Air-liquid interface cultures trigger a metabolic shift in intestinal epithelial cells (IPEC-1).

An improved oxygen availability in air-liquid interface (ALI) cultures of enterocytes of the small intestine seems to be primarily responsible for morphological, metabolic, and functional changes. Intestinal porcine epithelial cells 1 (IPEC-1) are less investigated and are rarely used as model for intestinal barrier but showed a profound change of cell shape during ALI cultivation. We aim to answer the following question: Are the observed morphological effects accompanied by changes in metabolic function? A microarray analysis of submerged culture (SMC) and ALI cultures identified 830 significantly regulated genes. Subsequent functional clustering revealed alterations in 31 pathways, with the highest number of regulated genes in metabolic pathways, carbon metabolism, glycolysis, and hypoxia-inducible factor (HIF) signaling. Furthermore, HIF-1α as a mediator of a metabolic switch between glycolysis and oxidative phosphorylation showed a trend of increased mRNA levels in ALI in contrast to a reduced nuclear HIF-1α content in the nucleus. Candidate genes of oxidative phosphorylation such as a mitochondrial marker exhibited enhanced mRNA levels, which was confirmed by western blot analysis. Cytochrome C oxidase (COX) subunit 5B protein was decreased in ALI, although mRNA level was increased. The oxidation of ferrocytochrome C to ferricytochrome C was used for detection of cytochrome C oxidase activity of isolated mitochondria and resulted in a trend of higher activity in ALI. Furthermore, quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. To evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenized cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In contrast, blocking with 2-desoxy-D-glucose (2DG) significantly reduced ATP content in ALI and SMC. These results indicate a metabolic shift in IPEC-1 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis.

On the distribution and metabolism of Fusarium-toxins along the gastrointestinal tract of endotoxaemic pigs.

The aim of this study was to investigate the potential modulatory effect of E. coli lipopolysaccharides (LPS) on residues of deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL), ß-zearalenol (ß-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and ß-zearalanol (ß-ZAL) after pre- or post-hepatic administration along the gastrointestinal axis. Fifteen barrows were exposed to a naturally mycotoxin contaminated diet (4.59 mg DON/kg feed and 0.22 mg ZEN/kg feed) and equipped with jugular (ju) and portal (po) catheters. On sampling day (day 29), the barrows were infused with LPS or a control fluid (LPS, 7.5 µg/kg body weight; control, 0.9% NaCl) either pre- or post-hepatically, resulting in three infusion groups: CONju-CONpo, CONju-LPSpo and LPSju-CONpo. At 195 min relative to infusion start (210 min post-feeding), pigs were sacrificed and content of stomach and small intestine (proximal, medial and distal part) as well as faeces were collected. In all LPS-infused animals, higher amounts of dry matter were recovered irrespective of LPS entry site suggesting a reduced gastric emptying and a decreased gastrointestinal motility under endotoxaemic conditions. DON metabolism in the gastrointestinal tract (GIT) remained unaltered by treatments and included an increase in the proportion of DOM-1 along the GIT, particularly from distal small intestine to faeces. Variables describing ZEN metabolism suggest a stimulated biliary release of ZEN and its metabolites in LPS-infused groups, particularly in the LPSju-CONpo group. In conclusion, the GIT metabolism of ZEN was markedly influenced in endotoxaemic pigs whereby a jugular induction of an acute phase reaction was more effective than portal LPS infusion hinting at a strong hepatic first-pass effect.

The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits ß5i/LMP7 (LMP, low molecular weight protein) or ß1i/LMP2 and ß5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit ß2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. ß2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies.

Correlation of CT-based bone mineralization with drilling-force measurements in anatomical specimens is suitable to investigate planning of trans-pedicular spine interventions.

This interdisciplinary study examined the relationship between bone density and drilling forces required during trans-pedicular access to the vertebra using fresh-frozen thoraco-lumbar vertebrae from two female body donors (A, B). Before and after biomechanical examination, samples underwent high-resolution CT-quantification of total bone density followed by software-based evaluation and processing. CT density measurements (n = 4818) were calculated as gray values (GV), which were highest in T12 for both subjects (GVmaxA = 3483.24, GVmaxB = 3160.33). Trans-pedicular drilling forces F (Newton N) were highest in L3 (FmaxB = 5.67 N) and L4 (FmaxA = 5.65 N). In 12 out of 13 specimens, GVs significantly (p < 0.001) correlated with force measurements. Among these, Spearman correlations r were poor in two lumbar vertebrae, fair in five specimens, and moderately strong in another five specimens, and highest for T11 (rA = 0.721) and L5 (rB = 0.690). Our results indicate that CT-based analysis of vertebral bone density acquired in anatomical specimens is a promising approach to predict the drilling force appearance as surrogate parameter of its biomechanical properties by e.g., linear regression analysis. The study may be of value as basis for biomechanical investigations to improve planning of the optimal trajectory and to define safety margins for drilling forces during robotic-assisted trans-pedicular interventions on the spine in the future.

Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

Helicobacter pylori induces type 4 secretion system-dependent, but CagA-independent activation of IκBs and NF-κB/RelA at early time points.

Colonization of the gastric epithelium by Helicobacter pylori induces the transcription factor nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) and the innate immune response. Virulent strains of H. pylori carry a cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS). Recent publications have shown controversial data regarding the role of the T4SS and the effector protein cytotoxin associated gene A (CagA), which becomes translocated by the T4SS into the eukaryotic epithelial cell, in H. pylori-induced NF-κB activation. Thus, this study analyses by using three different H. pylori strains (P1, B128 and G27) whether CagA is required to initiate activation of different molecules of inhibitors of kappa B (IκB) and the NF-κB transcription factor RelA. We provide experimental evidence that H. pylori induces phosphorylation of NF-κB inhibitors IκBα, IκBß and IκBɛ, and degradation of IκBα. Further, H. pylori stimulates phosphorylation of RelA at amino acids S536, S468 and S276, promotes DNA binding of RelA, and interleukin 8 (IL-8) gene expression in a T4SS-, but CagA-independent manner at early time points.

The major soyabean allergen P34 resists proteolysis in vitro and is transported through intestinal epithelial cells by a caveolae-mediated mechanism.

Soya is considered to be one of the eight most significant food allergens. Among the allergenic soya proteins determined to date, P34 has been identified as one of the immunodominant soya antigens. Sensitisation to a specific food antigen like P34 generally follows the transit of intact antigens across the intestinal barrier and usually occurs in infants, who are most susceptible to food allergies. In the present study, we used the intestinal epithelial cell line IPEC-J2, which was originally derived from the jejunum of a neonatal piglet, to recapitulate the infant intestinal epithelium and study the binding and uptake of P34 protein. P34 was partially resistant to degradation in an in vitro proteolysis assay. IPEC-J2 cells were able to endocytose intact P34, as shown by immunofluorescence and immunoelectronmicroscopy methods. P34 associated with lipid raft microdomains of IPEC-J2 cells, and disruption of caveolae/lipid raft microdomains using methyl-ß-cyclodextrin abolished P34 endocytosis, indicating that the observed endocytosis was mediated by caveolae. Using IPEC-J2 cells grown on Transwell filters, we further demonstrated that P34 is transported through the epithelial monolayer by transcytosis. Piglets frequently show hypersensitivity to soya antigens, and in this study, we show that healthy adult pigs with dietary exposure to soya protein mount an antibody response to soyabean protein P34, suggesting that this protein has entered the body, probably through gastrointestinal uptake. In summary, our data suggest that soya P34 resists proteolysis in the gastrointestinal tract and is transported through the intestinal epithelial barrier, thereby allowing sensitisation of immune cells in the sub-epithelial compartment.

[Anatomical aspects regarding the endoscopic release of the nervus interosseus anterior]. / Anatomische Aspekte zur endoskopischen Dekompression beim Interosseus-anterior-Syndrom.

BACKGROUND: Decompression of the anterior interosseous nerve can be performed in an open operative exploration or endoscopically. Using an endoscopic decompression superficial anatomical landmarks serve as reference point. The aim of the study was to determine the location of the distribution of the median nerve in relation to the elbow joint in order to facilitate preparation during endoscopic decompression. MATERIALS AND METHODS: The median nerve and the anterior interosseous nerve were dissected in 31 human specimens with regard to the elbow joint. The superficial anatomical landmark was the intercondyle line between the medial and lateral epicondyles. The distance between the origination of the anterior interosseous nerve of the median nerve was measured in relation to the intercondyle line. RESULTS: The anatomical preparation was done using 62 adult cadaveric upper extremities. 11 specimens were formalin fixed and 20 specimens were fresh frozen cadaveric upper extremities. The average of the intercondyle distance was 7.2 cm ± 0.5 (min. 5.8; max. 7.8). The anterior interosseous nerve originated from the median nerve in average 39 mm ± 18 (min. 8; max. 80) distal to the intercondyle line. In 12 cases the distance was within the first 2 cm. There was only a correlation between the length of the upper arm and the nerve junction. CONCLUSION: The anterior interosseous nerve originated from the median nerve in average 4 cm distal to the intercondyle line. Although there was a distribution under 2 cm in around 20 % of the cases. This is very important with regard to the endoscopically technique and should be considered.

IT support in emergency remote teaching in response to COVID-19.

Background: The COVID-19 pandemic hit the German education system unexpectedly and forced its universities to shift to Emergency Remote Teaching (ERT). The Data Integration Center (DIC) of the University Hospital Magdeburg and the Institute of Biometry and Medical Informatics (IBMI) has developed a concept based on existing structures that can be quickly implemented and used by the Medical Faculty at Otto von Guericke University. This manuscript focuses on the IT support for lecturers, which allows them to concentrate on teaching their lessons, although the authors are aware that this is only a small part of the entire subject. Additionally, there is a great awareness that ERT can never replace well-structured in-person classes. Concept: The key feature of the concept uses the well-working management system for all physical rooms of the university by designing a virtual video conference room for every physical room. This allows high interactivity for lectures and seminars while applying proven teaching methods. Additionally, a collaboration software system to document all lessons learned and a technical support team have been available for the teaching staff. Courses with a hands-on approach require more personal interaction than lectures. Therefore, the issues of practical trainings have not been solved with this concept, but been tackled by using questionnaires and minimizing contacts during attestations. Applied IT tools: The concept's requirements were met by Zoom Meetings, Confluence, HIS/LSF and Moodle. Discussion and Conclusion: The concept helped the lecturers to provide high-quality teaching for students at universities. Additionally, it allows for a dynamic response to new needs and problems. The concept will be reviewed as part of a higher Universal Design for Learning concept and may support lecturers in the following semesters in hybrid meetings with real and virtual attendees.

Does chronic dietary exposure to the mycotoxin deoxynivalenol affect the porcine hepatic transcriptome when an acute-phase response is initiated through first or second-pass LPS challenge of the liver?

The sensitivity of pigs to deoxynivalenol (DON) might be increased by systemic inflammation (SI), which also has consequences for hepatic integrity. Liver lesions and a dys-regulated gene network might hamper hepatic handling and elimination of DON whereby the way of initiation of hepatic inflammation might play an additional role. First and second-pass exposure of the liver with LPS for triggering a SI was achieved by LPS infusion via pre- or post-hepatic venous route, respectively. Each infusion group was pre-conditioned either with a control diet (0.12 mg DON/kg diet) or with a DON-contaminated diet (4.59 mg DON/kg diet) for 4 wk. Liver transcriptome was evaluated at 195 min after starting infusions. DON exposure alone failed to modulate the mRNA expression significantly. However, pre- and post-hepatic LPS challenges prompted transcriptional responses in immune and metabolic levels. The mRNAs for B-cell lymphoma 2-like protein 11 as a key factor in apoptosis and IFN-γ released by T cells were clearly up-regulated in DON-fed group infused with LPS post-hepatically. On the other hand, mRNAs for nucleotide binding oligomerization domain containing 2, IFN-α and eukaryotic translation initiation factor 2α kinase 3 as ribosomal stress sensors were exclusively up-regulated in control pigs with pre-hepatic LPS infusion. These diverse effects were traced back to differences in TLR4 signalling.

Course of macroscopic anatomy in Magdeburg under pandemic conditions.

Introduction and objectives: The Covid-19 pandemic has created major challenges for university teaching. At the beginning of the summer semester 2020, teaching at the Medical Faculty in Magdeburg was almost completely online. Also the course in macroscopic anatomy had to be replaced by virtual e-learning offers. Methods: Videos and photo presentations of the preparation steps and structures to be displayed were made available online. The reactions of the students showed very quickly that the three-dimensionality, the independent preparation and the haptics of the object to be studied make up a large part of this subject. Results and conclusions: Virtual e-learning offerings are a useful supplement to, but not a substitute for, active dissecting on body donors. By changing the course offerings in compliance with hygiene and distance rules, we were able to offer a classroom course again during the semester, which was expressly welcomed by the students.

Oral exposure of pigs to the mycotoxin deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction.

The sensitivity of pigs to deoxynivalenol (DON) might be influenced by systemic inflammation (SI) which impacts liver. Besides following acute-phase proteins, our aim was to investigate both the hepatic fractional albumin (ALB) synthesis rate (FSR) and the ALB concentration as indicators of ALB metabolism in presence and absence of SI induced by LPS via pre- or post-hepatic venous route. Each infusion group was pre-conditioned either with a control diet (CON, 0.12 mg DON/kg diet) or with a DON-contaminated diet (DON, 4.59 mg DON/kg diet) for 4 wk. A depression of ALB FSR was observed 195 min after LPS challenge, independent of feeding group or LPS application route, which was not paralleled by a down-regulated ALB mRNA expression but by a reduced availability of free cysteine. The drop in ALB FSR only partly explained the plasma ALB concentrations which were more depressed in the DON-pre-exposed groups, suggesting that ALB levels are influenced by further mechanisms. The abundances of haptoglobin, C-reactive protein, serum amyloid A, pig major acute-phase protein, fibrinogen and LPS-binding protein mRNA were up-regulated upon LPS stimulation but not accompanied by increases in the plasma concentrations of these proteins, pointing at an imbalance between synthesis and consumption.

Short chain regioselectively hydrolyzed scleroglucans induce maturation of porcine dendritic cells.

Branched beta-1,3/1,6-glucans (scleroglucan) were produced by cultivation of Sclerotium rolfsii ATCC 15205. Regioselective hydrolysis at the beta-1,3-linkage of the cell-free and purified polysaccharide was performed in borosilicate glass bottles at pH 5, 121 degrees C, and 1 bar for 72 h. The mixture was divided into four molar mass fractions by stepwise cross-flow filtration using different cutoffs. In vitro studies revealed that scleroglucan hydrolysates with a low molar mass of less than 5 kDa significantly stimulated the activation and maturation of porcine monocyte derived dendritic cells (MoDC) by upregulation of CD40 and CD80/86 as well as by reduction of antigen uptake. MoDC treated with low molar mass scleroglucan showed a considerable increase in the amounts of secreted proinflammatory cytokine tumor necrosis factor alpha and stimulated the proliferation of lymphocytes. Therefore, scleroglucan molecules of low molecular weight are able to induce activation and maturation of porcine DC, which are key initiators of inflammatory and adaptive immune responses, and could provide improved protection against infectious diseases.

Effects of deoxynivalenol-feed contamination on circulating LPS in pigs.

Low concentration of LPS can be detected in healthy mammals without triggering systemic inflammation. Here we analysed the influence of the mycotoxin deoxynivalenol (DON) on very low LPS concentrations and the role of DON in the physiology of pigs challenged with high artificial LPS dosage mimicking septic shock. Pigs were fed for 29 d with DON-contaminated (4.59 mg/kg feed) or control feed. Samples of control animals showed 6.6 ± 13.5 pg/ml LPS in portal and 3.1 ± 7.6 pg/ml LPS in jugular serum samples. In the DON fed group, 3.4 ± 7.2 pg/ml and 0.6 ± 0.8 pg/ml were detected. The differences were statistically not significant, indicating that DON is not a trigger for enhanced LPS transfer into the blood circulation. Next, pigs were challenged with 7.5 µg LPS/kg body mass via portal or jugular route. The application route did not significantly influence the LPS concentration. We expected higher circulating LPS concentrations in the presence of DON due to the additional stress of liver metabolism and reduced liver capacity to remove LPS from circulation. This scenario is supported by tendency. In summary, we found that DON is unlikely to influence LPS transfer in the gut; DON likely reduces the capacity for LPS removal in septic shock conditions.

Isolation of soybean protein P34 from oil bodies using hydrophobic interaction chromatography.

BACKGROUND: Soybeans play a prominent role in allergologic research due to the high incidence of allergic reactions. For detailed studies on specific proteins it is necessary to have access to a large amount of pure substance. RESULTS: In this contribution, a method for purifying soybean (Glycine max) protein P34 (also called Gly m Bd 30 K or Gly m 1) using hydrophobic interaction chromatography is presented. After screening experiments using 1 mL HiTrap columns, Butyl Sepharose 4 FF was selected for further systematic investigations. With this stationary phase, suitable operation conditions for two-step gradient elution using ammonium sulphate were determined experimentally. The separation conditions obtained in a small column could be scaled up successfully to column volumes of 7.5 and 75 mL, allowing for high product purities of almost 100% with a yield of 27% for the chromatographic separation step. Conditions could be simplified further using a onestep gradient, which gave comparable purification in a shorter process time. The identity of the purified protein was verified using in-gel digestion and mass spectrometry as well as immunological techniques. CONCLUSION: With the technique presented it is possible to produce, within a short timeframe, pure P34, suitable for further studies where an example antigen is needed.

Influence of carvacrol on proliferation and survival of porcine lymphocytes and intestinal epithelial cells in vitro.

Carvacrol, an essential oil compound of oregano and thyme, has potential applications as an alternative to antibiotic growth promoters in pig nutrition. Carvacrol is well known for its antibacterial effects, but it is unclear whether there are additional effects on the porcine immune system. In the present study, the influence of carvacrol on porcine blood lymphocytes was examined. The porcine enterocyte cell line IPEC-1 was examined for comparison. Carvacrol inhibited the proliferation of purified lymphocytes with an IC50 of 182+/-67 microM in MTT assays. This was confirmed by CFSE assay. The presence of monocytes in carvacrol-treated lymphocyte preparations had a protective effect on the lymphocytes, significantly raising the IC50 to 516+/-87 microM. FACS analysis of CFSE labelled lymphocyte subsets revealed that gammadelta T cells were less susceptible to carvacrol toxicity than CD4 and CD8 T cells. The reduced lymphocyte proliferation measured after carvacrol exposure was shown to be due to apoptotic cell death, as determined by annexin-V binding and caspase-3 activation. The observed effects were not specific for lymphocytes, since carvacrol similarly induced apoptosis and suppressed proliferation in the porcine enterocyte cell line IPEC-1.

Deoxynivalenol Affects Cell Metabolism and Increases Protein Biosynthesis in Intestinal Porcine Epithelial Cells (IPEC-J2): DON Increases Protein Biosynthesis.

Deoxynivalenol (DON) is a toxin found in cereals as well as in processed products such as pasta, and causes substantial economic losses for stock breeding as it induces vomiting, reduced feeding, and reduced growth rates in piglets. Oxidative phosphorylation, TCA-cycle, transcription, and translation have been hypothesized to be leading pathways that are affected by DON. We used an application of high and low glucose to examine oxidative phosphorylation and anaerobic glycolysis. A change in the metabolic status of IPEC-J2 was observed and confirmed by microarray data. Measurements of oxygen consumption resulted in a significant reduction, if DON attacks from the basolateral. Furthermore, we found a dose-dependent effect with a significant reduction at 2000 ng/mL. In addition, SLC7A11 and PHB, the genes with the highest regulation in our microarray analyses under low glucose supply, were investigated and showed a variable regulation on protein level. Lactate production and glucose consumption was investigated to examine the impact of DON on anaerobic glycolysis and we observed a significant increase in 2000 blhigh and a decrease in 2000 aphigh. Interestingly, both groups as well as 200 blhigh showed a significant higher de novo protein synthesis when compared to the control. These results indicate the direct or indirect impact of DON on metabolic pathways in IPEC-J2.

MIRACUM: Medical Informatics in Research and Care in University Medicine.

INTRODUCTION: This article is part of the Focus Theme of Methods of Information in Medicine on the German Medical Informatics Initiative. Similar to other large international data sharing networks (e.g. OHDSI, PCORnet, eMerge, RD-Connect) MIRACUM is a consortium of academic and hospital partners as well as one industrial partner in eight German cities which have joined forces to create interoperable data integration centres (DIC) and make data within those DIC available for innovative new IT solutions in patient care and medical research. OBJECTIVES: Sharing data shall be supported by common interoperable tools and services, in order to leverage the power of such data for biomedical discovery and moving towards a learning health system. This paper aims at illustrating the major building blocks and concepts which MIRACUM will apply to achieve this goal. GOVERNANCE AND POLICIES: Besides establishing an efficient governance structure within the MIRACUM consortium (based on the steering board, a central administrative office, the general MIRACUM assembly, six working groups and the international scientific advisory board), defining DIC governance rules and data sharing policies, as well as establishing (at each MIRACUM DIC site, but also for MIRACUM in total) use and access committees are major building blocks for the success of such an endeavor. ARCHITECTURAL FRAMEWORK AND METHODOLOGY: The MIRACUM DIC architecture builds on a comprehensive ecosystem of reusable open source tools (MIRACOLIX), which are linkable and interoperable amongst each other, but also with the existing software environment of the MIRACUM hospitals. Efficient data protection measures, considering patient consent, data harmonization and a MIRACUM metadata repository as well as a common data model are major pillars of this framework. The methodological approach for shared data usage relies on a federated querying and analysis concept. USE CASES: MIRACUM aims at proving the value of their DIC with three use cases: IT support for patient recruitment into clinical trials, the development and routine care implementation of a clinico-molecular predictive knowledge tool, and molecular-guided therapy recommendations in molecular tumor boards. RESULTS: Based on the MIRACUM DIC release in the nine months conceptual phase first large scale analysis for stroke and colorectal cancer cohorts have been pursued. DISCUSSION: Beyond all technological challenges successfully applying the MIRACUM tools for the enrichment of our knowledge about diagnostic and therapeutic concepts, thus supporting the concept of a Learning Health System will be crucial for the acceptance and sustainability in the medical community and the MIRACUM university hospitals.

The Fusarium toxin deoxynivalenol disrupts phenotype and function of monocyte-derived dendritic cells in vivo and in vitro.

The trichothecene mycotoxin deoxynivalenol (DON) causes systemic immuno-suppression in pigs and possibly also in humans after chronic dietary exposure. Since the outcome of every immune response is largely controlled by dendritic cells (DC), we hypothesised that a direct influence of DON on DC function might play a role in mediating DON immunotoxicity. To test this hypothesis, a 2x2 factorial design study was performed. Pigs were fed a control diet or a diet containing DON (DON-diet); monocyte-derived DC (MoDC) from these pigs were then treated with DON in vitro or left untreated. Phenotype and function of the MoDC were analysed. In vitro DON-treatment of MoDC from pigs fed the control diet resulted in a down-regulation of CD80/86 and CD40. This was associated with an activation of the mitogen-associated protein kinases ERK1/2 and JNK. The endocytic activity of MoDC was decreased after in vitro DON-exposure while their T cell stimulatory capacity was not altered. MoDC derived from pigs that had been fed the DON-diet failed to up-regulate MHC-II in response to LPS/TNFalpha. Dietary exposure of pigs to DON inhibited endocytosis of FITC-dextran by MoDC, but did not influence T cell stimulatory capacity. ERK1/2 and JNK were constitutively activated in MoDC from pigs fed the DON-diet. If MoDC derived from pigs fed the DON-diet were exposed to DON in vitro, this resulted in an up-regulation of MHC-II and CD80/86, but not CD40. In comparison to untreated MoDC from pigs fed DON-diet, endocytic capacity was further down-regulated, whereas mitogen-activated protein kinase activation was increased. In summary, DON disrupts porcine DC function in vitro and in vivo, which might contribute to the immunosuppressive effects of this mycotoxin.

Air-liquid interface enhances oxidative phosphorylation in intestinal epithelial cell line IPEC-J2.

The intestinal porcine epithelial cell line IPEC-J2, cultured under the air-liquid interface (ALI) conditions, develops remarkable morphological characteristics close to intestinal epithelial cells in vivo. Improved oxygen availability has been hypothesised to be the leading cause of this morphological differentiation. We assessed oxygen availability in ALI cultures and examined the influence of this cell culture method on glycolysis and oxidative phosphorylation in IPEC-J2 using the submerged membrane culture (SMC) and ALI cultures. Furthermore, the role of HIF-1 as mediator of oxygen availability was analysed. Measurements of oxygen tension confirmed increased oxygen availability at the medium-cell interface and demonstrated reduced oxygen extraction at the basal compartment in ALI. Microarray analysis to determine changes in the genetic profile of IPEC-J2 in ALI identified 2751 modified transcripts. Further examinations of candidate genes revealed reduced levels of glycolytic enzymes hexokinase II and GAPDH, as well as lactate transporting monocarboxylate transporter 1 in ALI, whereas expression of the glucose transporter GLUT1 remained unchanged. Cytochrome c oxidase (COX) subunit 5B protein analysis was increased in ALI, although mRNA level remained at constant level. COX activity was assessed using photometric quantification and a three-fold increase was found in ALI. Quantification of glucose and lactate concentrations in cell culture medium revealed significantly reduced glucose levels and decreased lactate production in ALI. In order to evaluate energy metabolism, we measured cellular adenosine triphosphate (ATP) aggregation in homogenised cell suspensions showing similar levels. However, application of the uncoupling agent FCCP reduced ATP levels in ALI but not in SMC. In addition, HIF showed reduced mRNA levels in ALI. Furthermore, HIF-1α protein was reduced in the nuclear compartment of ALI when compared to SCM as confirmed by confocal microscopy. These results indicate a metabolic switch in IPEC-J2 cultured under ALI conditions enhancing oxidative phosphorylation and suppressing glycolysis. ALI-induced improvement of oxygen supply reduced nuclear HIF-1α, demonstrating a major change in the transcriptional response.