Jens Werner

Molecular Control of Systemic Bile Acid Homeostasis by the Liver Glucocorticoid Receptor

Systemic bile acid (BA) homeostasis is a critical determinant of dietary fat digestion, enterohepatic function, and postprandial thermogenesis. However, major checkpoints for the dynamics and the molecular regulation of BA homeostasis remain unknown. Here we show that hypothalamic-pituitary-adrenal (HPA) axis impairment in humans and liver-specific deficiency of the glucocorticoid receptor (GR) in mice disrupts the normal changes in systemic BA distribution during the fasted-to-fed transition. Fasted mice with hepatocyte-specific GR knockdown had smaller gallbladder BA content and were more susceptible to developing cholesterol gallstones when fed a cholesterol-rich diet. Hepatic GR deficiency impaired liver BA uptake/transport via lower expression of the major hepatocyte basolateral BA transporter, Na(+)-taurocholate transport protein (Ntcp/Slc10a1), which affected dietary fat absorption and brown adipose tissue activation. Our results demonstrate a role of the HPA axis in the endocrine regulation of BA homeostasis through the liver GR control of enterohepatic BA recycling.

Outcome of surgery for pancreatic neuroendocrine neoplasms

The incidence of pancreatic neuroendocrine neoplasms (pNEN) is increasing. This study aimed to evaluate predictors of overall survival and the indication for surgery.

Complementary induction of immunogenic cell death by oncolytic parvovirus H-1PV and gemcitabine in pancreatic cancer

ABSTRACT Novel therapies employing oncolytic viruses have emerged as promising anticancer modalities. The cure of particularly aggressive malignancies requires induction of immunogenic cell death (ICD), coupling oncolysis with immune responses via calreticulin, ATP, and high-mobility group box protein B1 (HMGB1) release from dying tumor cells. The present study shows that in human pancreatic cancer cells (pancreatic ductal adenocarcinoma [PDAC] cells; n = 4), oncolytic parvovirus H-1 (H-1PV) activated multiple interconnected death pathways but failed to induce calreticulin exposure or ATP release. In contrast, H-1PV elevated extracellular HMGB1 levels by 4.0 ± 0.5 times (58% ± 9% of total content; up to 100 ng/ml) in all infected cultures, whether nondying, necrotic, or apoptotic. An alternative secretory route allowed H-1PV to overcome the failure of gemcitabine to trigger HMGB1 release, without impeding cytotoxicity or other ICD activities of the standard PDAC medication. Such broad resistance of H-1PV-induced HMGB1 release to apoptotic blockage coincided with but was uncoupled from an autocrine interleukin-1β (IL-1β) loop. That and the pattern of viral determinants maintained in gemcitabine-treated cells suggested the activation of an inflammasome/caspase 1 (CASP1) platform alongside DNA detachment and/or nuclear exclusion of HMGB1 during early stages of the viral life cycle. We concluded that H-1PV infection of PDAC cells is signaled through secretion of the alarmin HMGB1 and, besides its own oncolytic effect, might convert drug-induced apoptosis into an ICD process. A transient arrest of cells in the cyclin A1-rich S phase would suffice to support compatibility of proliferation-dependent H-1PV with cytotoxic regimens. These properties warrant incorporation of the oncolytic virus H-1PV, which is not pathogenic in humans, into multimodal anticancer treatments. IMPORTANCE The current therapeutic concepts targeting aggressive malignancies require an induction of immunogenic cell death characterized by exposure of calreticulin (CRT) as well as release of ATP and HMGB1 from dying cells. In pancreatic tumor cells (PDAC cells) infected with the oncolytic parvovirus H-1PV, only HMGB1 was released by all infected cells, whether nondying, necrotic, or succumbing to one of the programmed death pathways, including contraproductive apoptosis. Our data suggest that active secretion of HMGB1 from PDAC cells is a sentinel reaction emerging during early stages of the viral life cycle, irrespective of cell death, that is compatible with and complements cytotoxic regimens. Consistent induction of HMGB1 secretion raised the possibility that this reaction might be a general “alarming” phenomenon characteristic of H-1PVs interaction with the host cell; release of IL-1β points to the possible involvement of a danger-sensing inflammasome platform. Both provide a basis for further virus-oriented studies.

Somatic Mutations in Exocrine Pancreatic Tumors: Association with Patient Survival

KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application.

Irreversible electroporation of the pig kidney with involvement of the renal pelvis: technical aspects, clinical outcome, and three-dimensional CT rendering for assessment of the treatment zone.

PURPOSE To analyze irreversible electroporation (IRE) of the pig kidney with involvement of the renal pelvis. MATERIALS AND METHODS IRE of renal tissue including the pelvis was performed in 10 kidneys in five pigs. Three study groups were defined: group I (two applicators with parallel configuration; n = 11), group II (three applicators with triangular configuration; n = 2), and group III (six applicators with complex configuration; n = 3). After IRE and before euthanasia, pigs underwent contrast-enhanced computed tomography (CT). Technical aspects (radial distance of applicators, resulting mean current), clinical outcome (complications, blood samples), and three-dimensional CT rendering for assessment of the treatment zone (short axis, circularity) were assessed. RESULTS Radial distances of applicators were 14.3 mm ± 2.8 in group I, 12.3 mm ± 1.9 in group II, and 16.4 mm ± 3.5 in group III. Resulting mean currents were 25.7 A ± 6.5 in group I, 27.0 A ± 7.1 in group II, and 39.4 A ± 8.9 in group III. In group III, two perirenal hematomas were identified. There was no damage to the renal pelvis. During IRE, clinical blood parameters and cardiovascular markers did not change significantly. Short axis measurements were 20.6 mm ± 3.6 in group I, 31.9 mm ± 8.2 in group II, and 39.3 mm ± 2.4 in group III (P < .01 between groups). Circularity scores were 0.8 ± 0.2 in group I, 0.7 ± 0.1 in group II, and 0.7 ± 0.1 in group III, with a score of 1 indicating perfect roundness (P value not significant). CONCLUSIONS IRE of the pig kidney with involvement of the renal pelvis is feasible and safe. Size but not shape of the treatment zone is significantly affected by applicator configuration.

In vivo immunoregulatory properties of the novel mitochondria-targeted antioxidant SkQ1.

Reactive oxygen species (ROS) is a group of highly reactive oxygen-containing chemicals. ROS are essential for various biological functions, including cell survival and growth, proliferation and differentiation. At the same time ROS production is connected to a number of disorders, such as chronic inflammation, age-related diseases and cancers. In the immune system, ROS are involved in the defence of the host organism, immune response and immune regulation. One of the main sites of ROS generation in the cell is mitochondrial electron transport. In contrast to a number of traditional antioxidants, the novel mitochondria-targeted antioxidant SkQ1 exerts its antioxidant properties even in nanomolar concentrations. In this work, we investigated immunomodulatory properties of SkQ1 and demonstrated that treatment of mice with SkQ1 led to a decrease in percentage of CD8(+) T cells but not of CD4(+) T cells. We documented a decrease of a relative number of naïve T cells with a simultaneous increase in percentage of effector memory T cells. Central memory T cells had also a trend to be increased after SkQ1 treatment. In fraction of dendritic cells, we found an increase in percentage of plasmacytoid dendritic cells. In the case of myeloid cells, SkQ1 treatment decreased significantly the percentage of granulocytes. No effect of SkQ1 was observed on regulatory T cells, natural killer cells, natural killer T cells, as well as on freshly isolated CD8(+) T or CD4(+) T cells, indicating the indirect influence of SkQ1 on immune cells.

Effects of pantoprazole in experimental acute pancreatitis.

AIMS Oxidative stress with free radicals plays a crucial role in acute pancreatitis (AP). Pantoprazole (PPZ), widely used as a proton pump inhibitor, possesses reactivity towards hydroxyl radicals. The aim of the study was to examine the effect of PPZ on the course of experimental AP. MAIN METHODS Mild AP was induced in rats by caerulein (n=12). Severe AP was induced by infusion of glycodeoxycholic acid (10mM) into the pancreatic duct combined with caerulein (n=12). Both AP models were randomized to PPZ treatment (20mg/kg at baseline and after 12h) or placebo. Control animals received Ringer solution (n=6) without AP induction. After 24h severity of AP was examined by histology, enzyme levels, edema and inflammatory markers (myeloperoxidase, protein profiling). Furthermore, CD62P and CD31 for leukocyte and platelet activation were investigated. KEY FINDINGS Histology showed that PPZ treatment reduced tissue infiltration of inflammatory cells and acinar cell necrosis in severe AP. After PPZ treatment CD62P expression in mild AP and CD31 expression in severe pancreatitis decreased, indicating an inhibition of platelet activation. In mild and severe AP, PPZ significantly decreased amylase, LDH, edema and myeloperoxidase activity. Protein profile of pancreatic juice and serum revealed different spectra and less pancreatic juice proteins in PPZ treated groups indicating less acinar cell leakage. SIGNIFICANCE PPZ possesses anti-inflammatory in vivo properties and attenuates the course of AP. This is mediated via a reduced expression of inflammatory and adhesive proteins with a consecutive decrease in platelet and leukocyte activation as key steps in the pathogenesis of AP.

Neue Möglichkeiten in der retinalen Diagnostik mittels OCT-Angiografie

BACKGROUND Instruments for using OCT angiography (OCTA) in daily clinical practice have recently become available. The aim of this paper is to report the possibilities, advantages and limitations of OCTA in the clinical diagnosis of diseases of the posterior segment of the eye. PATIENTS/METHODS Patients with diabetic retinopathy, retinal vascular occlusions, and age-related macular degeneration who had been assigned to fluorescein angiography (FA) additionally underwent an AngioPlex™-OCTA examination, which captures a 6 × 6 mm scanning area centred on the fovea. If deemed necessary, 3 × 3 mm volume scans were created in regions of interest. The findings of FA and OCTA were correlated and compared. RESULTS The OCTA procedure took only a few seconds, was easily integrated into the standard OCT diagnostic imaging procedure, and delivered highly detailed, three dimensional images of the entire microvasculature of the retina and choroid. Microvascular changes, such as microaneurysms, intraretinal microvascular abnormalities, non-perfused areas, alterations in the foveal avascular zone (FAZ) and neovascularization were reliably detected. Overall, OCTA results were in good agreement with the results of the FA. Additionally, OCTA provided more detailed and three dimensional information and thus permitted a better assessment of the spatial extension of microvascular abnormalities. Due to OCTAs limited scanning area, vascular alterations in the mid-periphery were detected only if their location had already been determined by FA. Although OCTA does not show leakage, macular oedema can be diagnosed from OCTA, together with OCT thickness measurements. CONCLUSION OCTA provides important three dimensional information on vascular alterations and is already an indispensable diagnostic method. As the procedure takes just a few seconds and can be performed non-invasively, OCTA is well suited as a monitoring method and may allow early diagnosis. In this sense, prospective studies are required to determine precise OCTA analytical strategies for specific diseases. It is very likely that OCTA will revolutionise the diagnosis of retinal and choroidal diseases; however, it is not yet clear estimated to what extent it will replace FA.

Chondroitin Sulfate Proteoglycan CSPG4 as a Novel Hypoxia-Sensitive Marker in Pancreatic Tumors

CSPG4 marks pericytes, undifferentiated precursors and tumor cells. We assessed whether the shed ectodomain of CSPG4 (sCSPG4) might circulate and reflect potential changes in CSPG4 tissue expression (pCSPG4) due to desmoplastic and malignant aberrations occurring in pancreatic tumors. Serum sCSPG4 was measured using ELISA in test (n = 83) and validation (n = 221) cohorts comprising donors (n = 11+26) and patients with chronic pancreatitis (n = 11+20) or neoplasms: benign (serous cystadenoma SCA, n = 13+20), premalignant (intraductal dysplastic IPMNs, n = 9+55), and malignant (IPMN-associated invasive carcinomas, n = 4+14; ductal adenocarcinomas, n = 35+86). Pancreatic pCSPG4 expression was evaluated using qRT-PCR (n = 139), western blot analysis and immunohistochemistry. sCSPG4 was found in circulation, but its level was significantly lower in pancreatic patients than in donors. Selective maintenance was observed in advanced IPMNs and PDACs and showed a nodal association while lacking prognostic relevance. Pancreatic pCSPG4 expression was preserved or elevated, whereby neoplastic cells lacked pCSPG4 or tended to overexpress without shedding. Extreme pancreatic overexpression, membranous exposure and tissuehigh/seralow-discordance highlighted stroma-poor benign cystic neoplasm. SCA is known to display hypoxic markers and coincide with von-Hippel-Lindau and Peutz-Jeghers syndromes, in which pVHL and LBK1 mutations affect hypoxic signaling pathways. In vitro testing confined pCSPG4 overexpression to normal mesenchymal but not epithelial cells, and a third of tested carcinoma cell lines; however, only the latter showed pCSPG4-responsiveness to chronic hypoxia. siRNA-based knockdowns failed to reduce the malignant potential of either normoxic or hypoxic cells. Thus, overexpression of the newly established conditional hypoxic indicator, CSPG4, is apparently non-pathogenic in pancreatic malignancies but might mark distinct epithelial lineage and contribute to cell polarity disorders. Surficial retention on tumor cells renders CSPG4 an attractive therapeutic target. Systemic ‘drop and restoration’ alterations accompanying IPMN and PDAC progression indicate that the interference of pancreatic diseases with local and remote shedding/release of sCSPG4 into circulation deserves broad diagnostic exploration.

Specific CT 3D rendering of the treatment zone after Irreversible Electroporation (IRE) in a pig liver model: the “Chebyshev Center Concept” to define the maximum treatable tumor size

BackgroundSize and shape of the treatment zone after Irreversible electroporation (IRE) can be difficult to depict due to the use of multiple applicators with complex spatial configuration. Exact geometrical definition of the treatment zone, however, is mandatory for acute treatment control since incomplete tumor coverage results in limited oncological outcome. In this study, the “Chebyshev Center Concept” was introduced for CT 3d rendering to assess size and position of the maximum treatable tumor at a specific safety margin.MethodsIn seven pig livers, three different IRE protocols were applied to create treatment zones of different size and shape: Protocol 1 (n = 5 IREs), Protocol 2 (n = 5 IREs), and Protocol 3 (n = 5 IREs). Contrast-enhanced CT was used to assess the treatment zones. Technique A consisted of a semi-automated software prototype for CT 3d rendering with the “Chebyshev Center Concept” implemented (the “Chebyshev Center” is the center of the largest inscribed sphere within the treatment zone) with automated definition of parameters for size, shape and position. Technique B consisted of standard CT 3d analysis with manual definition of the same parameters but position.ResultsFor Protocol 1 and 2, short diameter of the treatment zone and diameter of the largest inscribed sphere within the treatment zone were not significantly different between Technique A and B. For Protocol 3, short diameter of the treatment zone and diameter of the largest inscribed sphere within the treatment zone were significantly smaller for Technique A compared with Technique B (41.1 ± 13.1 mm versus 53.8 ± 1.1 mm and 39.0 ± 8.4 mm versus 53.8 ± 1.1 mm; p < 0.05 and p < 0.01). For Protocol 1, 2 and 3, sphericity of the treatment zone was significantly larger for Technique A compared with B.ConclusionsRegarding size and shape of the treatment zone after IRE, CT 3d rendering with the “Chebyshev Center Concept” implemented provides significantly different results compared with standard CT 3d analysis. Since the latter overestimates the size of the treatment zone, the “Chebyshev Center Concept” could be used for a more objective acute treatment control.