Pascal Frei

Smoking cessation induces profound changes in the composition of the intestinal microbiota in humans.

Background The human intestinal microbiota is a crucial factor in the pathogenesis of various diseases, such as metabolic syndrome or inflammatory bowel disease (IBD). Yet, knowledge about the role of environmental factors such as smoking (which is known to influence theses aforementioned disease states) on the complex microbial composition is sparse. We aimed to investigate the role of smoking cessation on intestinal microbial composition in 10 healthy smoking subjects undergoing controlled smoking cessation. Methods During the observational period of 9 weeks repetitive stool samples were collected. Based on abundance of 16S rRNA genes bacterial composition was analysed and compared to 10 control subjects (5 continuing smokers and 5 non-smokers) by means of Terminal Restriction Fragment Length Polymorphism analysis and high-throughput sequencing. Results Profound shifts in the microbial composition after smoking cessation were observed with an increase of Firmicutes and Actinobacteria and a lower proportion of Bacteroidetes and Proteobacteria on the phylum level. In addition, after smoking cessation there was an increase in microbial diversity. Conclusions These results indicate that smoking is an environmental factor modulating the composition of human gut microbiota. The observed changes after smoking cessation revealed to be similar to the previously reported differences in obese compared to lean humans and mice respectively, suggesting a potential pathogenetic link between weight gain and smoking cessation. In addition they give rise to a potential association of smoking status and the course of IBD.

Combined effect of 25-OH vitamin D plasma levels and genetic Vitamin DReceptor (NR 1I1) variants on fibrosis progression rate in HCV patients

Decreased vitamin D levels have been described in various forms of chronic liver disease and associated with advanced fibrosis. Whether this association is a cause or consequence of advanced fibrosis remains unclear to date.

Smoking cessation alters intestinal microbiota: insights from quantitative investigations on human fecal samples using FISH.

Background:There has been a dramatic increase in investigations on the potential mechanistic role of the intestinal microbiota in various diseases and factors modulating intestinal microbial composition. We recently reported on intestinal microbial shifts after smoking cessation in humans. In this study, we aimed to conduct further microbial analyses and verify our previous results obtained by pyrosequencing using a direct quantitative microbial approach. Methods:Stool samples of healthy smoking human subjects undergoing controlled smoking cessation during a 9-week observational period were analyzed and compared with 2 control groups, ongoing smoking and nonsmoking subjects. Fluorescence in situ hybridization was applied to quantify specific bacterial groups. Results:Intestinal microbiota composition was substantially altered after smoking cessation as characterized by an increase in key representatives from the phyla of Firmicutes (Clostridium coccoides, Eubacterium rectale, and Clostridium leptum subgroup) and Actinobacteria (HGC bacteria and Bifidobacteria) as well as a decrease in Bacteroidetes (Prevotella spp. and Bacteroides spp.) and Proteobacteria (&bgr;- and &ggr;-subgroup of Proteobacteria). Conclusions:As determined by fluorescence in situ hybridization, an independent direct quantitative microbial approach, we could confirm that intestinal microbiota composition in humans is influenced by smoking. The characteristics of observed microbial shifts suggest a potential mechanistic association to alterations in body weight subsequent to smoking cessation. More importantly, regarding previously described microbial hallmarks of dysbiosis in inflammatory bowel diseases, a variety of observed microbial alterations after smoking cessation deserve further consideration in view of the divergent effect of smoking on the clinical course of Crohns disease and ulcerative colitis.

Protein tyrosine phosphatase N2 regulates TNFα-induced signalling and cytokine secretion in human intestinal epithelial cells

Objective The Crohns disease (CD) susceptibility gene, protein tyrosine phosphatase N2 (PTPN2), regulates interferon γ (IFNγ)-induced signalling and epithelial barrier function in T84 intestinal epithelial cells (IECs). The aim of this study was to investigate whether PTPN2 is also regulated by tumour necrosis factor α (TNFα) and if PTPN2 controls TNFα-induced signalling and effects in IECs. Methods T84 IECs were used for all cell studies. Protein levels were assessed by western blotting, mRNA levels by reverse transcription–PCR (RT–PCR) and cytokine levels by ELISA. PTPN2 knock-down was induced by small interfering RNA (siRNA). Imaging was performed by immunohistochemistry or immunofluorescence. Results TNFα treatment elevated PTPN2 mRNA as well as nuclear and cytoplasmic protein levels and caused cytoplasmic accumulation of PTPN2. Biopsy specimens from patients with active CD showed strong immunohistochemical PTPN2 staining in the epithelium, whereas samples from patients with CD in remission featured PTPN2 levels similar to controls without inflammatory bowel disease (IBD). Though samples from patients with active ulcerative colitis (UC) revealed more PTPN2 protein than non-IBD patients and patients with UC in remission, their PTPN2 expression was lower than in active CD. Samples from patients with CD in remission and responding to anti-TNF treatment also showed PTPN2 levels that were similar to those in control patients. Pharmacological inhibition of nuclear factor-κB (NF-κB) by BMS-345541 prevented the TNFα-induced rise in PTPN2 protein, independent of apoptotic events. PTPN2 knock-down revealed that the phosphatase regulates TNFα-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation, without affecting c-Jun N-terminal kinase (JNK), inhibitor of κB (IκB) or NF-κB phosphorylation. Loss of PTPN2 potentiated TNFα-induced secretion of interleukin 6 (IL-6) and IL-8. In TNFα- and IFNγ-co-treated cells, loss of PTPN2 enhanced protein expression of inducible nitric oxide synthase (iNOS). Conclusions TNFα induces PTPN2 expression in IECs. Loss of PTPN2 promotes TNFα-induced mitogen-activated protein kinase signalling and the induction of inflammatory mediators. These data indicate that PTPN2 activity could play a crucial role in the establishment of chronic inflammatory conditions in the intestine, such as CD.

Bilberry ingestion improves disease activity in mild to moderate ulcerative colitis — An open pilot study

BACKGROUND AND AIMS A significant fraction of patients with ulcerative colitis (UC) is not sufficiently controlled with conventional therapy or suffers from therapy related side effects. Anthocyanins, highly abundant in bilberries (Vaccinium myrtillus), were shown to have antioxidative and anti-inflammatory effects. We aimed to explore the therapeutic potential of bilberries in active UC. METHODS In an open pilot trial with a total follow-up of 9 weeks the effect of a daily standardized anthocyanin-rich bilberry preparation was tested in 13 patients with mild to moderate UC. Clinical, biochemical, endoscopic and histologic parameters were assessed. RESULTS At the end of the 6 week treatment interval 63.4% of patients achieved remission, the primary endpoint, while 90.9% of patients showed a response. In all patients a decrease in total Mayo score was detected (mean: 6.5 and 3.6 at screening and week 7, respectively; p<0.001). Fecal calprotectin levels significantly decreased during the treatment phase (baseline: mean 778 μg/g, range 192-1790 μg/g; end of treatment: mean 305 μg/g, range <30-1586 μg/g; p=0.049), including 4 patients achieving undetectable levels at end of treatment. A decrease in endoscopic Mayo score and histologic Riley index confirmed the beneficial effect. However, an increase of calprotectin levels and disease activity was observed after cessation of bilberry intake. No serious adverse events were observed. CONCLUSIONS This is the first report on the promising therapeutic potential of a standardized anthocyanin-rich bilberry preparation in UC in humans. These results clearly indicate a therapeutic potential of bilberries in UC. Further studies on mechanisms and randomized clinical trials are warranted.

Protein tyrosine phosphatase nonreceptor type 2 regulates autophagosome formation in human intestinal cells

Background: Autophagy is a process of central importance for maintaining cell homeostasis, survival, and the regulation of inflammation. Recent studies associated variants within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2), and autophagy genes, such as autophagy‐related 16‐like 1 (ATG16L1), with chronic inflammatory disorders, such as Crohns disease (CD). We show that PTPN2 regulates autophagy in human intestinal epithelial cells (IEC) and primary colonic lamina propria fibroblasts (CLPF). Methods: Protein analysis in IEC and CLPF was performed by western blotting. Autophagososme formation was assessed by LC3B immunofluorescence or immunohistochemistry. Human intestinal tissue samples were obtained from noninflammatory bowel disease (IBD) control or from CD patients and genotyped for disease‐associated PTPN2 or ATG16L1 variations. Results: Knockdown of PTPN2 causes impaired autophagosome formation and dysfunctional autophagy resulted in increased levels of intracellular Listeria monocytogenes (LM) and elevated IEC apoptosis in response to tumor necrosis factor (TNF) and interferon gamma (IFN‐&ggr;). Similar findings were observed in primary CLPF derived from CD patients carrying the CD‐associated PTPN2 variant. Presence of the ATG16L1 variant prevented the cytokine‐induced rise in PTPN2 protein, finally resulting in impaired LC3B‐II levels in IEC. Actively inflamed intestinal biopsies from CD patients carrying either ATG16L1 or PTPN2 genetic variants revealed aberrant LC3B expression patterns when compared with samples from non‐IBD control patients. Conclusions: Our results demonstrate that PTPN2 regulates autophagosome formation in human intestinal cells. We provide a model of how a dysfunction of the CD susceptibility genes, PTPN2 and/or ATG16L1, may contribute to the onset and perpetuation of chronic intestinal inflammation. (Inflamm Bowel Dis 2011;)

Analysis of risk factors for low bone mineral density in inflammatory bowel disease

Background/Aim: Several risk factors have been suggested for osteoporosis which frequently occurs in inflammatory bowel disease (IBD) patients. We studied prevalence and risk factors for reduced bone mineral density (BMD) in IBD patients at the University Hospital of Zürich, Switzerland. Methods: The BMD was determined by dual-energy X-ray absorptiometry at the lumbar spine and femoral neck in 88 IBD patients (55 with Crohn’s disease, 30 with ulcerative colitis, and 3 with indeterminate colitis). Z scores were obtained by comparison with age- and sex-matched normal values, and T scores by comparison with sex-matched healthy young adults. Osteopenia and osteoporosis were defined according to the WHO guidelines. Predictive factors for BMD were analyzed by group comparison and stepwise regression analysis. Results: Osteopenia was present in 43% of the patients at the lumbar spine and in 42% of them at the femoral neck. Osteoporosis was present in 14% of the patients at the lumbar spine and in 5% of them at the femoral neck. At the lumbar spine, stepwise regression analysis showed that body mass index, age, number of bowel resections, topic steroids, and azathioprine correlated with the Z scores. Cumulative steroid dose, topic steroids, age and bowel resection were found to be predictors for a pathological T score. At the femoral neck, regression analysis showed that body mass index, age, topic steroids, and azathioprine correlated with the Z scores. Only a low body mass index was a significant predictor for pathological femoral T scores. Conclusions: Osteopenia and osteoporosis are commonly found in IBD patients. Steroid treatment and bowel resection were significant risk factors for osteoporosis of the lumbar spine. However, disease-inherent factors also appear to confer a major risk, indicating that the BMD should be determined in all IBD patients, irrespective of steroid treatment.

Crohn's disease: small bowel motility impairment correlates with inflammatory-related markers C-reactive protein and calprotectin

To evaluate the correlation between the levels of C‐reactive protein (CRP), calprotectin, and small bowel motility in patients with Crohns disease assessed with MRI.

Interleukin-13 and transforming growth factor β synergise in the pathogenesis of human intestinal fistulae

Objective Epithelial to mesenchymal transition (EMT) seems to play an important role in the pathogenesis of fistulae, a common clinical complication of Crohns disease (CD). TGFβ and interleukin-13 (IL-13) have been correlated with the onset of EMT-associated organ fibrosis and high levels of TGFβ have been shown in transitional cells (TCs) lining CD fistula tracts. This study investigated whether IL-13 could be involved in the pathogenesis of CD-associated fistulae. Design Protein or mRNA levels in HT29 intestinal epithelial cells (IECs) or colonic lamina propria fibroblasts (CLPFs) were studied by western blotting or real-time PCR. CLPFs were isolated from non-inflammatory disease controls or patients with CD with or without fistulae and IL-13 levels were analysed in surgically removed fistula specimens by immunohistochemistry. Results TGFβ induced IL-13 secretion in CLPFs from patients with fistulising CD. In fistula specimens high levels of IL-13 were detected in TCs covering fistula tracts. In HT29 IEC monolayers, IL-13 induced SLUG and β6-integrin mRNA, which are associated with cell invasion. HT29 spheroids completely disintegrated when treated with TGFβ for 7 days, whereas IL-13-treated spheroids did not show morphological changes. Here, TGFβ induced mRNA expression of SNAIL1 and IL-13, whereas IL-13 elevated SLUG and β6-integrin mRNA. An anti-IL-13 antibody was able to prevent IL-13-induced SLUG expression in HT29 IECs. Conclusions TGFβ induces IL-13 expression and an EMT-like phenotype of IECs, while IL-13 promotes the expression of genes associated with cell invasion. These findings suggest that TGFβ and IL-13 play a synergistic role in the pathogenesis of fistulae and inhibition of IL-13 might represent a novel therapeutic approach for fistula treatment.

A common polymorphism in the ABCB11 gene is associated with advanced fibrosis in hepatitis C but not in non-alcoholic fatty liver disease

Chronic HCV (hepatitis C virus)-associated cirrhosis represents a major indication for liver transplantation. Bile acids contribute to hepatic stellate cell activation as a key event in fibrogenesis. The aim of the present study was to investigate the role of bile acids and polymorphisms in bile acid level-regulating genes on fibrosis progression. A total of 206 subjects with chronic HCV infection were included for ABCB11 (ATP-binding cassette, subfamily B, member II) 1331T>C and NR1H4 (nuclear receptor) -1G>T genotyping, 178 of which were analysed for fibrosis stage. Exclusion criteria were HBV (hepatitis B virus) or HIV coinfection, alcohol >40 g/day and morbid obesity. A total of 358 patients with NAFLD (non-alcoholic fatty liver disease) were genotyped for comparison with a non-viral liver disease. Caucasian individuals (n = 110), undergoing liver resection for focal hepatic metastasis, served as controls. The ABCB11 1331C allele was significantly overrepresented in HCV patients compared with controls {allelic frequency 62.9%; OR (odds ratio), 1.41 [95% CI (confidence interval), 1.012-1.965]}. Median plasma bile acid levels were not significantly increased in the CC compared with TT genotype [7.2 (1-110) μmol/l compared with 3.5 (1-61) μmol/l; values are medians (range). A significant association between the presence of cirrhosis and ABCB11 genotype (CC compared with CT or TT, P=0.047) was observed in the χ2 test and independent of other risk factors of age, gender, body mass index and disease duration in multivariate analysis (P = 0.010). No such association could be observed in fatty liver patients with regard to advanced fibrosis (F ≥ 2). The common ABCB11 1331CC genotype, which is present in 40% of HCV patients and renders the carrier susceptible to increased bile acid levels, is associated with cirrhosis.