Paul Ritschl focusses on "The Impact of Donor Derived Microparticles Following Solid Organ Transplantation". Mentors are Priv.-Doz. Dr. Schmelzle and Priv.-Doz Dr. Öllinger.
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Mathilde Feist and Paul Ritschl: Clinician Scientists
Paul Ritschl focusses on "The Impact of Donor Derived Microparticles Following Solid Organ Transplantation". Mentors are Priv.-Doz. Dr. Schmelzle and Priv.-Doz Dr. Öllinger.
Nanomolar sensing of NAD
Authors are P. Brunnbauer, A. Leder, C. Kamali, K. Kamali, E. Keshi, K. Splith, S. Wabitsch, P. Haber, G. Atanasov, L. Feldbrügge, I.M. Sauer, J. Pratschke, M. Schmelzle, and F. Krenzien.
Nicotinamide adenine dinucleotide (NAD), a prominent member of the pyridine nucleotide family, plays a pivotal role in cell-oxidation protection, DNA repair, cell signalling and central metabolic pathways, such as beta oxidation, glycolysis and the citric acid cycle. In particular, extracellular NAD+ has recently been demonstrated to moderate pathogenesis of multiple systemic diseases as well as aging. Herein we present an assaying method, that serves to quantify extracellular NAD+ in human heparinised plasma and exhibits a sensitivity ranging from the low micromolar into the low nanomolar domain. The assay achieves the quantification of extracellular NAD+ by means of a two-step enzymatic cycling reaction, based on alcohol dehydrogenase. An albumin modified revised simulated body fluid was employed as standard matrix in order to optimise enzymatic activity and enhance the linear behaviour and sensitivity of the method. In addition, we evaluated assay linearity, reproducibility and confirmed long-term storage stability of extracellular NAD+ in frozen human heparinised plasma. In summary, our findings pose a novel standardised method suitable for high throughput screenings of extracellular NAD+ levels in human heparinised plasma, paving the way for new clinical discovery studies.
Normothermic ex vivo machine perfusion
Authors are J. Gassner, M. Nösser, S. Moosburner, R. Horner, P. Tang, L. Wegener, D. Wyrwal, F. Claussen, R. Arsenic, J. Pratschke, I.M. Sauer, and N. Raschzok.
Normothermic ex vivo liver machine perfusion might be a superior preservation strategy for liver grafts from extended criteria donors. However, standardized small animal models are not available for basic research on machine perfusion of liver grafts. A laboratory-scaled perfusion system was developed consisting of a custom-made perfusion chamber, a pressure-controlled roller pump, and an oxygenator. Male Wistar rat livers were perfused via the portal vein for 6 hours using oxygenated culture medium supplemented with rat erythrocytes. A separate circuit was connected via a dialysis membrane to the main circuit for plasma volume expansion. Glycine was added to the flush solution, the perfusate, and the perfusion circuit. Portal pressure and transaminase release were stable over the perfusion period. Dialysis significantly decreased the potassium concentration of the perfusate and led to significantly higher bile and total urea production. Hematoxylin and eosin staining and immunostaining for ssDNA and activated caspase 3 showed less sinusoidal dilatation and tissue damage in livers treated with dialysis and glycine. While Kupffer cells were preserved, tumor necrosis factor α mRNA levels were significantly decreased by both treatments. For proof of concept, the optimized perfusion protocol was tested with DCD grafts, resulting in significantly lower transaminase release into the perfusate and preserved liver architecture compared to baseline perfusion.
Our laboratory-scale normothermic portovenous ex vivo liver perfusion system enables rat liver preservation for 6 hours. Both dialysis and glycine treatment were shown to be synergistic for preservation of the integrity of normal and DCD liver grafts.
Hendrik Napierala defended his thesis summa cum laude
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Tumor-stromal cross-talk in PDAC
Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant solid tumor with a dismal prognosis. The stroma component makes up to 90% of the tumor mass and is thought to be one of the main reasons for the tumor's high chemoresistance. Cancer associated fibroblasts (CAFs) have previously been identified to be the key stromal players. This is the first time we provide detailed in vitro experiments investigating tumor-stromal interactions when exposed to three well-known chemotherapeutic agents.
Monocultures, indirect and direct co-cultures of two PDAC cell lines (AsPC and Panc-1) and six primary patients derived CAFs were treated with gemcitabine, nab-paclitaxel and the γ-secretase-inhibitor (GSI) DAPT. The cell viability of each component was measured with XTT. Finally, IL-6 concentrations of the supernatants were analyzed.
On the contrary to PDAC cell lines, CAF monocultures hardly responded to any treatment which suggested that stroma (CAFs) itself is more resistant to standard chemo-treatments than the epithelial cancer cells. Moreover, only a weak chemotherapeutic response was observed in direct co-cultures of cancer cells with CAFs. A change in the morphology of direct co-cultures was accompanied with the chemoresistance. CAFs were observed to build cage-like structures around agglomerates of tumor cells. High levels of IL-6 were also associated with a reduced response to therapy. Indirect co-cultures make the tumor-stromal interaction more complex.
Prof. Marcus Bahra Charité W2 professor
Since 2014 he held an extraordinary professorship for Surgery at the Charité.
Wibke Schulte: BIH Charité Clinician Scientist
Barbara Kern: BIH Charité Clinician Scientist
