effects of an increased osmotic pressure and feeding conditions

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Literatur 3\. Material und Methoden 4\. Ergebnisse 5\. Diskussion 6\. Zusammenfassung / Summary 7\. Anhang LiteraturverzeichnisDie Untersuchung beschäftigt sich mit dem Effekt des luminalen osmotischen Druckes auf Eigenschaften der Pansenepithelien in Abhängigkeit von der Fütterung. Die Schafe wurden ca. 3 Wochen mit vier verschiedenen Diäten gefüttert: (1) Heu ad libitum, (2) 600 g Kraftfutter/d, (3) 1200 g Kraftfutter/d und (4) 1800 g Kraftfutter/d. Zusätzlich zum Kraftfutter wurde Heu ad libitum angeboten. Die Versuche wurden in vitro mit Hilfe der Ussing-Kammer-Methode an der isolierten Pansenschleimhaut durchgeführt. Dabei wurde die luminale Osmolarität in den Kontrollgruppen auf 300 mosmol/l eingestellt und in den Versuchsgruppen mit Mannit auf 375 bzw. 450 mosmol/l erhöht. 1\. Der Na-Nettotransport (Jnet Na) wurde insgesamt durch die Kraftfuttergaben erhöht. Dies ist auf eine Steigerung des Transports von mukosal nach serosal (Jms Na), wahrscheinlich auf eine Zunahme der Aktivität des apikalen Na/H-Austauschers zurückzuführen. 2\. Die Erhöhung der Osmolarität führte zu einem Anstieg der Leitfähigkeit (Gt) in allen Fütterungsgruppen. Diese Auswirkung war bei hohen Kraftfuttergaben deutlich geringer. Die Gt-Veränderungen sind bei Heu und 600 g Kraftfutter/d auf parazelluläre, bei hohen Kraftfuttergaben wahrscheinlich auf transzelluläre Effekte zurückzuführen. Die osmotische Resistenz des Epithels nimmt also mit steigenden Kraftfuttergaben zu. 3\. Der Na-Nettotransport wurde durch einen erhöhten osmotischen Druck reduziert, die Abnahme war am ausgeprägtesten bei hohen Kraftfuttergaben. 4\. In der Fütterungsgruppe Heu und 600 g Kraftfutter/d wurde der Na-Transport von serosal nach mukosal (Jsm Na) erhöht, in den Gruppen 1200 und 1800 g Kraftfutter/d veränderte sich Jsm Na dagegen nicht. 5\. In den Gruppen 1200 und 1800 g Kraftfutter/d wurde Jms Na durch den erhöhten osmotischen Druck vermindert. Diese Reduktion erfolgt wahrscheinlich durch eine Hemmung der Aktivität des Na/H-Austauschers. 6\. Die Ergebnisse werden im Hinblick auf ihre mögliche praktische Bedeutung diskutiert.The intention of this study was to test the possible dietary dependent effect of luminal osmotic pressure on characteristics of the ruminal epithelium. Sheep were kept under different dietary trials for at least three weeks: (1) hay ad libitum, (2)600 g concentrate/d, (3) 1200 g concentrate/d and (4) 1800 g concentrate/d. In addition to the concentrate, sheep were offered hay ad libitum. The experiments were conducted in vitro with isolated ruminal epithelial tissues using conventional Ussing chamber technique. In these experiments, luminal osmolarity was adjusted by mannitol to 300 mosmol/l in the control group and to 375 or 450 mosmol/l in the experimental groups. 1\. Concentrate feeding led to an increase of net Na transport (Jnet Na); this is due to the stimulation of the mucosal to serosal sodium transport (Jms Na) and most probably due to an increase in the apical Na/H exchanger activity. 2\. Under all dietary trials, increasing the luminal osmolarity resulted in an elevation of the tissue conductance (Gt). This increase in conductance was lowest at higher concentrate feeding. This modification of Gt could be attributed to paracellular effects under hay and 600 g concentrate dietary trials and probably to transcellular effects under higher concentrate dietary trials. Thus, the osmotic resistance of the epithelium seems to increase with higher concentrate intake. 3\. Increasing luminal osmotic pressure resulted in a reduction of net Na transport, this reduction was most pronounced at higher concentrate intake. 4\. Hay and 600 g concentrate/d feeding led to a stimulation of the serosal to mucosal Na flux (Jsm Na); however, higher concentrate intake (1200 and 1800 g concentrate/d) did not show any effect. 5\. At higher concentrate intake (1200 and 1800 g concentrate/d) increasing luminal osmotic pressure resulted in a reduction of the mucosal to serosal Na flux. This is probably due to the inhibition of the Na/H exchanger activity. 6\. The results are discussed regarding their possible practical meaning

The Inflammatory Response to Enterotoxigenic E. coli and Probiotic E. faecium in a Coculture Model of Porcine Intestinal Epithelial and Dendritic Cells

The gut epithelium constitutes an interface between the intestinal contents and the underlying gut-associated lymphoid tissue (GALT) including dendritic cells (DC). Interactions of intestinal epithelial cells (IEC) and resident DC are characterized by bidirectional crosstalk mediated by various factors, such as transforming growth factor-β (TGF-β) and thymic stromal lymphopoietin (TSLP). In the present study, we aimed (1) to model the interplay of both cell types in a porcine in vitro coculture consisting of IEC (cell line IPEC-J2) and monocyte-derived DC (MoDC) and (2) to assess whether immune responses to bacteria are altered because of the interplay between IPEC-J2 cells and MoDC. With regard to the latter, we focused on the inflammasome pathway. Here, we propose caspase-13 as a promising candidate for the noncanonical inflammasome activation in pigs. We conducted challenge experiments with enterotoxigenic Escherichia coli (ETEC) and probiotic Enterococcus faecium (E. faecium) NCIMB 10415. As potential mediators of IEC/DC interactions, TGF-β and TSLP were selected for analyses. Cocultured MoDC showed attenuated ETEC-induced inflammasome-related and proinflammatory interleukin (IL)-8 reactions compared with MoDC monocultures. Caspase-13 was more strongly expressed in IPEC-J2 cells cocultured with MoDC and upon ETEC incubation. We found that IPEC-J2 cells and MoDC were capable of releasing TSLP. The latter cells secreted greater amounts of TSLP when cocultured with IPEC-J2 cells. TGF-β was not modulated under the present experimental conditions in either cell types. We conclude that, in the presence of IPEC-J2 cells, porcine MoDC exhibited a more tolerogenic phenotype, which might be partially regulated by autocrine TSLP production. Noncanonical inflammasome signaling appeared to be modulated in IPEC-J2 cells. Our results indicate that the reciprocal interplay of the intestinal epithelium and GALT is essential for promoting balanced immune responses

Altered Cytokine Expression and Barrier Properties after In Vitro Infection of Porcine Epithelial Cells with Enterotoxigenic Escherichia coli and Probiotic Enterococcus faecium

The aim of the present study was to elucidate the effects of the probiotic feed additive Enterococcus faecium NCIMB 10415 (E. faecium) on porcine jejunal epithelial cells (IPEC-J2) during an in vitro challenge with enterotoxigenic Escherichia coli (ETEC). Cells were incubated with E. faecium, ETEC, or both, and the effects on barrier function and structure and intra- and intercellular signaling were determined. Coincubation with E. faecium abolished the ETEC- induced decrease in transepithelial resistance (Rt). No differences were seen in the expression levels of the intercellular connecting tight junction proteins examined. However, for the first time, a reorganization of the monolayer was observed in ETEC-infected cells but not in coincubated cells. ETEC induced an increase in cytotoxicity that was prevented by coincubation, whereas apoptosis rates were not affected by bacterial treatment. ETEC increased the mRNA expression and release of proinflammatory cytokines TNF-α, IL-1α, and IL-6 which could be prevented by coincubation for TNF-α mRNA expression and IL-6 protein. Likewise, cAMP concentrations elevated by ETEC were reduced in coincubated cells. These findings indicate a protective effect of the probiotic E. faecium on inflammatory responses during infection with ETEC

Cry1Ab treatment has no effects on viability of cultured porcine intestinal cells, but triggers Hsp70 expression.

In vitro testing can contribute to reduce the risk that the use of genetically modified (GM) crops and their proteins show unintended toxic effects. Here we introduce a porcine intestinal cell culture (IPEC-J2) as appropriate in vitro model and tested the possible toxic potential of Cry1Ab protein, commonly expressed in GM-maize. For comprehensive risk assessment we used WST-1 conversion and ATP content as metabolic markers for proliferation, lactate dehydrogenase release as indicator for cells with compromised membrane and transepithelial electrical resistance as parameter indicating membrane barrier function. The results were compared to the effects of valinomycin, a potassium ionophore, known to induce cytotoxic effects in most mammalian cell types. Whereas no toxicity was observed after Cry1Ab treatment, valinomycin induced a decrease in IPEC-J2 viability. This was confirmed by dynamic monitoring of cellular responses. Additionally, two dimensional differential in-gel electrophoresis was performed. Only three proteins were differentially expressed. The functions of these proteins were associated with responses to stress. The up-regulation of heat shock protein Hsp70 was verified by Western blotting as well as by enzyme-linked immunosorbent assay and may be related to a protective function. These findings suggest that the combination of in vitro testing and proteomic analysis may serve as a promising tool for mechanism based safety assessment

Effects of the Probiotic Enterococcus faecium and Pathogenic Escherichia coli Strains in a Pig and Human Epithelial Intestinal Cell Model

The aim of this study has been to elucidate the effect of the probiotic Enterococcus faecium NCIMB 10415 on epithelial integrity in intestinal epithelial cells and whether pre- and coincubation with this strain can reproducibly prevent damage induced by enterotoxigenic (ETEC) and enteropathogenic Escherichia coli (EPEC). Porcine (IPEC-J2) and human (Caco-2) intestinal epithelial cells were incubated with bacterial strains and epithelial integrity was assessed by measuring transepithelial electrical resistance (TEER) and mannitol flux rates. E. faecium alone increased TEER of Caco-2 cells without affecting mannitol fluxes whereas the E. coli strains decreased TEER and concomitantly increased mannitol flux rates in both cell lines. Preincubation with E. faecium had no effect on the TEER decrease induced by E. coli in preliminary experiments. However, in a second set of experiments using a slightly different protocol, E. faecium ameliorated the TEER decrease induced by ETEC at 4 h in IPEC-J2 and at 2, 4, and 6 h in Caco-2 cells. We conclude that E. faecium positively affected epithelial integrity in monoinfected Caco-2 cells and could ameliorate the damage on TEER induced by an ETEC strain. Reproducibility of the results is, however, limited when experiments are performed with living bacteria over longer periods

List of identified differentially expressed proteins in Cry1Ab treated IPEC-J2 cells.

a)<p>Theoretical isoelectric point (pI).</p>b)<p>Accession number in NCBI database.</p>c)<p>Average ratio (Cry1Ab treated/non-treated control) indicates the value derived from the normalized spot volume standardized against the intra-gel standard provided by DeCyder software analysis.</p>d)<p>Peptide coverage, the amount of the identified proteins that the peptides covered.</p

Effect of Cry1Ab on viability of IPEC-J2 cells.

<p>IPEC-J2 cells were incubated with indicated concentrations of Cry1Ab for 24 h and 48 h, respectively. Data are from one representative experiment out of 2 performed. (A) The Cell Proliferation Reagent WST-1 (Roche) was used to measure mitochondrial dehydrogenase activity of viable cells. Valinomycin was used as cell damaging control. Bars represent the mean +/−S.D. of 5 replicate wells. (B) The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to estimate the number of viable metabolically active cells based on quantitation of ATP. Bars represent the mean +/−S.D. of 5 replicate wells. (C) The CytoTox-ONE™ Homogenous Membrane Integrity Assay (Promega) was used for estimating the number of dead cells by release of LDH from damaged cells. Bars represent the mean +/−S.D. of 12 replicate wells.</p

Proteomic profiling of IPEC-J2 cells in response to Cry1Ab treatment as revealed by 2D DIGE analysis.

<p>Image shows one representative spot map of Cry1Ab treated IPEC-J2 cell extracts (n = 5) indicating spot boundaries of proteins, whose expression level is increased (red) or decreased (green) in comparison with the corresponding untreated controls (only medium) (P<0.05) as revealed by Decyder V.7.0 software. Spots marked with a number, correlating to the identified proteins in Table1.</p