E. coli Nissle 1917 Affects Salmonella Adhesion to Porcine Intestinal Epithelial Cells

BACKGROUND: The probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to interfere in a human in vitro model with the invasion of several bacterial pathogens into epithelial cells, but the underlying molecular mechanisms are not known. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the inhibitory effects of EcN on Salmonella Typhimurium invasion of porcine intestinal epithelial cells, focusing on EcN effects on the various stages of Salmonella infection including intracellular and extracellular Salmonella growth rates, virulence gene regulation, and adhesion. We show that EcN affects the initial Salmonella invasion steps by modulating Salmonella virulence gene regulation and Salmonella SiiE-mediated adhesion, but not extra- and intracellular Salmonella growth. However, the inhibitory activity of EcN against Salmonella invasion always correlated with EcN adhesion capacities. EcN mutants defective in the expression of F1C fimbriae and flagellae were less adherent and less inhibitory toward Salmonella invasion. Another E. coli strain expressing F1C fimbriae was also adherent to IPEC-J2 cells, and was similarly inhibitory against Salmonella invasion like EcN. CONCLUSIONS: We propose that EcN affects Salmonella adhesion through secretory components. This mechanism appears to be common to many E. coli strains, with strong adherence being a prerequisite for an effective reduction of SiiE-mediated Salmonella adhesion

New Platform Technology for Comprehensive Serological Diagnostics of Autoimmune Diseases

Antibody assessment is an essential part in the serological diagnosis of autoimmune diseases. However, different diagnostic strategies have been proposed for the work up of sera in particular from patients with systemic autoimmune rheumatic disease (SARD). In general, screening for SARD-associated antibodies by indirect immunofluorescence (IIF) is followed by confirmatory testing covering different assay techniques. Due to lacking automation, standardization, modern data management, and human bias in IIF screening, this two-stage approach has recently been challenged by multiplex techniques particularly in laboratories with high workload. However, detection of antinuclear antibodies by IIF is still recommended to be the gold standard method for antibody screening in sera from patients with suspected SARD. To address the limitations of IIF and to meet the demand for cost-efficient autoantibody screening, automated IIF methods employing novel pattern recognition algorithms for image analysis have been introduced recently. In this respect, the AKLIDES technology has been the first commercially available platform for automated interpretation of cell-based IIF testing and provides multiplexing by addressable microbead immunoassays for confirmatory testing. This paper gives an overview of recently published studies demonstrating the advantages of this new technology for SARD serology

Gentamicin sulphate permeation through porcine intestinal epithelial cell monolayer

Gentamicin is an aminoglycoside antibiotic widely used in combination with dimethyl sulphoxide (DMSO) in topical drug formulations. It is not known, however, whether DMSO can enhance the permeation of gentamicin through biological membranes, leading to oto- and nephrotoxic side effects. A simple and reliable high-performance liquid chromatographic (HPLC) method was applied for the quantitative determination of gentamicin collected from the apical and basolateral compartments of the porcine intestinal epithelial cell line IPEC-J2 cell monolayer using fluorometric derivatisation of the analyte with fluorenylmethyloxycarbonyl chloride (FMOC) prior to chromatographic run in the presence and absence of 1% DMSO. The lack of change in transepithelial electrical resistance (TER) demonstrated that gentamicin and 1% DMSO did not affect IPEC-J2 cell monolayer integrity via the disruption of cell membranes. Chromatographic data also ascertained that gentamicin penetration across the cell monolayer even in the presence of 1% DMSO was negligible at 6 h after the beginning of apical gentamicin administration. This study further indicates that the addition of this organic solvent does not increase the incidence of toxic effects related to gentamicin permeation

Effect of Fructooligosaccharide Metabolism on Chicken Colonization by an Extra-Intestinal Pathogenic Escherichia coli Strain

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains cause many diseases in humans and animals. While remaining asymptomatic, they can colonize the intestine for subsequent extra-intestinal infection and dissemination in the environment. We have previously identified the fos locus, a gene cluster within a pathogenicity island of the avian ExPEC strain BEN2908, involved in the metabolism of short-chain fructooligosaccharides (scFOS). It is assumed that these sugars are metabolized by the probiotic bacteria of the microbiota present in the intestine, leading to a decrease in the pathogenic bacterial population. However, we have previously shown that scFOS metabolism helps BEN2908 to colonize the intestine, its reservoir. As the fos locus is located on a pathogenicity island, one aim of this study was to investigate a possible role of this locus in the virulence of the strain for chicken. We thus analysed fos gene expression in extracts of target organs of avian colibacillosis and performed a virulence assay in chickens. Moreover, in order to understand the involvement of the fos locus in intestinal colonization, we monitored the expression of fos genes and their implication in the growth ability of the strain in intestinal extracts of chicken. We also performed intestinal colonization assays in axenic and Specific Pathogen-Free (SPF) chickens. We demonstrated that the fos locus is not involved in the virulence of BEN2908 for chickens and is strongly involved in axenic chicken cecal colonization both in vitro and in vivo. However, even if the presence of a microbiota does not inhibit the growth advantage of BEN2908 in ceca in vitro, overall, growth of the strain is not favoured in the ceca of SPF chickens. These findings indicate that scFOS metabolism by an ExPEC strain can contribute to its fitness in ceca but this benefit is fully dependent on the bacteria present in the microbiota

Loss of tolerance to gut immunity protein; glycoprotein 2 (GP2) is associated with progressive disease course in primary sclerosing cholangitis

Abstract Glycoprotein 2[GP2] is a specific target of pancreatic autoantibodies[PAbs] in Crohn’s disease(CD) and is involved in gut innate immunity processes. Our aim was to evaluate the prevalence and prognostic potential of PAbs in primary sclerosing cholangitis(PSC). Sixty-five PSC patients were tested for PAbs by indirect immunofluorescence and compared with healthy (n = 100) and chronic liver disease controls(CLD, n = 488). Additionally, a panel of anti-microbial antibodies and secretory (s)IgA levels were measured, as markers of bacterial translocation and immune dysregulation. PAbs were more frequent in PSC(46.2%) compared to controls(healthy:0% and CLD:4.5%), [P < 0.001, for each]. Occurrence of anti-GP2 antibody was 30.8% (20/65) and was exclusively of IgA isotype. Anti-GP2 IgA positive patients had higher sIgA levels (P = 0.021). With flow-cytometry, 68.4% (13/19) of anti-GP2 IgA antibodies were bound with secretory component, suggesting an active retro-transportation of anti-GP2 from the gut lumen to the mucosa. Anti-GP2 IgA was associated with shorter transplant-free survival [PLogRank < 0.01] during the prospective follow-up (median, IQR: 87 [9–99] months) and remained an independent predictor after adjusting for Mayo risk score(HR: 4.69 [1.05–21.04], P = 0.043). These results highlight the significance of gut-liver interactions in PSC. Anti-GP2 IgA might be a valuable tool for risk stratification in PSC and considered as a potential therapeutic target

Air–liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC)

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism’s internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air–liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro

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

Mucosal Autoimmunity to Cell-Bound GP2 Isoforms Is a Sensitive Marker in PSC and Associated With the Clinical Phenotype

Introduction: Zymogen granule glycoprotein 2 (GP2) was demonstrated as first autoimmune mucosal target in primary sclerosing cholangitis (PSC) associated with disease severity. Autoantibodies to four GP2 isoforms (aGP21−4) were found in patients with inflammatory bowel diseases but reactivity against specific GP2 epitopes has not been investigated in PSC yet. Hence, the prevalence of aGP21−4 and their association with the PSC phenotype for risk prediction were examined.Methods: GP2 isoforms were stably expressed as glycosylphosphatidyl - inositol-anchored molecules in the membrane of HEp-2 cells and used as autoantigenic targets in indirect immunofluorescence assay (IFA). aGP21−4 IgA and IgG were detected by IFA in 212 PSC patients of four European university hospitals and 145 controls comprising 95 patients with cystic fibrosis and 50 healthy subjects.Results: Combined aGP21 and aGP24 IgA testing with a sensitivity of 66.0% and a specificity of 97.9% resulted in the best diagnostic performance (Youden index: 0.64) regarding all aGP2 and combinations thereof. aGP24 IgA positivity is significantly associated with the presence of cirrhosis in PSC (p = 0.0056). Logistic regression revealed the occurrence of aGP21 IgA (odds ratio [OR] 1.38, 95% confidence interval [CI]: 1.03–1.86) and aGP24 IgA (OR 1.52, 95%CI: 1.07–2.15) along with male gender (OR 0.51, 95%CI: 0.27–0.97) and older age (OR 1.03 95%CI: 1.01–1.05) as significant risks for the concomitant presence of cirrhosis in PSC.Conclusions: Combined aGP21 and aGP24 IgA analysis is preferred to single aGP2 isoform analysis for sensitive PSC autoantibody testing. Positivity for aGP21 and aGP24 IgA is associated with cirrhosis in PSC and could be used for risk stratification

The Mycotoxin Deoxynivalenol Potentiates Intestinal Inflammation by Salmonella Typhimurium in Porcine Ileal Loops

Background and Aims: Both deoxynivalenol (DON) and nontyphoidal salmonellosis are emerging threats with possible hazardous effects on both human and animal health. The objective of this study was to examine whether DON at low but relevant concentrations interacts with the intestinal inflammation induced by Salmonella Typhimurium. Methodology: By using a porcine intestinal ileal loop model, we investigated whether intake of low concentrations of DON interacts with the early intestinal inflammatory response induced by Salmonella Typhimurium. Results: A significant higher expression of IL-12 and TNF alpha and a clear potentiation of the expression of IL-1 beta, IL-8, MCP-1 and IL-6 was seen in loops co-exposed to 1 mu g/mL of DON and Salmonella Typhimurium compared to loops exposed to Salmonella Typhimurium alone. This potentiation coincided with a significantly enhanced Salmonella invasion in and translocation over the intestinal epithelial IPEC-J2 cells, exposed to non-cytotoxic concentrations of DON for 24 h. Exposure of Salmonella Typhimurium to 0.250 mu g/mL of DON affected the bacterial gene expression level of a limited number of genes, however none of these expression changes seemed to give an explanation for the increased invasion and translocation of Salmonella Typhimurium and the potentiated inflammatory response in combination with DON. Conclusion: These data imply that the intake of low and relevant concentrations of DON renders the intestinal epithelium more susceptible to Salmonella Typhimurium with a subsequent potentiation of the inflammatory response in the gut