Leen Boesmans

Impaired Islet Function in Commonly Used Transgenic Mouse Lines due to Human Growth Hormone Minigene Expression

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on β cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic β cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the β cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.

Modulating the microbiota in inflammatory bowel diseases: prebiotics, probiotics or faecal transplantation?

Crohns disease (CD) and ulcerative colitis (UC) are the two major phenotypes of inflammatory bowel diseases (IBD) which constitute a spectrum of chronic, debilitating diseases characterised by a relapsing inflammation of the intestinal mucosal lining. Evidence from a variety of disciplines implicates the intestinal microbiota in the pathogenesis of idiopathic IBD and their complications, including pouchitis. Many studies have reported a dysbiosis in IBD, characterised by a decrease in diversity, a decreased abundance of some dominant commensal members (such as Clostridium IV and XIVa) and an increase in detrimental bacteria (such as sulphate reducing bacteria and Escherichia coli). Therapies such as prebiotics and probiotics aim to selectively manipulate the intestinal microbiota and have been evaluated as an attractive therapeutic option with few side effects. The multispecies product VSL#3 was found effective in preventing and maintaining remission in pouchitis, whereas both VSL#3 and E. coli Nissle were effective in maintaining remission in UC. A more drastic approach to restore the composition of the microbiota and correct the underlying imbalance is a faecal microbiota transplantation (FMT). FMT has been successfully applied to treat patients with even recalcitrant Clostridium difficile infection. Particularly in UC, the majority of studies suggest that FMT may be an effective treatment option although the evidence is still limited. It is anticipated that our increasing knowledge on the composition and function of the intestinal microbiota components will allow in the future for a better selection of highly performing bacteria with specific functions required for specific benefits.

Inflammation-Induced Downregulation of Butyrate Uptake and Oxidation Is Not Caused by a Reduced Gene Expression.

In ulcerative colitis (UC) the butyrate metabolism is impaired, leading to energy‐deficiency in the colonic cells. The effect of inflammation on the butyrate metabolism was investigated. HT‐29 cells were incubated with pro‐inflammatory cytokines (TNF‐α and/or IFN‐γ) for 1 and 24 h. Cells were additionally stimulated with butyrate to investigate its anti‐inflammatory potential. Butyrate uptake and oxidation were measured using 14C‐labeled butyrate. Gene expression of the butyrate metabolism enzymes, interleukin 8 (IL‐8; inflammatory marker) and villin‐1 (VIL‐1; epithelial cell damage marker) was measured via quantitative RT‐PCR. Significantly increased IL‐8 expression and decreased VIL‐1 expression after 24 h incubation with TNF‐α and/or IFN‐γ confirmed the presence of inflammation. These conditions induced a decrease of both butyrate uptake and oxidation, whereas the gene expression was not reduced. Simultaneous incubation with butyrate counteracted the reduced butyrate oxidation. In contrast, 1 h incubation with TNF‐α induced a significant increased IL‐8 expression and decreased butyrate uptake. Incubation with TNF‐α and/or IFN‐γ for 1 h did not induce cell damage nor influence butyrate oxidation. The inflammation‐induced downregulation of the butyrate metabolism was not caused by a reduced gene expression, but appeared consequential to a decreased butyrate uptake. Increasing the luminal butyrate levels might have therapeutic potential in UC. J. Cell. Physiol. 230: 418–426, 2015.

Mo1799 Increased Fecal Water Cytotoxicity in Patients With Ulcerative Colitis Is Associated With Low Levels of Short Chain Fatty Acids

Background: Many studies have focused on the fecal microbiota however it is now widely accepted that the gastrointestinal (GI) mucosa associated microbiota, rather than luminal content, is critically important in host-microbe interactions linked to health and disease. Mucosal biopsy is the most common sampling technique used to assess the mucosa associated microbiota. However, normal biopsy devices are designed to take samples for histologic assessment. Thus when biopsies are taken via working channels that are also used to aspirate luminal content it is highly likely that cross contamination occurs, that ultimately questions the validity of these samples. In view of this difficulty we have developed a novel device that allows targeted biopsies to be taken in any segment of the GI tract without cross contamination in an aseptic manner. Methods: Six patients undergoing upper GI endoscopy for iron deficiency were recruited with consent and ethical approval. In pilot experiments various prototypes of a sampling device were tested in vivo. Two final configurations of a sheath that covered miniature biopsy forceps were tested and compared with the normal single-use biopsy forceps (18 samples). Matched duodenal biopsy samples were collected from each patient, transferred individually into RNA later, and subject to gDNA extraction. Amplicon libraries spanning the V6-V8 region of the 16S rRNA gene were constructed, sequenced using the Illumina MiSeq platform, and analysed via the QIIME pipeline. Results: Microbial DNA, representing a diverse community, was observed in all duodenal samples obtained using the aseptic device, indicating this device is effective in sampling mucosa associated organisms present in the duodenum (Chao 1 estimated richness = 386, Goods coverage 99.8%). Assessment of the duodenal microbiota revealed a community dominated by the genera Streptococcus, Prevotella, Veillonella, Neisseria and Porphyromonas. There were substantial differences in the microbiota of samples obtained using the aseptic device and samples obtained with standard biopsy forceps. Only a low level of correlation (Pearsons r<0.6) between the matched biopsy pairs was seen across all bacterial phyla observed. Phylogenetic based (UniFrac) evaluation of beta-diversity revealed the aseptic samples clustered together and showed reduced inter-individual variability compared to those obtained with standard forceps. Conclusions: Sampling of duodenal biopsies with routine biopsy forceps resulted in greater microbial diversity as compared to samples acquired under aseptic conditions. This suggests cross contamination from other sites of the upper GI tract when utilising standard forceps. Based upon our data, aseptic techniques with a specific sampling device should be used to collect samples for studies that aim to define the mucosa associated microbiota.

Mo1800 Colonic Luminal Compounds Do Not Affect Butyrate Metabolism in Ulcerative Colitis

Background: Many studies have focused on the fecal microbiota however it is now widely accepted that the gastrointestinal (GI) mucosa associated microbiota, rather than luminal content, is critically important in host-microbe interactions linked to health and disease. Mucosal biopsy is the most common sampling technique used to assess the mucosa associated microbiota. However, normal biopsy devices are designed to take samples for histologic assessment. Thus when biopsies are taken via working channels that are also used to aspirate luminal content it is highly likely that cross contamination occurs, that ultimately questions the validity of these samples. In view of this difficulty we have developed a novel device that allows targeted biopsies to be taken in any segment of the GI tract without cross contamination in an aseptic manner. Methods: Six patients undergoing upper GI endoscopy for iron deficiency were recruited with consent and ethical approval. In pilot experiments various prototypes of a sampling device were tested in vivo. Two final configurations of a sheath that covered miniature biopsy forceps were tested and compared with the normal single-use biopsy forceps (18 samples). Matched duodenal biopsy samples were collected from each patient, transferred individually into RNA later, and subject to gDNA extraction. Amplicon libraries spanning the V6-V8 region of the 16S rRNA gene were constructed, sequenced using the Illumina MiSeq platform, and analysed via the QIIME pipeline. Results: Microbial DNA, representing a diverse community, was observed in all duodenal samples obtained using the aseptic device, indicating this device is effective in sampling mucosa associated organisms present in the duodenum (Chao 1 estimated richness = 386, Goods coverage 99.8%). Assessment of the duodenal microbiota revealed a community dominated by the genera Streptococcus, Prevotella, Veillonella, Neisseria and Porphyromonas. There were substantial differences in the microbiota of samples obtained using the aseptic device and samples obtained with standard biopsy forceps. Only a low level of correlation (Pearsons r<0.6) between the matched biopsy pairs was seen across all bacterial phyla observed. Phylogenetic based (UniFrac) evaluation of beta-diversity revealed the aseptic samples clustered together and showed reduced inter-individual variability compared to those obtained with standard forceps. Conclusions: Sampling of duodenal biopsies with routine biopsy forceps resulted in greater microbial diversity as compared to samples acquired under aseptic conditions. This suggests cross contamination from other sites of the upper GI tract when utilising standard forceps. Based upon our data, aseptic techniques with a specific sampling device should be used to collect samples for studies that aim to define the mucosa associated microbiota.