Organoide (Patient Derived Organoids, PDOs) können mit hoher Effizienz aus gastrointestinalen Tumoren gewonnen werden und zeigen hohe Übereinstimmung mit dem Ursprungsgewebe. Daher können sie mittels ex vivo Wirkstofftests zur Etablierung neuer Therapiestrategien nützlich sein.

RAS-Mutationen gehören zu den häufigsten Mutationen bei Darmkrebspatienten und haben therapeutische Konsequenzen. Der Transkriptionsfaktor PPARgamma spielt eine noch unbekannte Rolle bei der Entwicklung serratierter Läsionen der kolorektalen Karzinogenese, die durch Überexpression des humanen, mutierten KRAS Onkogens im Darmepithel der Maus ausgelöst werden kann.

Einleitung: Transgene KRAS-Mausmodelle entwickeln Läsionen der serratierten Histo-Morphologie der kolorektalen Karzinogenese. RAS induziert die Produktion von Chemo-/Zytokinen und fördert die maligne Transformation des Darmepithels. Das Immunsystem spielt dabei eine ambivalente Rolle, so kann es neben der Tumorkontrolle auch kolorektale Karzinome (KRK) fördern, indem tumorassoziierte Makrophagen (TAMs) immunsuppressive und tumorfördernde Wirkungen zeigen. Gleichzeitig haben Immuntherapien zur Behandlung maligner Erkrankungen große Bedeutung erlangt.

BACKGROUND and Aims: We and others have previously shown, that the hepatic endocannabinoid (EC) system becomes activated and plays an important role in liver fibrogenesis. However, cellular origin and mechanisms that lead to increased EC levels (e.g. 2-arachydonoyl glycerol, 2-AG) are unknown. We analysed expression of the major 2-AG-generating enzyme diacylglycerol lipase (DAGL)-beta and the main degrading enzyme monoacylglycerol-lipase (MGL) in different hepatic cell populations as well as in normal and fibrotic mouse livers at different timepoints after bile duct ligation (BDL). Methods: Primary mouse hepatocytes, hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) were isolated by collagenase perfusion or MACS, respectively. 2-AG was measured by LC/MS in mouse livers 2, 7 and 14 days after BDL or sham operation (n=6 each). Alpha-SMA, DAGL-beta, FAAH and MGL mRNA and protein expression were determined by qRT PCR and Westernblot. Results: Hepatic 2-AG levels were increasingly elevated >2.5-fold 14 days (p< 0.05) after BDL. Interestingly, mRNA and protein expression of the 2-AG production enzyme DAGL beta was highest in LSECs, second highest in HSCs and third highest in KCs, the degradation enzyme MGL was highest in LSECs and HSCs, respectively. Hepatocytes did not express notable levels of either enzyme. During the timecourse of fibrogenesis, mRNA and protein expression of DAGL-beta in whole liver tissue was significantly increased, whereas MGL expression decreased significantly, thus correlating with increasing 2-AG levels and alpha-SMA expression. Conclusion: In the liver, 2-AG is mainly produced and degraded by LSECs and HSCs. Elevated 2-AG levels during fibrogenesis result from increased production and decreased degradation. The data further points to LSECs or HSCs rather than hepatocytes or KCs being the most important hepatic cell populations in 2-AG metabolism in fibrogenesis.

Hintergrund/Ziele: Wir und andere konnten kürzlich zeigen, dass das hepatische Endocannabinoidsystem während der Fibrogenese aktiviert wird und hierbei eine wichtige Rolle spielt. Insbesondere werden Cannabinoid-Rezeptoren und die intrahepatischen Spiegel von Endocannabinoiden (EC, z.B. 2-Arachidonoylglycerol, 2-AG) hochreguliert. Allerdings sind die zelluläre Herkunft der EC sowie die Mechanismen, die zu deren erhöhten Spiegeln führen, unbekannt. Deshalb wurden die Expressionen des wichtigsten 2-AG-Produktionsenzyms Diacylglycerollipase (DAGL)-beta sowie der Abbauenzyme Monoacylglycerollipase (MGL) und Fettsäureamidhydrolase (FAAH) in verschiedenen hepatischen Zellpopulationen sowie in normalen und fibrotischen Mäuselebern zu verschiedenen Zeitpunkten nach Gallengangsligatur (BDL) analysiert.

Background & Aims: Mutation of Jagged-1 gene, a Notch ligand, leads to Alagille syndrome. However, patients with Alagille syndrome usually do not progress into serious liver fibrosis. In the present study, we investigated the role of Jagged-1 in liver fibrogenesis. Methods: Jagged-1 expression in diseased human and mouse livers were evaluated by immunohistochemistry and Western blot. The effect of a Jagged-1 was examined in hepatic stellate cells (HSCs). Results: Immunohistochemistry and Western blot analysis revealed that protein expression of Jagged-1 in liver tissues from 2-week bile duct ligated mice was significantly increased compared to sham operated mice. Consistently, enhanced Jagged-1 expression was found in 80 HBV and 65 HCV patients, when compared to 5 normal liver samples by immunohistochemistry. In vitro, a gamma-secretase inhibitor (GSI), representing a general inhibitor for Notch receptor signaling, markedly decreased TGF-beta induced mRNA expression of collagen I as well as protein expression of alpha-smooth muscle actin and connective tissue growth factor (CTGF) in rat HSCs. Furthermore, knock-down of Jagged-1 by using siRNA significantly decreased protein expression of collagen I and CTGF in CFSC cells, a cell line from CCl4-induced cirrhotic rat HSCs. Conclusions: Jagged-1 plays a crucial role in liver fibrogenesis possibly via influencing activation of HSCs and extracelluar matrix production.

POSTERSniche microdissected from healthy livers (Mann Whitney U test, p < 0.05).Immunohistochemistry confirmed the expression of BMI1 on the majority of activated LPCs in all types of liver disease, and intermittently on cholangiocytes. Conclusions:The results clearly show that BMI1 is a relevant marker of activated progenitor cells in the liver and may be involved in self-renewal of LPCs, as it is in the progenitor cells of other adult tissues.

Einleitung: Wir und andere konnten kürzlich zeigen, dass das hepatische Endocannabinoid (EC)-System während hepatischer Schädigung und Fibrogenese aktiviert wird. Insbesondere werden die Expression von Cannabinoidrezeptoren sowie die Spiegel der ECs Anandamid (AEA) und 2-Arachidonoyl-Glycerol (2-AG) heraufreguliert. Jedoch sind die Mechanismen, die zum intrahepatischen Anstieg der ECs während der Fibrogenese führen, bislang unbekannt.

Background: We have shown that the endcannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) selectively induce cell death in activated hepatic stellate cells (HSCs), but not in hepatocytes. In contrast to HSCs, hepatocytes highly express the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) that protects them from AEA-induced injury. However, the hepatic expression pattern and function of the 2-AG-degrading enzyme monoacyl glycerol lipase (MGL) has not been investigated. Methods: MGL expression was analyzed by western blot and real time PCR in primary rat hepatocytes and primary rat HSCs. MGL or FAAH activity was determined colorimetrically. Cell death was analyzed by LDH release, propidium iodide uptake and western blot for caspase 3- and PARP cleavage. MGL or FAAH overexpression was achieved by adenoviral infection. Results: In contrast to FAAH, MGL mRNA, protein and activity did not significantly differ in hepatocytes and HSCs. Hepatocytes which had been pretreated with the MGL inhibitor URB754 were not sensitized towards 2-AG-mediated death, indicating a minor role for MGL in the resistance against 2-AG. Moreover, MGL overexpression did not render HSCs resistant towards 2-AG. Conversely, FAAH overexpression prevented 2-AG-induced death in HSCs. Accordingly, 2-AG induced necrotic cell death in hepatocytes which had been pretreated with the FAAH inhibitor URB597 or depleted of GSH and 2-AG induced an increase in ROS production in hepatocytes which had been pretreated with the FAAH inhibitor URB597. Conclusion: The main 2-AG degrading enzyme MGL is not differentially expressed in hepatocytes or HSCs and does not appear to play a crucial role for the resistance of hepatocytes against 2-AG-induced cell death. In contrast, the high expression of the primary AEA-metabolizing enzyme FAAH in hepatocytes and its absence in HSCs accounts for the differential susceptibility of these hepatic cell populations towards cell death mediated by endocannabinoids.

Fragestellung: Die chronische Hepatitis C verläuft in Gegenwart von Risikofaktoren wie dem chronischen Alkoholkonsum und assoziierten erhöhten Blutkonzentrationen von Lipopolysaccharid (LPS) ungünstiger. In früheren Studien konnte zudem gezeigt werden, dass sowohl Alkohol als auch LPS die antivirale Therapie beeinträchtigen. Ziel der Arbeit war es, den Einfluss von Alkohol und/oder LPS auf die Replikation von HCV und auf die Interferonwirkung zu prüfen. Methoden: Nutzung des Replikon-Systems. Quantifizierung der HCV-Replikation durch Luciferase-Reporter-Gen. Berücksichtigung der Proliferation der Huh-7-Zellen mittels MTT-Test. Ergebnisse: In einer 72-Stunden-Kinetik fand sich die stärkste Replikation nach ca. 48 Stunden. Dieser Replikationsgipfel entfiel bei Stimulation mit LPS (p<0,05). Einzelstimulation mit LPS senkte die HCV-Replikation bei 10µg/ml auf 77% (p<0,01), unter 0,1µg/ml LPS auf 88% (p<0,01) und unter 0,001µg/ml auf 90% (p<0,05) der Kontrolle. Einzelstimulation mit 1 I.E./ml Interferon-alpha senkte die HCV-Replikation auf 42% (p<0,01) der Kontrolle. Einzelstimulation mit 50mM Ethanol steigerte die Replikation des HC-Virus auf 125% (p<0,01) des Kontrollwertes, mit 100mM Ethanol auf 133% der Kontrolle (p<0,01). Bei Doppelstimulation LPS+Interferon ergab sich bei 0,1µg/ml LPS und 1 I.E./ml Interferon-alpha ein Replikationsrückgang auf 43% (p<0,01) der Kontrolle, entsprechend 102% der IFN-Einzelstimulation. Bei Doppelstimulation LPS+Ethanol ergab sich bei 0,001µg/ml LPS und 50mM Ethanol ein Replikationsanstieg auf 127% (p<0,01) der Kontrolle, entsprechend 102% der Ethanol-Einzelstimulation. Schlussfolgerung: Die Replikation des HC-Virus wird durch LPS allein gehemmt. Die Stärke der Hemmung korreliert positiv mit der applizierten LPS-Konzentration. In Kombination mit Ethanol und/oder Interferon-alpha kann die Hemmwirkung von niedrigen Konzentrationen von LPS abgeschwächt werden oder im Einzelfall auch zu einer Zunahme der HCV-Replikation führen.

Einleitung: Hepatische Sternzellen (HSCs) sind die primären fibrogenen Zellen in der fibrotischen Leber. Selektive Apoptose von HSCs stellt einen neuen antifibrotischen Therapieansatz dar. Wir haben kürzlich gezeigt, dass das Endocannabinoid 2-Arachidonoylglycerol (2-AG) die Proliferation von HSCs blockieren sowie Apoptose induzieren kann. Hepatozyten sind jedoch gegenüber diesen Mechanismen resistent. Der exakte molekulare Mechanismus dieser selektiven Zelltod-Induktion in HSCs ist noch nicht geklärt.

Einleitung: Wir konnten kürzlich zeigen, dass das Endocannabinoid Anandamid (Arachidonoylethanolamid, AEA) die Proliferation von hepatischen Sternzellen (HSCs), den primären fibrogenen Zellen der Leber, blockieren sowie deren Apoptose induzieren kann. Hepatozyten sind jedoch gegenüber diesen Mechanismen resistent. Die Wirkungen weiterer Endocannabinoide wie z.B. N-Arachidonoyldopamin (NADA) wurden bislang noch nicht untersucht.

Hintergrund: Das Interleukin (IL)-18 Bindungsprotein (bp) ist ein natürlicher Inhibitor des proinflammatorischen IL-18. Die Regulation der IL-18 und IL-18bp Expression in intestinalen Epithelzellen ist unvollständig charakterisiert. Ein besseres Verständnis ist Voraussetzung für eine gezielte therapeutische Modifikation IL-18-vermittelter intestinaler Inflammation. Ziel der Studie war die Identifizierung von Signaltransduktionswegen, die an der IFN-γ-induzierten Expression von IL-18bp beteiligt sind.

Expression of IL-18 in intestinal epithelial cells (IEC) has been implicated in Th1 cell-mediated chronic intestinal inflammation and anti-tumor immunity. However, physiological regulatory factors have not been identified. Besides their effects on proliferation and restitution, immunomodulatory functions have been attributed to short chain fatty acids (SCFA). We investigated the effect of SCFA (butyrate, propionate, acetate) on expression of IL-18 in IEC in vitro and in vivo. Expression of IL-18 mRNA and protein in human carcinoma-derived HT-29 and Caco-2 cells was analyzed by reverse transcription-PCR and Western blot. Transcriptional regulation of IL-18 gene expression was determined by transient transfection of wild-type and mutated IL-18 promoter. Further, in vivo expression of IL-18 in the intestine from butyrate-treated and untreated mice was assessed by immunohistochemistry. IL-18 mRNA and the IL-18 protein were expressed in IEC, while IL-18 secretion was not observed. Butyrate and acetate increased intracellular IL-18 content in a time- and dose-dependent fashion. In contrast to proinflammatory stimuli butyrate potently activated the IL-18 promoter, indicating that IL-18 is regulated at the transcriptional level by SCFA. Furthermore, a 108-bp sequence in the proximal region was identified to be essential for IL-18 promoter activation by butyrate. As proof of principle butyrate effects were confirmed in vivo by demonstration of increased IL-18 protein expression in IEC from butyrate-treated mice. In conclusion, SCFA up-regulate IL-18 protein expression in IEC, suggesting a potential regulatory contribution of these luminal constituents to T cell mediated inflammatory and neoplastic intestinal conditions.

Background: We have previously reported that some avirulent Salmonella strains have the ability to attenuate inflammatory responses in colonized epithelial cells by inhibiting the IKBubiquitination step of the NF-KB pathway (Neish, AS et al.Science.289:1560).The eukaryotic or prokaryotic determinants that mediate this phenomenon are unknown.Objective: To investigate the mechanism by which anti-inflammatory Salmonella strains inhibit NF-KB activation.Methods: Model polarized intestinal epithelia were prepared by culturing T-84 cells on permeable supports.ELISA was used to measure IL-8 secretion from colonized monolayers.Activation of NF-KB was assessed by electrophoretic mobility shift assay (EMSA) and immunofluorescent microscopy.Results: Colonization of model epithelia with anti-inflammatory strains of Salmonella (PhoF ~ and S. pu//orum) attenuated IL-8 secretion in response to TNF-e, while virulent strains (wild type) or avirulent strains with structural defects in the type lit secretion apparatus (invA-, invG-) did not reduce TNF induced IL-8 secretion.In the presence of prokaryotic protein synthesis inhibitors (gentamicin or chloramphenicol), the anti-inflammatory effects on IL-8 secretion were not affected.When apically applied cultures of antHnflammatory strains were physically separated from the T-84 monolayer by a .22#m filter that allowed passage of putative soluble bacterial factors, the observed anti-inflammatory effects on epithelial IL-8 secretion were abolished, suggesting that intimate contact is required.By EMSA, antiinflammatory strains prevented TNF-induced NF-KB activation.Immunofluorescent microscopy revealed that the p65 subunit of NF-KB formed aggregates in cells colonized by the antiinflammatory Salmonella strains but not in cells colonized by wild-type or type III secretiondeficient strains.Conclusions: Anti-inflammatory effects of nonpathogenic Salmonella require bacterial-epithelial contact, are protein synthesis-independent, and likely involve type litmediated secretion.Furthermore, anti-inflammatory bacteria may block NF-KB translocation by inducing formation of inactive NF-KB aggregates.

Surveillance endoscopy for BE remains contentious.Audit of our Barrett's database was undertaken to assess the incidence of dysplasia.The database includes patients on active follow-up from 1989 and subsequent new diagnoses of BE.Definition of BE: either ~3cm columnar epithelium ::' :: specialised intestinal metaplasia (SIM) or <3cm with SIM.Information was derived from case notes, endoscopy and histology records.Excluding patients presenting with esophageal cancer, 149 patients were identified of which 9 had documented low grade dysplasia (LGD) and 3 high grade dysplasia (HGD).An additional 6 patients were thought to have LGD but this was down-graded to regenerative changes after histological review, and none progressed to frank dysplasia.104 (70%) of patients were put on active annual surveillance.See table .LGD was diagnosed at the following surveillance endoscopies: 3 at 1st, 2 at each of 2nd and 3rd, 1 at each of 4th and 5th.LGD resolved in 5 of 9 patients; follow-up 1-5 years.It persisted with no progression in 3; follow-up 1-3 years. 1 patient was diagnosed with LGD and a co-existing squamous cell carcinoma within the BE at his 4th surveillance endoscopy.All patients with HGD had it at initial presentation.1 had an esophagectomy, 1 remains on 6 monthly surveillance, and 1 developed adenocarcinoma after 2 years follow-up (not surgically fit).The only common finding in this cohort of 104 surveillance patients was LGD which frequently regressed or remained static.Only 2 patients developed cancer, (one an unusual squamous cancer within the BE) giving an incidence of 1:145 patient years of follow-up.

Patient derived organoids (PDOs) closely resemble individual tumor biology and allow testing of small molecules ex vivo. To systematically dissect compound effects on 3D organoids, we developed a high-throughput imaging and quantitative analysis approach. We generated PDOs from colorectal cancer patients, treated them with >500 small molecules and captured >3 million images by confocal microscopy. We developed the software framework SCOPE to measure compound induced re-organization of PDOs. We found diverse, but re-occurring phenotypes that clustered by compound mode-of-action. Complex phenotypes were not congruent with PDO viability and many were specific to subsets of PDO lines or were influenced by recurrent mutations. We further analyzed specific phenotypes induced by compound classes and found GSK3 inhibitors to disassemble PDOs via focal adhesion signaling or that MEK inhibition led to bloating of PDOs by enhancing of stemness. Finally, by viability classification, we show heterogeneous susceptibilities of PDOs to clinical anticancer drugs.