Sara Bogaert

The need for transparency and good practices in the qPCR literature

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.

Hydroxylase Inhibition Abrogates TNF-α–Induced Intestinal Epithelial Damage by Hypoxia-Inducible Factor-1–Dependent Repression of FADD

Hydroxylase inhibitors stabilize hypoxia-inducible factor-1 (HIF-1), which has barrier-protective activity in the gut. Because the inflammatory cytokine TNF-α contributes to inflammatory bowel disease in part by compromising intestinal epithelial barrier integrity, hydroxylase inhibition may have beneficial effects in TNF-α–induced intestinal epithelial damage. The hydroxylase inhibitor dimethyloxalylglycin (DMOG) was tested in a murine model of TNF-α–driven chronic terminal ileitis. DMOG-treated mice experienced clinical benefit and showed clear attenuation of chronic intestinal inflammation compared with that of vehicle-treated littermates. Additional in vivo and in vitro experiments revealed that DMOG rapidly restored terminal ileal barrier function, at least in part through prevention of TNF-α–induced intestinal epithelial cell apoptosis. Subsequent transcriptional studies indicated that DMOG repressed Fas-associated death domain protein (FADD), a critical adaptor molecule in TNFR-1-mediated apoptosis, in an HIF-1α–dependent manner. Loss of this FADD repression by HIF-1α-targeting small interfering RNA significantly diminished the antiapoptotic action of DMOG. Additional molecular studies led to the discovery of a previously unappreciated HIF-1 binding site in the FADD promoter, which controls repression of FADD during hypoxia. As such, the results reported in this study allowed the identification of an innate mechanism that protects intestinal epithelial cells during (inflammatory) hypoxia, by direct modulation of death receptor signaling. Hydroxylase inhibition could represent a promising alternative treatment strategy for hypoxic inflammatory diseases, including inflammatory bowel disease.

Effects of vagus nerve stimulation on pro- and anti-inflammatory cytokine induction in patients with refractory epilepsy

The role of the vagus nerve in controlling and modulating inflammatory responses under physiological conditions has been investigated. The purpose of this study is to assess changes in the immunological state evoked by vagus nerve stimulation in humans, by measuring cytokines produced by peripheral blood mononuclear cells (PBMC). We compared induction of IL-1beta, IL-6, IL-8, IL-10 and TNF-alpha by lipopolysaccharide (LPS)-stimulated PBMC which were isolated from patients treated with vagus nerve stimulation for refractory epilepsy. We observed a significant decrease in IL-8 induction by LPS-stimulated PBMC after 6 months of vagus nerve stimulation in comparison to the pre-stimulation state. No significant changes were seen in the induction of IL-1beta, TNF-alpha, IL-6 or IL-10. The present study shows that cytokine induction by PBMC isolated from patients with refractory epilepsy is altered by long-term vagus nerve stimulation.

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

Background Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously. Methodology/Principal Findings Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohns disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa. Conclusions/Significance Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.

Differential mucosal expression of Th17-related genes between the inflamed colon and ileum of patients with inflammatory bowel disease

BackgroundImmunological and genetic findings implicate Th17 effector cytokines in the pathogenesis of inflammatory bowel disease (IBD). Expression of Th17 pathway-associated genes is mainly studied in colonic disease. The present study assessed the mRNA expression levels of Th17 effector cytokines (IL17A, IL17F, IL21, IL22 and IL26) and genes involved in differentiation (IL6, IL1B, TGFB1, IL23A and STAT3) and recruitment of Th17 cells (CCR6 and CCL20) by quantitative real-time PCR analysis of colonic and ileal biopsies from 22 healthy control subjects, 26 patients with Crohns disease (CD) and 12 patients with ulcerative colitis (UC). Inflammation was quantified by measuring expression of the inflammatory mediators IL8 and TNF.ResultsEvaluation of mRNA expression levels in colonic and ileal control samples revealed that TNF, TGFB1, STAT3 and CCR6 were expressed at higher levels in the ileum than in the colon. Expression of all the Th17 pathway-associated genes was increased in inflamed colonic samples. The increased expression of these genes was predominantly observed in samples from UC patients and was associated with more intense inflammation. Although increased expression of IL17A, IL17F, IL21 and IL26 was detected in inflamed ileal samples, expression of the indispensable Th17 cell differentiation factors TGFB1 and IL23A, the signaling molecule STAT3 and the Th17 recruitment factors CCR6 and CCL20 were unchanged.ConclusionsOur findings suggest that immune regulation is different in colonic and ileal disease, which might have important consequences for therapeutic intervention.

CARD15 variants determine a disturbed early response of monocytes to adherent-invasive Escherichia coli strain LF82 in Crohn's disease.

Caspase activation and recruitment domain 15 (CARD15) and Toll‐like receptor 4 (TLR4) are respectively intracellular and membrane‐bound receptors for bacterial cell wall components [respectively muramyl dipeptide (MDP) and lipopolysaccharide (LPS)]. Polymorphisms in CARD15 and TLR4 have been linked with Crohns disease (CD). Adherent‐invasive Escherichia coli (AIEC) strains with particular adhesion and invasion characteristics have been specifically associated with CD ileal mucosa. The aim of this study was to investigate the functional impact of these polymorphisms on monocytes in patients with CD, in response to MDP, LPS and AIEC strain LF82. Monocytes were isolated from 40 patients with CD using magnetic cell sorting, stimulated with LPS or MDP or infected with AIEC. IL‐1β, IL‐6, IL‐8, IL‐10, IL‐12 and tumour necrosis factor alpha induction was assessed using quantitative real time–polymerase chain reaction, Cytometric Bead Array and ELISA. Bacterial intracellular survival and replication was assessed using a gentamicin protection assay. Results were linked with the presence of CARD15 and TLR4 polymorphisms. Monocytes of patients with CARD15 polymorphisms showed an early reduced cytokine response (IL‐1β, IL‐6 and IL‐10) to infection with AIEC, which was restored after 20 h. A gene–dose effect was seen, comparing wild‐types, heterozygotes and homozygotes. We found no differences in intracellular survival and replication of AIEC. Heterozygous carriage of TLR4 polymorphisms did not influence monocyte response. In conclusion, patients with CD carrying CARD15 polymorphisms show a disturbed early inflammatory monocyte response after infection with AIEC strain LF82. For the first time, a functional defect was detected in single heterozygous carriers. These findings reflect the potential role of a genetically altered host response to disease‐related bacteria in the pathogenesis of CD.

M2045 Reduced Metallothionein Expression in Colonic Crohn's Disease: Evidence for a New Disease-Modifying Gene

Author Block D. Laukens 1 , H. Peeters 1 , A. Waeytens 1 , S. Bogaert 1 , B. Vander Cryssen 1 , D. Elewaut 1 , F. De Keyser 1 , H. Mielants 1 , C. Cuvelier 1 , K. Knecht 2 , P. Van Hummelen 3 , J. Del-Favero 4 , E. Remaut 5 , M. De Vos 1 , P. Rottiers 5 ; 1 Ghent University Hospital, Gent, BELGIUM, 2 Applied Maths BVBA, Sint-Martens-Latem, BELGIUM, 3 Flanders Institute for Biotechnology, Gent, BELGIUM, 4 Flanders Institute for Biotechnology, Antwerpen, BELGIUM, 5 Ghent University, Gent, BELGIUM.