Alfred Koenigsrainer

Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system.

BACKGROUND & AIMS Neural stem and progenitor cells from the enteric nervous system have been proposed for use in cell-based therapies against specific neurogastrointestinal disorders. Recently, enteric neural progenitors were generated from human neonatal and early postnatal (until 5 years after birth) gastrointestinal tract tissues. We investigated the proliferation and differentiation of enteric nervous system progenitors isolated from human adult gastrointestinal tract. METHODS Human enteric spheroids were generated from adult small and large intestine tissues and then expanded and differentiated, depending on the applied cell culture conditions. For implantation studies, spheres were grafted into fetal slice cultures and embryonic aganglionic hindgut explants from mice. Differentiating enteric neural progenitors were characterized by 5-bromo-2-deoxyuridine labeling, in situ hybridization, immunocytochemistry, quantitative real-time polymerase chain reaction, and electrophysiological studies. RESULTS The yield of human neurosphere-like bodies was increased by culture in conditional medium derived from fetal mouse enteric progenitors. We were able to generate proliferating enterospheres from adult human small or large intestine tissues; these enterospheres could be subcultured and maintained for several weeks in vitro. Spheroid-derived cells could be differentiated into a variety of neuronal subtypes and glial cells with characteristics of the enteric nervous system. Experiments involving implantation into organotypic intestinal cultures showed the differentiation capacity of neural progenitors in a 3-dimensional environment. CONCLUSIONS It is feasible to isolate and expand enteric progenitor cells from human adult tissue. These findings offer new strategies for enteric stem cell research and future cell-based therapies.

Circadian Expression of Clock- and Tumor Suppressor Genes in Human Oral Mucosa

Purpose: Circadian rhythms are daily oscillations of multiple biological processes driven by endogenous clocks. Imbalance of these rhythms has been associated with cancerogenesis in humans. To further elucidate the role circadian clocks have in cellular growth control, tumor suppression and cancer treatment, it is revealing to know how clock genes and clock-controlled genes are regulated in healthy humans. Materials and Methods: Therefore comparative microarray analyses were conducted investigating the relative mRNA expression of clock genes throughout a 24-hour period in cell samples obtained from oral mucosa of eight healthy diurnally active male study participants. Differentially expressed selected genes of interest were additionally evaluated using qRT-PCR. Results: Microarray analysis revealed 33 significant differentially regulated clock genes and clock- controlled genes, throughout a one day period (6.00h, 12.00h, 18.00h, 24.00h). Hereof were 16 clock genes and 17 clock- controlled genes including tumor suppressor- and oncogenes. qRT-PCR of selected genes of interest, such as hPER2, hCRY1, hBMAL1, hCCRN4L and hSMAD5 revealed significant circadian regulations. Conclusion: Our study revealed a proper circadian regulation profile of several clock- and tumor suppressor genes at defined points in time in the participants studied. These findings could provide important information regarding genes displaying the same expression profile in the gastrointestinal tract amounting to a physiological expression profile of healthy humans. In the future asynchronous regulations of those genes might be an additional assistant method to detect derivations distinguishing normal from malignant tissue or assessing risk factors for cancer.

Percutaneous management of a hepatic artery aneurysm: bleeding after liver transplantation.

In this artical we present an unusual case of hepatic artery aneurysm bleeding due to a hepatic artery thrombosis after liver transplantation. The patient developed a recurrent hepatic artery thrombosis leading to severe graft failure in four consecutive liver transplantations. While being evaluated for a fifth transplant, stabilization of the clinical situation was attempted by interventional therapy. The first intervention was to place a stent into the hepatic artery to prevent further ischemic damage. This failed to improve graft function, but unfortunately led to the development of a pseudoaneurysm at the distal end with a subsequent rupture into the biliary tree. Bleeding was treated successfully by direct puncture and coil embolization of the aneurysm. In addition, the patient demonstrated a hemodynamically relevant portal vein stenosis on the CT scan. Stenting of the portal vein markedly improved graft function. After extensive investigations, a paroxysmal nocturnal hemoglobinuria was found to be the underlying cause of the recurrent hepatic artery thrombosis. Here we suggest that hepatic artery aneurysm bleeding is a rare but potentially fatal complication that can be successfully treated by percutaneous coil embolization. Additionally, we propose that stenting of the portal vein can lead to a significant improvement of the graft perfusion even though the hepatic artery remained occluded.

Successful Coil Embolization of a Ruptured Gastroduodenal Artery Aneurysm

Aneurysms of the gastroduodenal artery are rare. Reported here is the case of a 60-year-old woman suffering from the covered rupturing of a twin aneurysm of the gastroduodenal artery. The patient presented herself in the surgical emergency unit with abdominal discomfort. Diagnostics showed free fluid in the abdominal cavity together with anemia of 9.9 g/dL. A computed tomography scan and an angiography revealed the covered rupturing of a twin aneurysm of the gastroduodenal artery, which was treated by endovascular coiling of the gastroduodenal and pancreaticoduodenal arteries. The patients hemoglobin level remained stable after treatment, and she was released from the hospital after 18 days. Visceral artery aneurysms are rare. Although endovascular therapy is preferred in cases involving active bleeding, surgery remains the primary therapy in those cases in which bleeding becomes uncontrollable.

Abstract A115: Cancer vaccine development for hepatocellular carcinoma – HEPAVAC

The HEPAVAC Consortium aims to develop a highly innovative, novel cancer vaccine approach for hepatocellular carcinoma (HCC). The international project consortium consists of 9 European Partners from academia and the biotech industry with complementary and substantial expertise in developing immunotherapeutic strategies to treat cancer. The project has started in September 2013 and is supported by the European Commission9s 7th Framework Program (www.hepavac.eu). HCC/normal adjacent tissue matched samples have been collected for HLA immunopeptidome analysis. 17 HCC samples from HLA-A*02+ patients and 15 samples from HLA-A*24+ patients have been analysed by mass spectrometry (LC-MS/MS). RNA-expression profiles have been established for 12 HCC samples. HLA-presentation/expression of peptides and mRNA on primary HCC samples are compared to n>140 normal tissue samples from relevant organs (including heart, brain, lung, kidney, liver, nerve, skin etc.) from Immatics9 database. A total of 9051 HLA-A*02-restricted different tumor-associated peptides (TUMAPs) have been identified from HLA-A*02+ samples, while a total of 3286 different HLA-A*24-restricted TUMAPs have been identified from HLA-A*24+ samples. Of these, 33 HLA-A*02+ TUMAPS and 33 HLA-A*24+ TUMAPs are currently in the process of validation, including peptide synthesis, immunogenicity testing and pharmaceutical evaluation. Most promising TUMAP candidates show selective expression only in HCC samples and no expression in normal tissues. In parallel, more than 6600 HLA-DR TUMAPs have been identified in Hep3B cells transfected with CIITA as well as an average of 1500 HLA-DR TUMAPs have been identified in HCC samples. Additional HLA-DR TUMAPs derived by distinct CIITA-transfected cell lines with different HLA-DR haplotypes are under scrutiny. The discovery phase of the HEPAVAC project is proceeding according to the proposed timelines. HCC-specific epitopes to be included in the vaccine cocktail will be selected in the coming weeks for GMP production. In parallel, preclinical studies assessing the formulation and combination of the immunological RNA-based adjuvant (RNAdjuvant®) with peptide cocktails are underway. Citation Format: Luigi Buonaguro, Sarah Kutscher, Roberto Accolla, Yuk T. Ma, Regina Heidenreich, Francesco Izzo, Alfred Koenigsrainer, Markus Loeffler, Phillip Mueller, Andrea Mayer, Hans-Georg Rammensee, Bruno Sangro, Sven Francque, Danila Valmori, Toni Weinschenk, Harpreet Singh-Jasuja. Cancer vaccine development for hepatocellular carcinoma – HEPAVAC. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A115.