L. Van Lommel

Insulin crystallization depends on zinc transporter ZnT8 expression, but is not required for normal glucose homeostasis in mice

Zinc co-crystallizes with insulin in dense core secretory granules, but its role in insulin biosynthesis, storage and secretion is unknown. In this study we assessed the role of the zinc transporter ZnT8 using ZnT8-knockout (ZnT8−/−) mice. Absence of ZnT8 expression caused loss of zinc release upon stimulation of exocytosis, but normal rates of insulin biosynthesis, normal insulin content and preserved glucose-induced insulin release. Ultrastructurally, mature dense core insulin granules were rare in ZnT8−/− beta cells and were replaced by immature, pale insulin “progranules,” which were larger than in ZnT8+/+ islets. When mice were fed a control diet, glucose tolerance and insulin sensitivity were normal. However, after high-fat diet feeding, the ZnT8−/− mice became glucose intolerant or diabetic, and islets became less responsive to glucose. Our data show that the ZnT8 transporter is essential for the formation of insulin crystals in beta cells, contributing to the packaging efficiency of stored insulin. Interaction between the ZnT8−/− genotype and diet to induce diabetes is a model for further studies of the mechanism of disease of human ZNT8 gene mutations.

Mucosal gene signatures to predict response to infliximab in patients with ulcerative colitis

Background and aims: Infliximab is an effective treatment for ulcerative colitis with over 60% of patients responding to treatment and up to 30% reaching remission. The mechanism of resistance to anti-tumour necrosis factor α (anti-TNFα) is unknown. This study used colonic mucosal gene expression to provide a predictive response signature for infliximab treatment in ulcerative colitis. Methods: Two cohorts of patients who received their first treatment with infliximab for refractory ulcerative colitis were studied. Response to infliximab was defined as endoscopic and histological healing. Total RNA from pre-treatment colonic mucosal biopsies was analysed with Affymetrix Human Genome U133 Plus 2.0 Arrays. Quantitative RT-PCR was used to confirm microarray data. Results: For predicting response to infliximab treatment, pre-treatment colonic mucosal expression profiles were compared for responders and non-responders. Comparative analysis identified 179 differentially expressed probe sets in cohort A and 361 in cohort B with an overlap of 74 probe sets, representing 53 known genes, between both analyses. Comparative analysis of both cohorts combined, yielded 212 differentially expressed probe sets. The top five differentially expressed genes in a combined analysis of both cohorts were osteoprotegerin, stanniocalcin-1, prostaglandin-endoperoxide synthase 2, interleukin 13 receptor alpha 2 and interleukin 11. All proteins encoded by these genes are involved in the adaptive immune response. These markers separated responders from non-responders with 95% sensitivity and 85% specificity. Conclusion: Gene array studies of ulcerative colitis mucosal biopsies identified predictive panels of genes for (non-)response to infliximab. Further study of the pathways involved should allow a better understanding of the mechanisms of resistance to infliximab therapy in ulcerative colitis. ClinicalTrials.gov number, NCT00639821.

Progesterone rise on HCG day in GnRH antagonist/rFSH stimulated cycles affects endometrial gene expression

Premature progesterone rise during gonadotrophin-releasing hormone (GnRH) antagonist cycles for IVF is a frequent phenomenon and has been associated with lower pregnancy and implantation rates. This study evaluated endometrial gene expression on the day of oocyte retrieval according to the concentration of serum progesterone on the day of human chorionic gonadotrophin (HCG) administration in GnRH-antagonist/recombinant FSH IVF cycles with fresh embryo transfer. Endometrial biopsies (n=14) were analysed with Affymetrix HG U133 Plus 2.0 Arrays. Patients were divided into three groups according to their progesterone serum concentration on the day of HCG administration: ≤ 0.9 ng/ml (group A), 1-1.5 ng/ml (group B) and >1.5 ng/ml (group C). Gene expression analysis showed a small number of significantly differentially expressed probe sets between groups A and B (five up/23 down in B) and a large difference between groups B and C (607 up/212 down; P ≤ 0.05, fold change ≥ 1.4). Validation was performed with quantitative real-time PCR on selected genes. As far as is known, this is the first study to demonstrate a distinct difference in endometrial gene expression profile between patients with a progesterone serum concentration above and below the threshold of 1.5 ng/ml on the day of HCG administration.

Discovery of molecular pathways mediating 1,25-dihydroxyvitamin D3 protection against cytokine-induced inflammation and damage of human and male mouse islets of Langerhans.

Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.

Testing the hypothesis of tissue selectivity

Motivation: Finding genes that are preferentially expressed in a particular tissue or condition is a problem that cannot be solved by standard statistical testing procedures. A relatively unknown procedure that can be used is the intersection–union test (IUT). However, two disadvantages of the IUT are that it is conservative and it conveys only the information of the least differing target tissue–other tissue pair. Results: We propose a Bayesian procedure that quantifies how much evidence there is in the overall expression profile for selective over-expression. In a small simulation study, it is shown that the proposed method outperforms the IUT when it comes to finding selectively expressed genes. An application to publicly available data consisting of 22 tissues shows that the Bayesian method indeed selects genes with functions that reflect the specific tissue functions. The proposed method can also be used to find genes that are underexpressed in a particular tissue. Availability: Both MATLAB and R code that implement the IUT and the Bayesian procedure in an efficient way, can be downloaded at http://ppw.kuleuven.be/okp/software/BayesianIUT/. Contact: katrijn.vandeun@psy.kuleuven.be

P067 Molecular profiling of early Crohn's disease reveals a prominent role for WNT5A.

[Verstockt, S.; Cleynen, I.] Katholieke Univ Leuven, Dept Human Genet, Leuven, Belgium. [Van der Goten, J.; Vancamelbeke, M.; Verstockt, B.; Rutgeerts, P.; Ferrante, M.; Vermeire, S.; Arijs, I.] Katholieke Univ Leuven, Dept Clin & Expt Med, Translat Res Ctr Gastrointestinal Disorders, Leuven, Belgium. [Van Lommel, L.; Schuit, F.] Katholieke Univ Leuven, Dept Cellular & Mol Med, Gene Express Unit, Leuven, Belgium. [Arijs, I.] Hasselt Univ, Fac Med & Life Sci, Hasselt, Belgium. [Arijs, I.] Jessa Hosp, Hasselt, Belgium.

P096 Analysis of the tryptophan metabolism and indoleamine 2,3-dioxygenase expression (IDO) in patients with inflammatory bowel disease before and after infliximab treatment

inflammatory bowel disease (IBD) patients and their clinical significance. Methods: Anti-IFI16 antibodies titers were measured in a cohort of 75 IBD patients’ sera, including ulcerative colitis (UC, n = 27) and Crohn’s disease (CD, n = 48), alongside with 182 healthy controls (HC), by means of an in house ELISA kit. Sera from those patients undergoing Infliximab (IFX) therapy were also collected before the first 5 drug infusions. Clinical data were collected and statistical analysis was performed using Student’s t test or Fisher’s exact test, as appropriate. Results: Significantly higher levels of anti-IFI16 autoantibodies were observed in IBD patients compared to HC (mean levels ±SEM: 109.7±16.9U/ml vs. 52.9±11.2U/ml, p < 0.0001). Fifteen patients (20%) displayed anti-IFI16 and there was a trend towards a higher prevalence in UC vs. CD (25.9% vs. 16.6%). No correlation with biochemical or clinical disease activity was found. Anti-IFI16 levels were monitored for the first 5 drug infusions in a subgroup of 37 patients (12 UC and 25 CD) undergoing IFX therapy. The percentage of anti-IFI16 positive patients increased during IFX therapy from 18.9% to 70.2%. Interestingly, patients presenting anti-IFI16 antibodies at baseline or developing them during IFX therapy were more likely to be primary or secondary non-responder to IFX than those negative (34% vs. 0%, respectively; p = 0.03).

P046 Differential expression of microRNAs in inflamed colonic mucosa of patients with ulcerative colitis

Conclusions: IFX acts locally at mucosa level, as shown by the decrease in inflammatory infiltrate and cytokines contents. The effect of IFX is partially dependent on direct effect on epithelial cells. IFX seems to ameliorate epithelial cell healing by blocking the detrimental capacity of TNF-a pre-treated fibroblast on intestinal epithelial cells. More data are necessary to unravel molecular pathways of these observations, in order also to clarify mechanisms of loss of responses to it as well as new targets for potential new therapies.

1 - Colonic mucosal expression of barrier genes in patients with inflammatory bowel disease before and after first infliximab treatment

1 Colonic mucosal expression of barrier genes in patients with inflammatory bowel disease before and after first infliximab treatment I. Arijs1 *, L. Van Lommel2, K. Van Steen3, G. De Hertogh4, R. Quintens2, G. Van Assche1, S. Vermeire1, K. Geboes4, F. Schuit2, P. Rutgeerts1. 1Department of Gastroenterology, University Hospital Gasthuisberg, Leuven, Belgium, 2Gene Expression Unit, Katholieke Universiteit Leuven, Leuven, Belgium, 3Department of Electrical Engineering and Computer Science (Montefiore Institute), University of Liege, Liege, Belgium, 4Department of Morphology and Molecular Pathology, University Hospital Gasthuisberg, Leuven, Belgium