle treatedshock, whereas d-JNKI-1 largely prevented LDH relase (4375±1195IU/L, p<0.05). The tissue damage observed in hematoxillin-eosinstained livers (i.e. cell swelling, coagulative necrosis) was bluntedafter d-JNKI-1 pretreatment. Leukocyte infiltration increased after H/R in the vehicle treated group, and was largely diminished after d-JNKI-1 preatreatment. Serum IL-6 levels rose after vehicle treated H/R compared to sham operation, IL-6 release was attenuated after d-JNKI-1 pretreatment.Conclusion: H/R induces a systemic inflammatory response andleads to end organ damage. The changes observed are mediated, atleast in part, by JNK. Therefore, JNK inhibition may provide clinicalbenefit in patients after resuscitated blood loss. JNK protects againstorgan damage after H/R.

104Influence of the age, gender and extraction siteon the colony number of mesenchymal stem cellsRobyn Tewksbury, Caroline Seebach, Dirk Henrich,Kerstin Wilhelm, Ingo MarziDepartment of Trauma Surgery, J.W. Goethe University Hospital,Frankfurt/Main, GermanyBackground: The application of mesenchymal stem cells (MSC) forbone regeneration is an expanding field of current research. In animalstudies, MSC have been used successfully for bone repair and thusoffer a therapeutic option for the treatment of bone defects in humansas well. The cells can be expanded ex vivo and reimplanted locally inthe bone defect, resulting in improved healing processes. For thesuccessful application of MSC, it must be determined under whichcircumstances they can optimally be cultivated and expanded.Therefore, our goal was to investigate the influence of age, genderand localisation in trauma patients with respect to MSC expansion.Materials and methods: MSC were isolated from bone marrowderived from a punction of either the pelvic crest bone (n=31) or tibia(n=3) of volunteers using standard procedures. The cells were seededin a density of 1x106 cells per cm². After 14 days of incubation withMesenCult® Basal Medium for Human Stem Cells, adherent MSCcolonies were stained using Diff-Quik® Staining Set. Finally, thetissue culture flasks were photographed and the colony number andsize were determined. This study was approved by the local ethicscommittee.Results: MSC obtained from iliac crest of young male volunteersresulted in the highest numbers of colonies compared to MSCobtained from the iliac crest of female volunteers and older malepatients (table). Moreover, MSC derived from young male patientstended to form larger colonies. MSC obtained from other locationsthan the iliac crest (tibia, n=3) yielded a distinctly lower number ofMSC colonies.

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Table: Group assignment and colony number. * indicates signifi-cance vs. indicated groups

Gender Age [years] n=34 Colony number Statistics[mean ± SEM]

Female >40 4 21.7±6.2 ——Female <40 8 21.6±4.3 ——Male >40 13 32±8.8 ——Male <40 9 65.7±11.3 * ↑ vs. male

>40 years* ↑ vs. female<40 years

Conclusion: Our data show that age, gender and presumably theextraction site play important roles in the number of MSC colonieswhich can be yielded from a single bone marrow aspirate. Due to thehigher incidence of fractures and impaired bone healing in older(predominantly female) patients, it is important to explore methodsof better treatment. Our results suggest the necessity to improve theculture conditions for the future implementation of autolog ex vivoexpanded MSC, especially for older (predominantly female) patients.

105A new surgical model of hepatectomy in pigsChristian Thiel, Martin Schenk, Karolin Knubben, Klaus Dietrich,Matthias H. Morgalla*, Ruth Ladurner, Horst D. Becker,Alfred KönigsrainerDepartment of General, Visceral and Transplantation Surgery, and*Department of Neurosurgery, University Hospital, Tuebingen,GermanyIntroduction: Animal models are suitable to simulate acute liverfailure. The established surgical methods for hepatectomy in pigsusing en-bloc-resection of the vena cava require a temporaryextracorporeal bypass and total clamping of the inferior vena cava.These steps lead to severe depression of circulation and activation ofthe coagulation system. Therefore postoperative survival and over-allsurvival is impaired.Methods: En-bloc hepatectomy including retrohepatic vena cavawas performed in twenty female pigs (30-41 kg). Using end-to-sideanastomosis between the vena cava above the right renal vein, theportal vein above the confluence, and the intrathoracic vena cavawith a three-way vascular prosthesis, blood flow maintainedconstantly stabile during hepatectomy by performing only partialclamping of the vessels. Venous stasis of the intestine organs wasalso avoided. After completion and release of the bypass a gentlehepatectomy with minimal blood loss was performed.Results: Using this new surgical technique makes it possible toavoid a total clamping of the mayor veins, so that maximal intra-andpostoperative hemodynamic stability can be reached. Duringoperation measured hemodynamic parameters like heart rate, meanarterial pressure, central venous pressure, SO2 oximetry stayedextremely stabile.Postoperative survival time was 100% after 12 hours, 95% after 24hours. Maximum survival time was 88.5 hours; mean survival timewas 50 hours. All animals died because of multiple organ failure,particularly lungs and kidneys were affected, which could bedemonstrated in histology.Conclusion: This new surgical technique permits a total hepatec-tomy with minimal blood loss and stabile circulation without usingan extracorporeal bypass. Relevant venous obstruction is alsoavoided. The labile organism of pigs is less stressed, which could beshown in better postoperative outcome and overall survival time.

106The suppression of IL-1��-production of monocytesin multiple trauma depends on injury severityand on the presence of abdominal injuryThomas Thuemmel, Kevin Brauns, Csaba Biro, Mark Lehnert,Dirk Henrich, Felix Walcher, Stefan Müller, Ingo MarziDepartment of Trauma Surgery,Johann-Wolfgang-Goethe-University, Frankfurt/Main, GermanyBackground: The dysfunction of cells of the innate immune systemis expected to be an important reason for organ dysfunction inpatients with severe trauma. However, data on monocytic function ina mixed trauma population including slightly injured patients islimited. Accordingly, we studied monocyte activity depending on theseverity of injury in the first five days after trauma.Methods: Prospective clinical study, duration 12 months. Freshblood of patients admitted via our emergency room (>18 years) wasobtained on admission, followed by daily blood samples over fivedays. The monocyte activity was assessed using whole bloodstimulation with LPS [10 μg/ml]. As surrogate of monocyte activitythe IL-1β-concentration in the supernatant was measured usingELISA-technique (LPS-response). Based on the injury severity score(ISS) the patients were allocated to 5 subgroups: I=ISS 1-8, (n=5);II=ISS 9-15, (n=13); III=ISS 16-24, (n=11); IV=ISS 25-40, (n=16);V=ISS 41-75, (n=12). The Wilcoxon-test was used for statisticalevaluation. The results were presented as mean±SEM. A p-valueless than 0.05 was considered significant.Results: Already on admission, the monocyte activity was rapidlyand significantly reduced depending on the increase of injuryseverity. Even slightly injured persons (group I) showed a significantsuppression of the LPS-response on admission compared to healthyvolunteers (Tab. 1). The recovery of the monocyte activity also de-pends on the injury severity. Whereas the LPS-response of slightlyinjured patients (ISS≤8) reached the values of healthy volunteers onthe second day after trauma, the monocytic activity was still reducedin the groups with more severe injuries. A significant recovery of theLPS-response was observed on day four in group II. In patients withISS≥25 partial recovery of monocyte function evolved 6 days afteradmission. Male patients show significantly higher monocyteactivity than female patients, independent from ISS (12951 pg/mlvs 8056 pg/ml, p<0.05). Moreover, the abdominal injuries lead to asignificant decrease of the monocytic LPS-response (no abdominalinjury: 11962±1137 pg/ml vs abdominal injury: 6151±740 pg/ml, p<0.05).

Table 1: LPS-response on the day of admission

Group IL-1β [pg/ml]

I 13657±7682II 18201±3555III 8887±1825IV 13182±7369V 3485±978healthy 37556±4783volunteers

Conclusion: The present study provides evidence that i) even mildtrauma substantially impairs LPS-induced monocytic IL-1β-produc-tion; ii) the presence of abdominal injury enhances suppression of theLPS-response and that iii) the recovery of monocyte functionstrongly depends on the degree of the initial injury. Further study iswarranted to i) elucidate if neurohumeral factors play a role in thechanges seen after mild injury; ii) examine the influence of highlevels of extracellular ubiquitinase on monocytic function and to iii)check if abdominal trauma leads to an induction of classical LPS-tolerance in monocytes, possibly due to impaired gut barrier functionwith release of LPS-tolerance inducing substances.

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107Differential gene expression pattern of human monocytesafter gram positive and gram negative bacterial challengeSven K. Tschöke1, Lyle L. Moldawer2, Wolfgang Ertel1,Andreas Oberholzer11Department of Trauma and Reconstructive Surgery,Charité – University Hospitals Berlin, Campus Benjamin Franklin,Berlin, Germany2Department of Surgery, University of Florida College of Medicine,Gainesville, FL, USABackground: Primary or secondary microbial invasion leads to theinduction of varying pattern of mediator signaling, which major in-fluences the extent of the inflammatory response and ultimately de-fines the overall clinical outcome. In the present study,we attempted toevaluate the genome wide gene expression by isolated peripheralhuman blood monocytes (as predominant innate immune effectorcells) in response to either aGram-negative orGram-positive stimulus.Patients and methods: Human peripheral blood monocytes wereisolated from three healthy subjects and were then stimulated witheither 1 μg/ml LPS (Gram-negative bacterial cell wall component) or0.1% wt/vol heat-killed Staphylococcus aureus (SAC: heat-killedGram-positive bacteria) for 2 hours and 24 hours, respectively.Genome wide expression analysis was performed using theAffymetrix HG-U133 plus 2.0 oligonucleotide micro-array.Results: The hybridization intensity signals of 1445 probe sets (1092annotated genes) significantly differed in the 2 hour stimulationgroup (FDR <0.001), and 6017 probe sets (4018 annotated genes) inthe 24 hour stimulation group, respectively. In general, geneexpression in both timepoints demonstrated a predominant down-regulation including genes involved in various protein metabolismand stress-induced defense processes. A large number of distinctivelyregulated genes suggested pathognomonical properties to either theGram-negative or a Gram-positive stimulus.Conclusions: The complex molecular interrelations underlying thephysiological manifestation of sepsis appear to be pathogen-specific.Thus, in addition to general critical care treatment strategies, septicpatients may require novel diagnostic approaches and customtailored therapies based upon distinct molecular interactions inducedby either Gram-negative or Gram-positive bacteria. In addition, earlymonitoring of genetic or cellular immune activities may supportpreventative strategies in the critically ill patient and help guidemaximum sufficient care.

108Surface-modificated vascular prostheses:which animal model is suitable for investigations?Torsten Ueberrueck, Thorsten WahlersDepartment of Cardiothoracic and Vascular Surgery,Friedrich-Schiller University, Jena, GermanyBackground: Despite the fact that there are several animal modelsexisting, it remains unclear which approach has the optimal basis totest the biocompatibility of vascular prostheses (VP).Material and method:We performed a comparative analysis of twodifferent animal models. Commercially available polyester VP(n=12) were implanted into the infrarenal porcine aorta or ovinecarotid artery. Surgical handling, patency rate and especially healingbased of macroscopic, microscopic and immunohistochemical resultsas well as costs were analysed over a three months period.Results: Handling and operating time (63±10 vs. 76±16 min;p=0.125) did not differ significantly. In both experimental groups oneVP was occluded (patency rate of 83.7%). The sheep model hasshown better integration of the VP (in the sheep group all prostheseswere integrated complete, in the pig group two were complete undfour were incomplete respectively). Only in the pig a completeendothelialisation of the VPs was observed in contrast to the sheep

model where only the anastomosis area did show circular endothe-lialisation. The thickness of neointima did not differ significantly inthe region of the proximal anastomosis (pig vs. sheep in µm; 1413.2±873.2 vs. 674.5±275.5; p=0.134), but did differ significantly in themiddle of VP (1188.2±472.0 vs. 118.9±222.9; p=0.004) and of thedistal anastomosis (2272.8±871.6 vs. 676.2± 99.1; p=0.014).Immunohistochemically, only periprosthetic Ki67 was significantlyreduced (28.7±9.9 vs. 6±0.9%; p=0.002) in the sheep. Expenses forthe two animal models were comparable.Conclusions: In contrast to the sheep, the endothelialisation of VPcould be analysed more efficient in the porcine model. However thiscould be accompanied by a higher occlusion rate. On the basis of theobserved lower rate of neointimal hyperplasia, the sheep model isrecommended for long term follow up studies, whereas the porcinemodel is to take into considerations for short term studies.

109CdX-2 as a potent marker in the developmentof intestinal metaplasia of the esophagusDaniel Vallböhmer1, Steve DeMeester2, Jan Brabender1,Jeffrey H. Peters2, Kathleen D. Danenberg2, Paul M. Schneider1,Arnulf H. Hölscher1, Peter V. Danenberg3, Tom R. DeMeester21Klinik für Viszeral- und Gefäßchirurgie, Universität zu Köln,Köln, Germany2Department of Surgery, University of Southern California, LosAngeles CA, USA3USC/Norris Comprehensive Cancer Center, University of SouthernCalifornia, Los Angeles CA, USABackground: The pathogenesis of Barrett’s epithelium is poorlyunderstood. Evidence suggests that the metaplastic process begins bythe transformation of distal squamous epithelium to cardiac mucosawhich subsequently becomes intestinalized. The homeobox geneCdX-2 has been shown to be an important transcriptional regulator ofembryonic differentiation and maintenance of adult intestinal typeepithelium. The aim of this study was to evaluate the gene-expressionlevels of CdX-2 in squamous, cardiac and Barrett’s epithelium at thegastroesophageal junction.Patients and methods: Biopsies were obtained from the gastro-esophageal junction in patients with symptoms of GERD. Histolo-gical classifications of epithelial types included: 1) normal squamous(n=62); 2) cardiac mucosa (n=19); 3) oxynto-cardiac mucosa (n=14);4) intestinal metaplasia (n=15) and 5) duodenum (n=26) as acolumnar control. After laser capture microdissection, gene expres-sion levels of CdX-2 were measured by quantitative real-time PCR.Results: CdX-2 gene expression for each epithelial type is shown[Table]. Consistent with its known function, levels were highest induodenal and nearly absent in squamous mucosa. There was astepwise increase in CdX-2 expression from cardiac to oxynto-cardiacand to Barrett’s epithelium. Expression levels of cardiac and oxynto-cardiac mucosa were 40-70 times higher and Barrett’s mucosa 600times higher than that of squamous epithelium.Conclusions:Relative expression of the homeobox geneCdX-2, knownto induce differentiation of intestinal type epithelium, confirms a stepwiseprocess in the development of Barrett’s esophagus. CdX-2 may be apotential biomarker to detect the early transition to Barrett’s esophagus.

Mucosa N CdX-2, median p-value vs.(25th-75th percent.) squamous

Squamous 62 0.01 (0.01–0.05)Cardiac 19 0.4 (0.3–0.7) p<0.01Oxynto- 14 0.76 (0.2–1.1) p<0.01cardiacBarrett’s 15 6.72 (3.9–8.08) p<0.01Duodenum 26 39.64 (25.9-55.2) p<0.01

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110Initial posttraumatic translocation of the transcriptionfactor STAT1 is reduced in trauma patientsfacing an unfavorable outcomeH. Vester1, J. Stegmaier1, K. Ruckdeschel2, C. Kirchhoff 1,V. Bogner, J. Heesemann2, W. Mutschler1, P. Biberthaler11Chirurgische Klinik und Poliklinik,Ludwig-Maximilians-Universität, München, Germany2Max-von-Pettenkofer-Institut, Ludwig-Maximilians-Universität,München, GermanyBackground: Severe injury induces Systemic Inflammatory Re-sponse Syndrome and Multiple Organ Failure (MOF) which ispartially orchestrated by post-traumatic monocyte dysfunction. Inthis context, the recently characterized transcription factor SignalTransducers and Activators of Transcription (STAT1) seem to play apivotal role in animal models after burn injury and in murine LPSinduced sepsis. However, the dynamic of posttraumatic translocationof the transcription factor STAT1 into the nucleus of monocytesobtained from multiple injured patients remain unclear, so far.Therefore, the aim of this study was to analyze STAT1 translocationinto the nuclear compartment in monocytes of multiple injuredpatients in the very early posttraumatic period and compare theobtained results between patients of favorable or unfavorableoutcome, respectively.Material: Twenty-five patients with an Injury Severity Score (ISS)above 16 points were enrolled. Blood samples were drawn onadmission within 90 min after the traumatic event and at 6, 12, 24,48, and 72 h after injury. STAT1 translocation of monocytic nuclearprotein was analyzed by Electrophoretic Mobility Shift Assay(EMSA). The results were quantified by densitometry and are givenin [arbitrary units] by mean±SEM. In addition, monocytes of healthyvolunteers were analysed either native (negative control) or after LPSstimulation as positive control, respectively. Statistical analysis wasperformed by ANOVA on ranks and SNK-test.Results: The mean ISS was 40±2 points. 18 Patients survived, sevendied in the posttraumatic period. In the complete collective of traumapatients (n=25), nuclear translocation of STAT1 was significantlyincreased during the whole observation period (on admission: 398±81; 6h: 490±113; 12 h: 488±124; 24h: 417±90; 48h: 479±87; 72 h:501±120) as compared to the native control (259±34; p<0.05). De-ceased patients had a significant reduced nuclear translocation ofSTAT1 6h (223±59 vs. 594±150; p<0.05) and 12h (171±26 vs. 612±164; p<0.05) as compared to the survivors, respectively.Conclusion:Within this pilot study we demonstrate for the first timethe measurement of transcription factor STAT1 translocation into thenuclear compartment of monocytes obtained from multiple injurepatients in the very early posttraumatic period. Although trauma ingeneral seem to increase transcriptional activity of STAT1, patientsfacing an unfavourable outcome presented lower activity thansurvivors. Currently, several follow-up studies are on their way toclarify the potential biological consequence of these results byanalysis of reporter genes etc.

111Posttraumatic osteomyelitis: generation of osteoclastsunder proinflammatory conditions as possiblemechanism of septic looseningChristof Wagner1, Sabine Zimmermann2, Gerald Zimmermann1,Andreas Wentzensen1, Volkmar Heppert1, G. Maria Hänsch21Klinik für Unfall- und Wiederherstellungschirurgie,Berufsgenossenschaftliche Unfallklinik Ludwigshafen,Ludwigshafen, Germany2Institut für Immunologie, Universität Heidelberg, Heidelberg,Germany

Background: Posttraumatic osteomyelitis is a chronic inflammation,leading to tissue destruction and eventually to osteolysis by not yetknown mechanisms. We hypothesize that the local inflammatoryprocess which occurs in consequence of the bacterial infection andthe infiltrating polymorphonuclear neutrophils (PMN) creates amicroenvironment that induces the differentiation of blood mono-cytes to osteoclasts, the latter known for their bone resorbingcapacity.Patients and methods: The cytokine profile of PMN recovered bylavage of the infected site of patients with posttraumatic osteomye-litis was determined by real-time PCR. Based on these results, themononuclear cells of healthy donors were cultivated in the presenceof interleukin (IL)-8. Fusion of cells and differentiation was observedmicroscopically; in addition, monocyte- and osteoclast-specificantigens were identified by indirect immunofluorescence or substrateconversion, respectively.Results: Among the cytokines tested, IL-8 was prevalent in thelocally infiltrated PMN recovered from the lavage. Therefore, itseffect on mononuclear cells was assessed in vitro. Mononuclear cellswere isolated from the peripheral blood of healthy donors andcultivated in medium containing IL-8 (10 µg/ml). After 24 h, the non-adherent cells were removed; the culture medium was replaced every4 to 5 days, always substituted with IL-8. After 2 weeks, a few cellswith typical characteristics of osteoclasts appeared; their numberincreased with time. In parallel, the number of monocytes positive forCD14 declined. By 6 to 8 weeks, the osteoclasts were predominant,while the remaining monocytes accumulated in clusters. Theosteoclasts remained viable for up to 12 weeks.Conclusion: Proinflammatory cytokines, such as IL-8, induce thedifferentiation of peripheral blood monocytes to cells with osteoclast-like characteristics. Thus, it is feasible that the local inflammatoryresponse caused by the infiltrated PMN creates a microenvironmentpromoting the generation of bone resorbing cells which in turncontributes to osteolysis and septic loosening.Acknowledgement: This work was supported by the DeutscheForschungsgemeinschaft (DFG) grant Wa 1623/1-1.

112Surgical trauma: cellular mechanisms of mechanicalstretch activation in intestinal muscularismacrophages and smooth muscle cellsSven Wehner PhD., Silke Schuchtrup, Andreas Hirner M.D.,Joerg C. Kalff M.D.Department of Surgery, University of Bonn, Bonn, GermanyBackground: Mechanical trauma is the inevitable consequence ofabdominal surgical procedures that result in a strong inflammation ofthe whole intestine and finally in postoperative ileus. Previousinvestigations of our group demonstrate that the activation of residentintestinal muscularis macrophages is a key event in this localinflammation process. The aims of this study was 1) to determine ifintestinal macrophages are directly activated by mechanical traumaand hence are the primary trigger in ileus formation or 2) ifcoexistence (coculture) of macrophages and smooth muscle cells isrequired for stretch induced macrophage activation and the sub-sequent inflammation.Methods: For in vitro cell stretch experiments we generated a novelmurine cell line (iMAC-M7) by retroviral v-myc transfection incultures of intestinal muscularis macrophages. Characterisation ofthis cell line was performed by analysis of classical macrophageproperties by FACS and immunofluorescence. iMAC-M7 cells andprimary smooth muscle cells (SMC) were stretched on siliconmembranes (FlexCell® system) in a continuous or interruptedmanner for in vitro imitation of surgical trauma. Cellular activationwas determined by quantitative PCR of COX-2, MIP-1a and LFA-1in macrophages and COX-2 in SMC.

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Results: In this study we show that the novel cell line iMAC-M7 ischaracterised by F4/80, CD11b, and CD14 expression and also by ahigh phagocytosis rate and a strong MHC-II dependent T-cellactivation. Mechanical stretch of the intestinal cell cultures lead todifferent results depending on stretch-method and cell type. SMCshowed an early 7-fold COX-2 upregulation after 3 hours continuousstretch while iMAC-M7 cells did not show any changes in MIP-1a,LFA-1 or COX-2 gene expression. In contrast to continuous stretchmacrophages were slightly activated, however firstly after 24 hours(3-fold COX-2 and LFA-1 expression), by interrupted stretch. Inaddition, slight macrophage activation was detected by 2-fold MIP-1a expression in coculture of primary muscularis cell cultures(macrophages and smooth muscle cells).Conclusion:Wewere able -for the first time- to generate an intestinalmuscularis macrophage cell line. Our in vitro stretch results suggestthat mechanical stretch directly activates smooth muscle cells andmacrophages in a time- and stretch dependent manner. Due to theearly stretch mediated activation of SMC but not macrophages inpure cell cultures we postulate an initial role for SMC in formation ofileus that is mediated by a subsequent activation of resident muscu-laris macrophages leading to the strong underlying inflammation.

113Influence of human cytomegaloviruson the malignancy of prostate tumor cellsEva Weich, Jindrich Cinatl, Wolf-Dietrich Beecken, Tobias Engl,Dietger Jonas, Roman BlahetaKlinik für Urologie und Kinderurologie, Zentrum der Chirurgie,J. W. Goethe-Universitätsklinik, Frankfurt/Main, GermanyBackground: Based upon immunohistochemical data, humancytomegalovirus (HCMV) infection might be associated with tumorprogression and malignancy. However, there has been no concreteevidence of a causal link between HCMV infection and prostatecancer dissemination. Using an in vitro cell culture model, weinvestigated the influence of HCMV on prostate tumor progressionand invasion.Methods: Prostate adenocarcinoma (PC3) cells were infected withHCMV strain Hi (PC3Hi) or transfected with immediate early genes 1(IE1) and 2 (IE2). Adhesion capacity of PC3Hi to endothelial cellmonolayers or to extracellular matrix proteins (collagen, fibronectin,laminin) was measured and compared to adhesion behaviour of non-infected controls (PC3). Adhesion receptors of the ß1-integrin familyand the oncogene proteins c-myc, p53, p73, PTEN, P-PTEN, Drg-1and PPARγ were evaluated by flow-cytometry, western-blot and RT-PCR (PC3Hi versus PC3).Results: Adhesion experiments revealed a significant increase of thebinding capacity of PC3Hi to endothelial cells or to extracellularmatrix proteins, compared to controls. IE1 and IE2 were found not tobe responsible for this effect. Furthermore, PC3Hi exhibited higherß1-integrin surface level as well as ß1-integrin protein content thancontrol PC3 cells. Transfection of PC3 with IE1 and IE2 induced up-regulation of c-myc, p53, p73, PTEN and P-PTEN and down-regulation of PPARγ.Conclusion: HCMV infection of prostate tumor cells is accompa-nied by an increased cellular adhesion potency, ß1-integrin expres-sion and modulation of oncogenic proteins. Therefore, we concludethat HCMV infection promotes prostate tumor malignancy anddissemination.

114Cytokine activity in sera of patientswith delayed fracture healingGerald Zimmermann1, Christof Wagner1, Stefan Weiss2,Maria Hänsch3, Andreas Wentzensen1, Sabine Zimmermann31Unfallchirurgie, BG-Unfallklinik, Ludwigshafen, Germany2Orthopädische Klinik, Universität Heidelberg, Schlierbach,Germany3Institut für Immunologie, Universität Heidelberg, Heidelberg,GermanyBackground: Transforming growth factor (TGF)ß and bonemorphogenic proteins (BMP) are well crucial for fracture healing.In a previous study we showed that systemic TGFß levels weredecreased in patients with delayed fracture union; BMPs, in contrast,were not detected in the serum by ELISA. In the present study weaddressed the question whether serum of patients with fractures oflong bones contained a proliferation-inducing activity.Patients and methods: From patients with either normal (group 1)or delayed fracture healing (group 2), or from patients with non-healing fractures, who were treated with local implantation of BMP-7(group 3) serum samples were collected 1,2 or 4 weeks after thefracture, and again 1 year; the latter serves as baseline values. Therewere 10 patients in each group matched for age, sex, fracturelocalisation and fixation. The functional activity of the sera sampleswere tested using a model cell line CCL64, which either proliferatesin response to certain cytokines, or differentiates, as seen by growtharrest. The cells were kept in minimal medium for growth arrest andsynchronisation and then suspended in cell culture mediumsupplemented with 10 % patients serum and cell proliferation wasmeasured after 72 h by incorporation of 3H-thymidine. TGFß wasmeasured in the sera samples by ELISA.Results: Without substantial variation, in all patients, the systemicTGFß level was enhanced within the first two weeks when comparedto baseline; then the TGFß level declined with a deeper drop down inpatients with delayed union. A mitogenic activity for CCL64 wasonly found in the plasma of the patients with non-union fractures,particularly with the sera samples obtained 2 weeks after the fracture(160 %). Quite in contrast, plasma from patients having receivedBMB-7 rather inhibited the proliferation of CCL64; again mostobviously seen in the ‘2 week sample’. Sera of patients with normalfracture healing had a minor inhibitory effect on the CCL64proliferation.Conclusions: In patients with non-healing fractures a mitogenicactivity is found in the serum, while the serum from patients who hadreceived BMP-7 for therapy of such non-union fractures, containedan entity inducing growth arrest and supposedly differentiation of theCCL64. This leads to the conclusion that (a) the systemic peak TGFßlevels do not directly correlate with fracture healing; (b) thatdifferentiation, rather than proliferation, is the decisive factor forfracture healing, and (c) that BMP-7 might exerts its effects byinducing differentiation.

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Author index(The numbers are the abstract numbers)

Adili F 92Aleksic M 1Alini M 32Allgayer H 69, 73Alves F 76, 77Ammerpohl O 35, 79, 81Amon M 2Aslam R 5August C 42

Bachem MG 97Baerlecken N 3Bahde R 42Baldus SE 15Baumgart M 4Becker H 4, 33Becker HD 59, 105Beckert S 5Beecken W-D 84, 113Beißer M 6Beier JP 30Benjamin LC 43Berger H 38Bergert H 7, 8, 56Bhargava S 48Biberthaler P 6, 13, 25, 57, 72,75, 101, 110

Biller T 73Biro C 16, 106Blaheta R 84, 113Böcker W 10Bockhorn M 9Bogdanski D 12Bogner V 6, 13, 25, 57, 72, 75,101, 110

Böhner H 11Boos AM 14Borges J 3Börner T 62Borsello T 103Brabender J 15, 109Braumüller ST 97Brauns K 16, 106Broelsch CE 9, 58Broich K 27Broll R 23, 40, 91Bruch HP 23Bruch H-P 40, 91Brückel M 18Bruckner U 50Brüggemann A 17Brunkwall J 1Bruns CJ 18, 20, 49, 52, 93Bruns H 28Brunski JB 68Büchler MW 86, 87Bucsky B 2Buettner O 100Buhr HJ 34, 48, 78, 83

Čamaj P 18Carvalho C 53Celik I 19, 21, 45, 54

Chi H 58Cinatl J 113Coerper S 5Conrad C 20, 49Cooper-White J 22Currey J 68Czerny C 103

Dahmen U 58Dammann P 9Dandri M 82Danenberg KD 109Danenberg PV 109Dauner M 88Davies N 29DeMeester S 109DeMeester TR 109Detlev Saeger H 8DeToni E 18Dietl K-H 42Dietrich K 59, 105Dietz C 19, 21, 54Dietz K 99Difilippantonio MJ 33Dirsch O 58Docheva D 10, 90Dolderer J 22Doser M 67, 99Dubiel W 66Duchrow M 23Duda GN 55Dudda M 24

Eckstein H-H 38Egana T 70Egea-Alonso V 10Egginger C 25Eisenach PA 26Eitenmüller I 27Engl T 113Engl T 84Ern C 90Ertel W 107Ertel W 47Esenwein SA 24Esenwein SA 36

Farke S 23Farrahi F 5Felmerer G 14Fennel M 52Fiegel HC 28Findlay M 22Fisch K 63Fischlein T 29Fittkau M 29Foerster VT 30Freitag P 102Friedrich M 23Friedrichs R 11Frilling A 9Fuchs S 55Fuchs-Moll G 102

Gaßmann P 63Gawenda M 1Gebhard F 50Gebhard MM 86, 87Germann G 22Ghadimi BM 4, 33Gliemroth J 17Gogolewski S 32Goralski M 9Görg S 17Grad S 32Grade M 33Graeb C 93Grau V 37, 41, 94, 102Green T 52Greschus S 41Gretz N 76, 77Gröne J 34Gross W 86, 87Grünewald P 9Grützmann R 35, 77, 79, 81Grützner KU 73Guba M 18, 49, 52, 93

Haak M 31Haasters F 90Habel U 11Habler O 74, 80Haerle M 99Haier J 63Hamacher J 95Hanke M 38Hänsch GM 111Hänsch M 114Harder Y 2, 95, 96Hauser J 36Hauser M 36Heckenkamp J 1Hecker A 37Heeschen C 49, 93Heesemann J 101, 110Heiden E 34Heidenreich S 42Heider P 38Heidrich B 76, 77Heil M 27, 39Heinmöller E 4Heise M 51Hellmuth M 47Helms JA 68Hennig S 21Henrich D 16, 89, 103, 104,106

Henschler R 98Heppert V 111Hermann K 34Hetfeld B 66Hilbert M 40Hinz S 79Hinzmann B 34Hirner A 62, 112Hirschburger M 41Hoerbelt R 43, 44

Hoffmann S 45Hölscher AH 15, 109Holstein JH 46Hölzen JP 42Holzhüter S 28Hopf H 5Horch RE 100Horisberger K 31Hornung HM 73Hostmann A 47Hotz B 48, 78Hotz HG 48, 78Huber S 20, 49Huber-Lang M 50Huber-Lang MS 97Hunt T 5Husmann I 51Huss R 20

Ischenko I 52, 93

Jauch K-W 18, 20, 49, 52, 93Johnston DR 43, 44Jonas D 84, 113Junker D 53

Kabelitz D 79Kalff JC 62, 112Kalina U 19Kalthoff H 26, 35, 79, 81Kanse SM 21Kanz KG 75Karakas E 19, 54Kasper G 55Kaufmann PM 28Kaun M 17Kelch S 83Kerkadze V 86, 87Kersting S 7, 8, 56Kertscho H 74, 80Kim J-B 68Kirchhoff C 6, 13, 25, 57, 72,75, 101, 110

Kirkpatrick CJ 61Kisker O 19Klein C 98Kleinert R 58Klöppel G 35, 79, 81Kluge A 27Kluth D 28Kluth J 28Knaebel HP 86, 87Kneser U 28, 100Knoch K-P 7, 8, 56Knöferl MW 97Knubben K 59, 105Koeller M 36Kolios L 61Köller M 12, 60, 64Königsrainer A 5, 59, 67, 88,105

Korn K 81Koscielny A 62

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Kostin S 39Krasnyanska J 63Kratz K 83Krauß E 81Kreimeier U 25, 57Kremer B 79Krengel T 70Kroll V 60, 64Krueger U 65Krüger S 70Krump-Konvalinkova V 10Kuchenbuch T 41Kummer W 37Kutscha-Lissberg F 24

Ladurner R 59, 105Landes J 6, 25, 72Lang D 42Langelotz C 66Langer F 95Laout M 80Laschke MW 53Lauscher P 74Lau-Werner U 73Laven H 6Lehnert M 16, 103, 106Lembert N 67Lendlein A 83Lengyel E 69Leucht P 68Leupold JH 69, 73Liersch T 33Lindenmaier W 70Linti C 88Lips KS 37Liu F 70Lizdenis P 86Loniewska D 74Luetgehetmann M 82Lüttges J 35

Ma PX 82Machens H-G 2, 17, 70Madrahimov N 58Madrahimova F 58Madsen JC 43, 44Mailänder P 17Manekeller S 71Marzi I 16, 89, 103, 104, 106Matz M 72Matziolis G 55Maurer GD 73Meder S 23Meier J 74, 80Meisterfeld R 7, 8Menger MD 2, 46, 53, 95, 96Metzger R 15Minor T 71Moldawer LL 107Momeni A 3Morgalla MH 59, 105Morrison W 22Mueller T 75Muhr G 12, 24, 36, 60, 64Müller H-W 99

Müller S 106Musholt P 45Musholt T 45Mussack T 57Mutschler W 6, 10, 13, 25, 57,72, 75, 90, 101, 110

Mziaut H 56

Nagy P 82Nau U 94Neff J 76, 77Neuhaus P 51Niedergethmann M 76, 77Nies H 20Niess H 49Noltze A 17Nüllen H 85

Oberg H 79Oberholzer A 47, 107Obert M 41Ode A 55Ohmann C 11Ohnolz J 100Oppermann E 84Otto SD 78Ouwendijk J 7

PadbergW 37, 41, 43, 44, 94, 102Paku S 82Pape A 74, 80Pappou E 10Papyan A 93Perl M 97Perka C 55Peters JH 109Petersen J 82Pfeil U 37Pilarsky C 35, 77, 79, 81Planck H 88Plaß R 94Pohlemann T 46Pollok J-M 82Pommer A 24Post S 31, 69, 73, 76, 77Prokofiev D 9

Qing H 58

Raluy LP 79Reißfelder C 83Relja B 84Rewerk S 85Ried T 33Ries C 10Riester T 85Ritter R 92Ritz JP 83Röder C 79Rogiers X 82Rosenthal A 34Rösner H 99Roßmann O 10Roth B 28Ruckdeschel K 101, 110

Rücker M 53Rüschoff J 4Rüster B 98Ryschich E 86, 87

Sachs DH 43, 44Sader R 98Saeger HD 7, 8, 35, 56, 79, 81Sandmann W 11Schackert HK 35, 79, 81Schaefer DJ 100Schäfer H 26Schäfer U 54Schäfers H-J 95, 96Schaller H-E 99Schaper W 27, 39Schenk M 59, 88, 105Scherzed A 89Scheuenstuhl H 5Schewe DM 73Schieker M 10, 90Schildhauer TA 12, 60, 64Schlaak JF 9Schlamp A 91Schlosshauer B 99Schmandra TC 92Schmid G 49, 93Schmidmaier G 51Schmidt A 94Schmidt J 86, 87Schmits R 96Schmitz-Rixen T 27, 39, 92Schneider F 11Schneider PM 15, 109Scholz H 65Schramm R 95, 96Schreiber H 50Schuchtrup S 112Schulte-Eversum C 99Schütz C 6Schwandner O 40, 91Schwenk W 66Schwerdel F 80Seebach C 89, 104Seifried E 98Seitz DH 97Seitz O 98Settmacher U 51Shakibaei M 90Shojy T 43, 44Simon R 33Sinis N 99Sipos B 79Solimena M 7, 8, 56Soulaiman M 54Spiegel HU 42Stangenberg L 100Stark GB 3, 14, 30, 100Steche M 80Stegmaier J 6, 13, 57, 72, 75,101, 110

Stern-Straeter J 100Sucke J 102Sun-Young Lee V 103Sürücü O 19, 21, 54

Tenholt M 92Tewksbury R 104Tewksbury R 89Thiel C 59, 105Thuemmel T 16, 106Torio-Padron N 3Török É 82Traupe H 41Tribulova S 39Trost N 22Tschöke SK 47, 107Tuischer J 55

Ueberrueck T 108Ungefroren H 26, 79Unger R 61

Vallböhmer D 15, 109Varma S 33Vester H 101, 110Viebahn R 88von Gerstenbergk-HelldorffB 31

Wagner C 111, 114Wahlers T 108Waidmann M 67Walcher F 16, 106Ward PA 50Warnecke-Eberz U 15Wedel M 80Wehner S 112Weich E 113Weimer T 21Weiss M 50Weiss S 114Weiss W 38Weitz-Schmidt G 96Wentzensen A 111, 114Werther M 4Wesch D 79Weyand M 29Wiemer J 34Wierer M 90Wildgruber M 38Wilhelm K 89, 104Willeke F 31, 76, 77Wimmer MA 32Winkler M 85Wolf O 38Wunderlich A 45

Yezhelyev M 52

Zanow J 65Zerial M 81Zielke A 45Zilla P 29Zimmermann G 111, 114Zimmermann S 111, 114Zipfel A 88Zwißler B 74Zwissler B 80

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