Einleitung: HGF (Hepatocyte Growth Factor) ist ein pleiotropes Zytokin, das eine wichtige Bedeutung für die Regeneration von Hepatozyten besitzt. Im Mausmodell konnte gezeigt werden, dass HGF die Todesrezeptor-vermittelte Apoptose von Hepatozyten inhibiert.

Background: Local treatment of HCC induces inflammatory reactions and tumor specific immune responses, which may contribute to a favorable clinical outcome. Whether this holds true for systemic treatment with kinase inhibitors has not been studied. Hence, we have investigated cytokine profiles of liver and HCC tissue in presence of Sorafenib to analyse its immune modulatory capacity.

AIM:The aim of this trial was to evaluate the impact of conversion from a calcineurin-inhibitor (CNI)-based immunosuppressive regimen to mycophenolate mofetil (MMF) and reduced-dose CNI on long-term renal function and survival in a series of 63 liver transplant patients with CNI-induced renal dysfunction.METHODS:CNI dosage was significantly tapered after introduction of 2,000 mg MMF per day. Renal function was assessed by determination of serum creatinine levels and calculated creatinine clearance (CCl). The impact of relevant clinical parameters on renal function and survival post-conversion was analyzed by univariate and multivariate analysis.RESULTS:At 60 months post-conversion, mean creatinine level had significantly declined from 197.2±58.3 μmol/l at baseline to 160.0±76.5 μmol/l, and mean CCl has significantly increased from 38.4±13.4 ml/min at baseline to 47.9±21.1 ml/min (p<0.001), respectively. Forty-six patients (73.1%) demonstrated sustained renal response to modified immunosuppression. Full-dose MMF medication (p=0.006) and the early conversion (p=0.02) were identified as independent predictors of persistent renal function improvement. Sustained renal response to MMF plus reduced-dose CNI was identified as the most relevant independent promoter of long-term survival (hazard ratio 6.9). Five-year survival rate post-conversion was 93.9% in renal responders and 64.3% in renal non-responders (log rank<0.001).CONCLUSIONS:Sustained renal response to MMF and CNI dose reduction promotes long-term survival in liver transplant patients with CNI-induced renal dysfunction.

Die Ergebnisse nach simultaner Nieren-Pankreas-Transplantation (NPTx) und nach orthotoper Lebertransplantation (LTx) haben sich die letzten Jahre nicht zuletzt dank moderner Immunsuppression verbessert. Inzwischen gehört die NPTx zur etablierten Therapie bei Patienten mit Typ-I-Diabetes und terminaler Niereninsuffizienz. Patienten nach erfolgreicher NPTx haben nicht nur eine höhere Lebenserwartung, sondern auch eine bessere Lebensqualität. Nach NPTx liegen die 1-Jahres-Transplantatüberlebensraten >85%, Abstoßungsraten <10% sind erreichbar. Bei der LTx wird die Immunsuppression Grund- und Begleiterkrankungen des Patienten angepasst; dabei kommt der Nephroprotektion durch die MELD-Allokation höchste Bedeutung zu, da das Serumkreatinin eine entscheidende Rolle bei der Berechnung der Dringlichkeit zur LTx spielt. Calcineurininhibitoren werden daher oft verzögert und niedriger dosiert eingesetzt. Darüber hinaus ist eine steroidfreie Immunsuppression heute klinische Realität. Der Entwicklung neuer, nicht nephrotoxischer, nicht diabetogener Substanzen ohne gastrointestinale Nebenwirkungen darf gespannt entgegengesehen werden.

Purpose: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1 α . The effects of PHD3 in tumour growth are largely unknown. Experimental design: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model. Results: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo , PHD3 inhibited tumour growth by abrogation of tumour angiogenesis. Conclusion: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

Nach Pankreasresektion bei duktalem Pankreasadenokarzinom (Pa-Ca) überleben manche Patienten nur einige Monate, obwohl die 5-Jahresüberlebsrate bei über 20% liegt. Die Ursache hierfür ist nicht bekannt, wenngleich einige tumoreigene Faktoren diskutiert werden. Eine kurative Resektion, Grading und TNM-Stadium sind bekannte postoperative Überlebens-Prädiktoren. Präoperative Faktoren zur Einschätzung des Überlebens sind nicht verfügbar. In eine elektronische Datenbank wurden 460 Patienten die zwischen 10/2001 und 02/2007 aufgrund eines Pa-Ca reseziert wurden prospektiv aufgenommen. Zwei Gruppen von Patienten wurden definiert: Patienten die <12 Monate nach Resektion überlebten (n=119) und Patienten die mindestens 24 Monate überlebten (n=95). Präoperative Faktoren die mit dem Kurz- oder Langzeitüberleben assoziiert waren wurden mittels logistischer Regression identifiziert und durch eine Univarianz- und Multivarianzanalyse an der Gesamtgruppe überprüft (Log-Rank-Test, Cox-Model). Geschlecht, BMI, Magenausgangssymptomatik, Ikterus, Diabetes mellitus, ASA-Score, Hämoglobin, Kreatinin, Harnstoff, Bilirubin, Cholinesterase, Gamma-GT, Albumin und die Prothrombinzeit zeigten keine Korrelation mit dem Überleben. Dagegen war Alter >70 Jahre, C-reaktives Protein ≥25mg/l, präoperatives CA19–9 ≥500U/ml, CEA ≥15ng/ml und mäßiger bis starker präoperativer Schmerz mit signifikant schlechterem Überleben in der multivarianten Analyse assoziiert. In einer stratifizierten Überlebensanalyse gemäß der Anzahl dieser Risikofaktoren betrug das mediane Überleben und die 3-Jahres Überlebensrate der Patienten ohne (n=184), mit einem (n=171) oder mit mehr als einem (n=105) dieser Risikofaktoren 27 Monate und 27,8%, 18 Monate und 16,8%, beziehungsweise 11 Monate und 19% (p<0.0001). Die Hazard Ratios für einen Risikofaktor und für mehr als einen Risikofaktor stiegen auf 1,6 beziehungsweise 2,5 an verglichen mit Patienten ohne Risikofaktor. Die identifizierten klinischen Marker können präoperativ die Überlebenszeit von Patienten die für eine Resektion eines duktalen Pankreasadenokarzinom geplant sind vorhersagen, und somit bei der präoperativen Entscheidungsfindung hinsichtlich der Auswahl der Patienten die von einem chirurgischen Eingriff hinsichtlich des Überlebens profitieren, helfen.

Little is known about the factors that enable the mobilisation of human mesenchymal stem cells (MSC) from the bone marrow into the blood stream and their recruitment to and retention in the tumour. We found specific migration of MSC towards growth factors present in pancreatic tumours, such as PDGF, EGF, VEGF and specific inhibitors Glivec, Erbitux and Avastin interfered with migration. Within a few hours, MSC migrated into spheroids consisting of pancreatic cancer cells, fibroblasts and endothelial cells as measured by time-lapse microscopy. Supernatant from subconfluent MSC increased sprouting of HUVEC due to VEGF production by MSC itself as demonstrated by RT-PCR and ELISA. Only few MSCs were differentiated into endothelial cells in vitro , whereas in vivo differentiation was not observed. Lentiviral GFP-marked MSCs, injected in nude mice xenografted with orthotopic pancreatic tumours, preferentially migrated into the tumours as observed by FACS analysis of green fluorescent cells. By immunofluorescence and intravital microscopic studies, we found the interaction of MSC with the endothelium of blood vessels. Mesenchymal stem cells supported tumour angiogenesis in vivo , that is CD31 + vessel density was increased after the transfer of MSC compared with siVEGF-MSC. Our data demonstrate the migration of MSC toward tumour vessels and suggest a supportive role in angiogenesis.

Genetic modification of human bone marrow mesenchymal stem cells (MSC) is highly valuable for their exploitation in basic science and therapeutic applications, for example in cancer. We present here a new, fast and easy-to-use method to enrich a functional population of lentiviral (LV)-transduced MSC expressing enhanced green fluorescent protein (eGFP). We replaced the eGFP gene by a fusion gene of puromycin acetyltransferase and eGFP. Upon LV gene transfer and puromycin selection, we quickly obtained a pure transduced MSC population, in which growth, differentiation capacity and migration preferences were not compromised. Furthermore, we are the first to report the migration velocity of MSC among which 30% were moving and velocity of about 15 μm h −1 was not altered by LV transduction. Manipulated MSC underwent senescence one passage earlier than non-transduced cells, suggesting the use for therapeutic intervention in early passage numbers. Upon tail vein application in nude mice, the majority of LV-transduced MSC could be detected in human orthotopic pancreatic tumor xenografts and to a minor extent in mouse liver, kidney and lung. Together, LV transduction of genes to MSC followed by puromycin selection is a powerful tool for basic research and improves the therapeutic prospects of MSC as vehicles in gene therapy.

Benigne und niedrigmaligne Tumoren des Pankreaskorpus können in der Regel durch Standardoperationen wie eine erweiterte partielle Pankreatikoduodenektomie oder eine erweiterte Pankreaslinksresektion reseziert werden. Der unvermeidbare Verlust von großen Teilen gesunden Pankreasparenchyms bei diesen beiden Operationsverfahren kann zu einer endokrinen und exokrinen Pankreasinsuffizienz führen.

Cell membrane disruption take place in different cell systems under physiological and pathological conditions. We tested the hypothesis that disruption of acinar cell membranes take place in the onset of acute pancreatitis.

Hintergrund: Zellmembranverletzungen finden in Zellsystemen unter physio- und pathologischen Situationen statt. Eine stattgefundene Membranverletzung kann durch den Einsatz von extrazellulären (EZR) Tracern, die intrazellulär (IZR) nicht vorkommen, bestätigt werden. Treten Zellmembranverletzungen auf, können EZR-Bestandteile in den IZR-Raum gelangen und dabei negative Milieu-Veränderungen hervorrufen. Ziel der Studie war es zu analysieren, ob Zellmembranverletzungen bei der akuten Pankreatitis eine Rolle spielen.

Surgical resected tumours are often stored for hours in the clinic upon transfer to the bench leading to apoptosis of tumour cells making them no longer suitable for molecular analysis and diagnostic procedures. The way out of this problem may be a new oxygen-enriched solution (OES). We tested this agent using surgical resections of carcinomas of lung, rectum and pancreas. Immediately after resection, one part of each individual tumour was stored in PBS and the other part in OES, and the content of viable or dead cells was determined by trypan blue exclusion and MTT-assay. We found that OES keeps tumour cells up to 3 days and longer more viable than PBS and reduces the percentage of dead cells without inducing therapy resistance and affecting the outcome of experimental procedures. Thus, storing freshly resected tumours in OES may save time for tumour transfer and initiation of experiments.

Treatment options for ductal adenocarcinoma of the pancreas are limited by early lymphatic spread, but the lymphatic vessels in pancreatic carcinoma have not been studied to date. Here, we present a histomorphological analysis of lymphatic vessels in pancreatic cancer resection specimens. Both intratumoral and peritumoral tissue were devoid of active lymphangiogenesis. Intratumoral lymphatics were frequently collapsed and non-functional, whereas peritumoral lymphatic vessels were enlarged, and numerous lymphatic vessels were seen in metastases. In addition, we screened pancreatic cancer tissue and pancreatic carcinoma cell lines for mRNA expression of the lymphangiogenic growth factor, VEGF-C; its receptor, VEGFR-3/flt4; and Prox1, a transcription factor essential for embryonic development of both lymphatic vessels and the pancreatic bud. VEGF-C was abundantly expressed in pancreatic cancer tissue and -cell lines and VEGFR-3/flt4 was expressed in cancer stromal cells. Prox1 was strongly expressed in the normal exocrine pancreas but significantly reduced in pancreatic cancer specimens from patients with short survival rates. Well-differentiated cell lines displayed higher levels of Prox1 mRNA than poorly differentiated ones. These results suggest that active lymphangiogenesis is not required for lymphovascular spread of pancreatic cancer. VEGF-C may promote local tumor growth via paracrine signaling to stromal cells expressing VEGFR-3 and support the entry of cancer cells into peritumoral lymphatics. Furthermore, loss of Prox1 function may be a driving force behind pancreatic carcinoma progression.

TNF-related apoptosis-inducing ligand (TRAIL) zeigt potente Anti-Tumor-Wirkung nach systemischer Applikation in-vitro und in-vivo, ohne die bekannten Nebenwirkungen anderer TNF-Familienmitglieder, wie TNF und CD95L in Phase I/II Studien aufzuweisen. Allerdings sind mehr als 50% der Tumoren resistent für TRAIL-induzierte Apoptose. Behandlung resistenter Tumorzellen mit verschiedenen Chemotherapeutika sensitiviert die meisten gastrointestinalen Tumorzellen für TRAIL-induzierte Apoptose. Allerdings gibt es eine große Kontroverse über potentielle toxische Effekte von TRAIL Liganden auf primäre humane Hepatozyten im Gegensatz zu murine Hepatozyten, insbesondre in der Kombination mit Chemotherapeutika. Unabhängig von den verschiednen TRAIL Formen gibt es sich widersprechende Ergebnisse was die Sensitivität von primären Hepatozyten gegenüber TRAIL angeht.

Bei 30% der Patienten mit chronischer Pankreatitis findet sich eine entzündliche Pankreaskopfvergrößerung. Eine Operationsindikation ergibt sich meist durch ein massives Schmerzsyndrom, Choledochusobstruktion, Duodenalobstruktion, Pankreasgangobstruktion oder Obstruktion der großen retropankreatischen Gefäße. In randomisiert kontrollierten Studien konnten vergleichbare Ergebnisse für die duodenumerhaltende Pankreaskopfresektion nach Beger und die Frey Operation bei diesen Patienten gezeigt werden. Bei genauer Betrachtung dieser Studien scheint die Durchtrennung der Bauchspeicheldrüse über der Pfortader, wie bei der Beger-Operation, sowie die komplette Längseröffnung des Pankreasganges, wie bei der Frey-Operation, nicht notwendig für die guten Ergebnisse beider Operationsmethoden zu sein. Durch die Kombination der Vorteile beider Operationen wurde eine neue, einfachere Methode, die Zentrale Pankreaskopfresektion, entwickelt.

The HER2/neu oncogene is overexpressed in up to 70% of human pancreatic cancer specimens when compared to normal pancreatic tissue. This cell surface receptor can be targeted specifically by the neutralizing antibody Herceptin. Herceptin has been successfully used in combination with other chemotherapeutic agents in breast cancer, a cancer in which only 30% of patients harbor elevated HER2/neu levels. In the present study, we investigated the therapeutic efficacy of Herceptin in combination with gemcitabine and docetaxel. Gemcitabine is currently the standard chemotherapeutic agent used to treat pancreatic cancer. In contrast, docetaxel, a taxane, is only just being investigated in pancreatic cancer. Tumor cell resistance to taxanes is at least in part mediated by the HER2/NEU oncogene. We have previously characterized HER2/NEU expression in human pancreatic cancer cell lines and studied the anti-tumor activity of Herceptin monotherapy in vitro and in vivo. In the present study, combination therapy resulted in a dramatic improvement of animals bearing human pancreatic cancer xenografts. Furthermore, metastasis and production of ascites was lower when a combination of these three agents was used. We conclude that, as with breast cancer, the anti-tumor activity of Herceptin may be improved by combination with taxanes or gemcitabine.

OBJECTIVE:To analyze the potential role of the Notch signaling pathway in pancreatic cancer angiogenesis and invasion.BACKGROUND:Angiogenesis, pain, and early neuroinvasion are clinical features of pancreatic cancer. Blood vessels and nerves develop together and use common routes through the organism. The Notch pathway (Notch-1/4, Jagged-1/2, Delta-1) appears crucial in this process. The current study analyzed the Notch pathway in pancreatic cancer and characterized its angiogenic and invasive effects.METHODS:Five PaCa cell lines were cultured for the in vitro experiments. Real-time quantitative RT-PCR was done to quantify mRNA expression in 31 human PaCa specimens, and immunohistochemistry was used to localize protein expression within tumor specimens. Activation of the Notch signaling was done by transfection of PaCa cells with a constitutive active Notch-1 mutant (Notch-IC). Overexpression of Jagged and Delta was achieved by transfection of full-length cDNA. Spheroid assays were used to study angiogenesis and ELISAs to measure VEGF, bFGF, and angiogenin expression. Matrigel invasion assays were used to analyze tumor cell invasion.RESULTS:Notch-3 and Notch-4 mRNA were significantly (P < 0.001) overexpressed in PaCa. Immunohistochemistry revealed protein accumulation of Notch-1 as well. All ligands were significantly up-regulated. A positive immunosignal of ligands was seen in nerves, blood vessels, and ductal tumor cells. Transfection of PaCa cells with the constitutive active Notch-IC mutant and with Jagged-1 revealed increased levels for VEGF. Concomitantly, recombinant Jagged-1 increased sprouting of endothelial cells in the spheroid assay.CONCLUSION:The Notch pathway most likely regulates neurovascular development in pancreatic cancer. Activation of this signaling pathway by constitutive Notch-1 mutants and by Jagged-1 causes an angiogenic and invasive tumor phenotype. Specific blockade of Notch signaling may therefore be beneficial for patients with pancreatic cancer.

CD95 (APO-1/Fas)-mediated apoptosis of hepatocytes plays a central role in the pathophysiology of various human liver diseases. Hepatocyte growth factor (HGF) was shown to exert antiapoptotic functions in rodent hepatocytes. We previously showed that primary human hepatocytes (PHH) are a valuable tool for the investigation of apoptotic processes in liver cells. In this study, we analyzed the influence of HGF on CD95-mediated apoptosis of PHH and its molecular determinants. HGF significantly inhibited CD95-mediated apoptosis of PHH as well as cleavage of caspase-8 and poly (ADP-ribose)polymerase. HGF transcriptionally induced the expression of the anti-apoptotic Bcl-2 family member myeloid cell leukemia-1 (Mcl-1). In contrary, HGF did not alter the expression levels of Bcl-2 or Bcl-x(L). HGF activated survival pathways such as the phosphatidylinositol-3 kinase (PI3K)/Akt pathway, the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK and the signal transducer and activator of transcription 3 (STAT3) pathway. Notably, HGF triggered serine(727)--but not tyrosine(705)--phosphorylation of STAT3. Pretreatment of PHH with the PI3K inhibitor LY294002 as well as adenoviral transduction of dominant negative Akt1 prevented HGF-mediated Mcl-1 induction and reversed the antiapoptotic effects of HGF. In conclusion, HGF confers survival of PHH by activation of the PI3K/Akt pathway. PI3K/Akt activation by HGF results in the induction of antiapoptotic proteins such as Mcl-1. Thus, application of HGF may be a therapeutic approach to prevent CD95-mediated hepatocellular damage in human liver diseases.