Gene Regulation of Intestinal Porcine Epithelial Cells IPEC-J2 Is Dependent on the Site of Deoxynivalenol Toxicological Action

The intestinal epithelial cell layer represents the border between the luminal and systemic side of the gut. The decision between absorption and exclusion of substances is the quintessential function of the gut and varies along the gut axis. Consequently, potentially toxic substances may reach the basolateral domain of the epithelial cell layer via blood stream. The mycotoxin deoxynivalenol (DON) is a Fusarium derived secondary metabolite known to enter the blood stream and displaying a striking toxicity on the basolateral side of polarised epithelial cell layers in vitro. Here we analysed potential mechanisms of apical and basolateral DON toxicity reflected in the gene expression. We used the jejunum-derived, polarised intestinal porcine epithelial cell line IPEC-J2 as an in vitro cell culture model. Luminal and systemic DON challenge of the epithelial cell layer was mimicked by a DON application from the apical or basolateral compartment of membrane inserts for 72 h. We compared the genome-wide gene expression of untreated and DON-treated IPEC-J2 cells with the GeneChip® Porcine Genome Array of Affymetrix. Low basolateral DON (200 ng/mL) application triggered 10 times more gene transcripts in comparison to the corresponding apical application (2539 versus 267) despite the intactness of the challenged cell layer as measured by transepithelial electrical resistance. Analysis of the regulated genes by bioinformatic resource DAVID identified several groups of biochemical pathways modulated by concentration and orientation of DON application. Selected genes representing pathways of the cellular metabolism, information processing and structural design were analysed in detail by quantitative PCR. Our findings clearly show that apical and basolateral challenge of epithelial cell layers trigger different gene response profiles paralleled with a higher susceptibility towards basolateral challenge. The evaluation of toxicological potentials of mycotoxins should take this difference in gene regulation dependent on route of application into account

Vulnerability of Polarised Intestinal Porcine Epithelial Cells to Mycotoxin Deoxynivalenol Depends on the Route of Application

BACKGROUND AND AIMS: Deoxynivalenol (DON) is a Fusarium derived mycotoxin, often occurring on cereals used for human and animal nutrition. The intestine, as prominent barrier for nutritional toxins, has to handle the mycotoxin from the mucosa protected luminal side (apical exposure), as well as already absorbed toxin, reaching the cells from basolateral side via the blood stream. In the present study, the impact of the direction of DON exposure on epithelial cell behaviour and intestinal barrier integrity was elucidated. METHODS: A non-transformed intestinal porcine epithelial cell line (IPEC-J2), cultured in membrane inserts, serving as a polarised in vitro model to determine the effects of deoxynivalenol (DON) on cellular viability and tight junction integrity. RESULTS: Application of DON in concentrations up to 4000 ng/mL for 24, 48 and 72 hours on the basolateral side of membrane cultured polarised IPEC-J2 cells resulted in a breakdown of the integrity of cell connections measured by transepithelial electrical resistance (TEER), as well as a reduced expression of the tight junction proteins ZO-1 and claudin 3. Epithelial cell number decreased and nuclei size was enlarged after 72 h incubation of 4000 ng/mL DON from basolateral. Although necrosis or caspase 3 mediated apoptosis was not detectable after basolateral DON application, cell cycle analysis revealed a significant increase in DNA fragmentation, decrease in G0/G1 phase and slight increase in G2/M phase after 72 hours incubation with DON 2000 ng/mL. CONCLUSIONS: Severity of impact of the mycotoxin deoxynivalenol on the intestinal epithelial barrier is dependent on route of application. The epithelium appears to be rather resistant towards apical (luminal) DON application whereas the same toxin dose from basolateral severely undermines barrier integrity

Evolutionary History of the Clostridium difficile Pathogenicity Locus

The symptoms of Clostridium difficile infection are caused by toxins expressed from its 19kb pathogenicity locus (PaLoc). Stable integration of the PaLoc is suggested by its single chromosomal location and the clade-specificity of its different genetic variants. However, the PaLoc is variably present, even among closely related strains, and thus resembles a mobile genetic element. Our aim was to explain these apparently conflicting observations by reconstructing the evolutionary history of the PaLoc. Phylogenetic analyses and annotation of the regions spanning the PaLoc were performed using C. difficile population-representative genomes chosen from a collection of 1,693 toxigenic (PaLoc present) and non-toxigenic (PaLoc absent) isolates. Comparison of the core genome and PaLoc phylogenies demonstrated an eventful evolutionary history, with distinct PaLoc variants acquired clade-specifically after divergence. In particular, our data suggest a relatively recent PaLoc acquisition in clade 4. Exchanges and losses of the PaLoc DNA have also occurred, via long homologous recombination events involving flanking chromosomal sequences. The most recent loss event occurred ~30 years ago within a clade 1 genotype. The genetic organisation of the clade 3 PaLoc was unique in containing a stably integrated novel transposon (designated Tn6218), variants of which were found at multiple chromosomal locations. Tn6218 elements were Tn916-related, but non-conjugative, and occasionally contained genes conferring resistance to clinically relevant antibiotics. The evolutionary histories of two contrasting, but clinically important genetic elements were thus characterised: the PaLoc, mobilised rarely via homologous recombination, and Tn6218, mobilised frequently through transposition

The role of innate Immunity and host specificity in Salmonella infection in vitro

Salmonellen sind bedeutende Zoonoseerreger und führen zu schweren Infektionskrankheiten sowohl beim Menschen als auch beim Tier. In der vorliegenden Arbeit wurden vergleichende Salmonella-Invasionstudien mit jeweils zwei bereits etablierten Zellarten (Epithel und Makrophagen) aus je drei verschiedenen Wirtsystemen (Schwein, Maus, Mensch) durchgeführt. Hierzu wurden sowohl wirtsadaptierte als auch nichtwirtsadaptierte S. enterica spp. enterica Serovare (S. Typhimurium, S. Choleraesius, S. Dublin und S. Enteritidis) genutzt. In der vorliegenden Arbeit wurde mittels Infektionsversuchen gezeigt, dass die wirtsadaptierten Serovare ein verlangsamtes Wachstum und eine verminderte Invasivität in dem entsprechenden Wirtssystem aufwiesen, wie S. Choleraeuis in porcinen Zellen und S. Typhimurium in murinen Zellen. Darüber hinaus zeigte die PMA-Aktivierung von Makrophagen einen deutlichen Einfluss auf die Invasivität und die intrazelluläre Vermehrungsrate der vier Salmonella-Serovare. Ein infektionsbedingter Zellverlust konnte in dieser Arbeit nach 24h p. i. nur durch S. Typhimurium Infektion, nicht jedoch bei einer Infektion mit S. Choleraesuis beobachtet werden. Somit war der Zelluntergang serovarspezifisch. S. Typhimurium zeigte darüber hinaus in murinen Makrophagen und S. Choleraesuis in porcinen Zellen das Bildnis ruhender oder sich nur schlecht vermehrender intrazellulärer Erreger, dies wiederum trägt zur systemischen Verbreitung des Erregers im Wirt bei. Somit war die intrazelluläre Überlebensrate der Salmonellen hingegen wirtsspezifisch und nicht serovarabhängig, dies zeigte sich durch den Vergleich zweier Untersuchungsmethoden (KbE und GFP). Um diesen Wirt-Erreger-Zusammenhang näher zu beleuchten, wurden NF-κB-Aktivierungsversuche durchgeführt. Hierbei konnte nachgewiesen werden, dass die wirtsspezifischen bzw. wirtsadaptierten Serovare gegenüber den nicht-wirtsadaptierten Erregern eine verminderte Luciferaseaktivität aufwiesen (S. Choleraesuis in porcinen Zellen und S. Typhimurium in murinen Zellen). Darüber hinaus wurde nachgewiesen, dass die bakterielle Zellwand im Vergleich zum LPS ein stärkerer NF-κB-induzierender Faktor ist und dass S. Typhimurium die zelleigene Immunantwort des Wirtes minimieren konnte. Diese ermittelten Ergebnisse zeigten, dass Gastroenteritisauslösende Salmonellen wie die nicht-wirtsadaptierte Serovare S. Enteritidis oder S. Typhimurium (Mensch, Schwein) über eine schnellere intrazelluläre Vermehrungsrate und über eine hohe NF-κB-Antwort ihre eigene Eliminierung durch das Wirtsimmunsytem fördern. Die wiederum Septikämie- auslösenden Erreger wie S. Choleraesuis (Schwein) und S. Typhimurium (Maus) führten in den Zellen zu einer geringeren immunologischen Zellantwort und zu einem geringen intrazellulären Wachstum. Dies ermöglicht den Erregern, die Immunabwehrmechanismen des Wirtes zeitweise zu umgehen und fördert eine effektivere Verbreitung der Erreger im Wirtsorganismus. Die Fähigkeit der Erreger, systemisch zu streuen, ist somit auch direkt verknüpft mit deren Fähigkeit, in Makrophagen eines bestimmten Wirtes zu überleben. So gesehen sind mononukleäre Phagozyten eines Wirtes eine essentielle Barriere und Bestandteil der Wirtspezifität der Salmonella-Serovare. Da der Wirt einen essentiellen Einfluss auf den Ausgang einer Infektion besitzt, wurden im zweiten Teil dieser Arbeit die porcinen NOD-Proteine der angeborenen Immunantwort untersucht, um damit eine Grundlage für weitere wissenschaftliche Arbeiten auf dem Gebiet der Salmonellenwirtsspezifität zu schaffen. Die erhobenen Daten zeigten, dass porcines NOD höhere Homologien mit dem humanen NOD als humanes NOD mit dem murinen NOD besitzen. Darüber hinaus zeigte die LRR-Domäne Unterschiede zwischen der porcinen NOD1-Sequenz und der humanen NOD1-Sequenz, dies lässt eine Wirtsspezifität auf der Ebene der PGN-Detektion vermuten. Darüber hinaus zeigte die LRR-Domäne von NOD zwischen zweier Schweinerassen SNPs auf, was auf eine verstärkte Variabilität der PRR in der Schweinepopulation hinweist. Diese Sequenzunterschiede könnten ein bedeutender Faktor sein, bei der Resistenzentwicklung oder der Empfindlichkeit bestimmter Rassen auf Krankheiten. Weiterführende Arbeiten zum Zusammenspiel der NOD- Proteine wird Erkenntnisse nicht nur hinsichtlich der bakteriellen Wirtszellantwort geben, sondern auch Möglichkeiten des pharmakologischen Modulierens bieten.Salmonella serovars are important zoonotic pathogens, and can cause severe infections in both humans and animals. In the study presented here, comparative in vitro infection studies were performed in two, established cell types (epithelia and macrophage) derived from three different host species origins (porcine, murine and human). Both host-adapted and broad host-range S. enterica spp. enterica serovars (S. Typhimurium, S. Choleraesius, S. Dublin und S. Enteritidis) were used for the infection studies. In this study, it was shown that the host-adapted serovars showed reduced growth and invasion of the pertinent host cells, e.g. S. Choleraesuis in porcine host cells and S. Typhimurium in murine host cells. In addition, it was shown that phorbol myristic acid (PMA) activation of macrophage results in large effects on the invasion and intracellular growth of the four Salmonella serovars. A Salmonella infection-dependent host cell loss was found to occur 24 hours post-infection only for S. Typhimurium-infected cells but not for S. Choleraesuis, indicating that the induction of host cell death was serovar- specific. Furthermore, the observation that S. Typhimurium and S. Choleraesuis showed static or poor intracellular growth in murine and porcine host cells, respectively, coupled with the known systemic proliferation of these serovars in their respective host species suggests a correlation of this growth characteristic contributing to systemic infections. That the intracellular survival of the Salmonella serovars is host-specific and not serovar-dependent was shown by comparison of two different methodologies (CFU and GFP). In order to clarify this host-pathogen interaction more closely, studies on the activation of host cell NF-κB in response to infections were carried out. In this work it was found that host-restricted or hostadapted serovars showed reduced lucifearse activation of their respective host cells compared to the broad host-range serovars. In addition, it was shown that the bacterial cell wall was a much more potent activator of NF-κB as compared to LPS, and that S. Typhimurium was capable of minimizing the host cell´s immune response. These results indicate that gastroenteritis-causing Salmonella such as the non-host- adapted or broad hostrange serovars S. Enteriditis or S. Typhimurium which have a more rapid intracellular growth rate and a higher NF-κB response in human and porcine hosts, contribute to their own elimination through the host immune system. Serovars causing septicemia such as S. Choleraesuis or S. Typhimurium in porcine and murine hosts, respectively, showed low intracellular growth and reduced host cell immune responses. It is suggested that this latter observation permits the pathogens to evade (at least for a short time) the host immune response, allowing a more effective dissemination of the pathogen in the host. However, the ability for the pathogen to spread systemically is also directly related to its ability to surivive within the macrophage of a particular host. The mononuclear phagocytes (macrophage) of the host therefore play an essential role as both barrier and component of the host-specificity of Salmonella serovars. Since the host also plays an essential role on the outcome of an infection, in the second half of this work, the porcine NOD proteins involved in the innate immune response were also investigated in order to provide a basis for future research on Salmonella host specificity. The data show that the porcine NOD proteins show a much higher homology with the human NOD proteins than the human NOD proteins show to murine NOD proteins. In addition, the LRR domains of the porcine NOD1 protein showed differences compared to the human NOD1, suggesting the recognition of PGN might play a role in host-specificity. Furthermore, the LRR domains among two different crosses of swine also showed single nucleotide polymorphisms (SNPs), possibly indicating variability among the swine populations. These sequence differences might represent an important factor involved in resistance or sensitivity to infections. Future work concerning the interplay between the NOD proteins could provide additional information not only with regard to the host cell response to bacteria, but also provide possibilities for pharmacological modulation of this response

Suppression of Acoustic Resonances in BST-Based Bulk-Ceramic Varactors by Addition of Magnesium Borate

This work presents a method for reducing acoustic resonances in ferroelectric barium strontium titanate (BST)-based bulk ceramic varactors, which are capable of operation in high-power matching circuits. Two versions of parallel-plate varactors are manufactured here: one with pure BST and one with 10 vol-% magnesium borate, Mg₃B₂O₆ (MBO). Each varactor includes a 0.85-mm-thick ferroelectric layer. Acoustic resonances that are present in the pure BST varactor are strongly suppressed in the BST-MBO varactor and, hence, the Q-factor is increased over a wide frequency range by the addition of small amounts of a low-dielectric-constant (LDK) MBO. Although the tunability is reduced due to the presence of non-tunable MBO, the increased Q-factor extends the varactor’s availability for low-loss and high-power applications

Western blot of tight junction proteins ZO-1 and claudin-3 in IPEC-J2 cells treated with deoxynivalenol (DON).

<p>Cells were grown on inserts and incubated for 24, 48 or 74 hours with DON (0 or 2000 ng/mL) applied from apical or basolateral side in complete medium. ZO-1 (225 kDa) and claudin-3 (22 kDa) expression was analysed by immunoblotting. The housekeeping protein GAPDH (37 kDa) was used as loading control.</p

Cellular distribution of the tight junction protein claudin-3 (CLDN-3) in IPEC-J2 monolayers treated with deoxynivalenol (DON).

<p>Cells were grown on inserts and incubated for 24, 48 or 74 hours either without DON (upper panel) or with 2000 ng/mL DON applied from apical (middle panel) or basolateral side (lower panel) in complete medium. Monolayers were stained for the tight junction associated protein claudin-3 and nuclei stained with DAPI, then detected by immunofluorescence microscopy. All micrographs were taken under identical exposure time and in the centre of each membrane. Micrographs are representative for 3 separate experiments with similar results. Scale bar  =  50 µm.</p

The systemic inflammatory response to Clostridium difficile infection.

The systemic inflammatory response to Clostridium difficile infection (CDI) is incompletely defined, particularly for patients with severe disease.Analysis of 315 blood samples from 78 inpatients with CDI (cases), 100 inpatients with diarrhea without CDI (inpatient controls), and 137 asymptomatic outpatient controls without CDI was performed. Serum or plasma was obtained from subjects at the time of CDI testing or shortly thereafter. Severe cases had intensive care unit admission, colectomy, or death due to CDI within 30 days after diagnosis. Thirty different circulating inflammatory mediators were quantified using an antibody-linked bead array. Principal component analysis (PCA), multivariate analysis of variance (MANOVA), and logistic regression were used for analysis.Based on MANOVA, cases had a significantly different inflammatory profile from outpatient controls but not from inpatient controls. In logistic regression, only chemokine (C-C motif) ligand 5 (CCL5) levels were associated with cases vs. inpatient controls. Several mediators were associated with cases vs. outpatient controls, especially hepatocyte growth factor, CCL5, and epithelial growth factor (inversely associated). Eight cases were severe and associated with elevations in IL-8, IL-6, and eotaxin.A broad systemic inflammatory response occurs during CDI and severe cases appear to differ from non-severe infections