Petr Ricanek

Evaluation of disease activity in IBD at the time of diagnosis by the use of clinical, biochemical, and fecal markers

Abstract Objective. The present population based adult cohort was part of a new prospective study of patients with inflammatory bowel disease (IBD) in South-Eastern Norway, the Inflammatory Bowel South-Eastern Norway II study, investigating disease characteristics in an attempt to improve our knowledge regarding factors related to early clinical phenotype and disease activity. Material and methods. Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days were examined at the local hospital. Colonoscopy with biopsies was performed and blood and stool samples were taken. Results. In ulcerative colitis (UC) patients, the median Simple Clinical Colitis Activity Index (SCCAI) was 4 (range 0–10) in mild and 6 (range 0–14) in patients with moderate or severe endoscopic activity of inflammation (p = 0.002). The calprotectin concentration in feces was significantly related to the SCCAI (p = 0.034) and the Mayo endoscopic subscore (p = 0.031). There was a significant association between the C-reactive protein (CRP) value, leucocytes and thrombocytes and the SCCAI, but only leucocytes were significantly associated with the Mayo endoscopic subscore. In Crohns disease (CD) patients, there was no statistical significant association between the Harvey-Bradshaw Index (HBI) and the endoscopic grade of mucosal inflammation (p = 0.8). The calprotectin concentration in feces was significantly related to the endoscopic activity score (p = 0.004), but not to the HBI (p = 0.5). HBI was significantly related to the CRP value (p = 0.047) and thrombocytes (p = 0.03). Conclusions . In UC, both biochemical and fecal markers are related to disease activity and extent of disease, whereas in CD, the fecal calprotectin concentration is a reliable marker of mucosal affection, but not for systemic disease activity.

Biosimilar infliximab (CT-P13) in the treatment of inflammatory bowel disease: A Norwegian observational study

Objective: To assess the efficacy, tolerability, and safety of CT-P13 (Remsima®) in patients with Crohn’s disease (CD) or ulcerative colitis (UC). Methods: This was a prospective observational study performed in a single center in Norway. Patients with CD (n = 46) or UC (n = 32) received CT-P13 (5 mg/kg) by intravenous infusion at weeks 0, 2, and 6. Efficacy end points included remission at week 14, measured by a Harvey-Bradshaw Index score of ≤4 or partial Mayo score of ≤2. Levels of the inflammatory markers C-reactive protein and calprotectin were measured. Adverse events up to week 14 were also recorded. Results: Seventy-nine percent of CD and 56% of UC patients achieved remission at week 14. Significant reductions in C-reactive protein and calprotectin occurred between baseline and week 14. There were no unexpected adverse events reported during the study. Conclusion: CT-P13 is efficacious and well tolerated in patients with CD or UC.

Dominant Fecal Microbiota in Newly Diagnosed Untreated Inflammatory Bowel Disease Patients

Our knowledge about the microbiota associated with the onset of IBD is limited. The aim of our study was to investigate the correlation between IBD and the fecal microbiota for early diagnosed untreated patients. The fecal samples used were a part of the Inflammatory Bowel South-Eastern Norway II (IBSEN II) study and were collected from CD patients (n = 30), UC patients (n = 33), unclassified IBD (IBDU) patients (n = 3), and from a control group (n = 34). The bacteria associated with the fecal samples were analyzed using a direct 16S rRNA gene-sequencing approach combined with a multivariate curve resolution (MCR) analysis. In addition, a 16S rRNA gene clone library was prepared for the construction of bacteria-specific gene-targeted single nucleotide primer extension (SNuPE) probes. The MCR analysis resulted in the recovery of five pure components of the dominant bacteria present: Escherichia/Shigella, Faecalibacterium, Bacteroides, and two components of unclassified Clostridiales. Escherichia/Shigella was found to be significantly increased in CD patients compared to control subjects, and Faecalibacterium was found to be significantly reduced in CD patients compared to both UC patients and control subjects. Furthermore, a SNuPE probe specific for Escherichia/Shigella showed a significant overrepresentation of Escherichia/Shigella in CD patients compared to control subjects. In conclusion, samples from CD patients exhibited an increase in Escherichia/Shigella and a decrease in Faecalibacterium indicating that the onset of the disease is associated with an increase in proinflammatory and a decrease in anti-inflammatory bacteria.

Reduced expression of aquaporins in human intestinal mucosa in early stage inflammatory bowel disease

Objectives The aim of this study was to investigate the relationship between aquaporin (AQP) water channel expression and the pathological features of early untreated inflammatory bowel disease (IBD) in humans. Methods Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days, were examined at the local hospital. Colonoscopy with biopsies was performed and blood samples were taken. Patients who did not meet the diagnostic criteria for IBD and who displayed no evidence of infection or other pathology in the gut were included as symptomatic non-IBD controls. AQP1, 3, 4, 5, 7, 8, and 9 messenger RNA (mRNA) levels were quantified in biopsies from the distal ileum and colon by quantitative real-time polymerase chain reaction. Protein expression of selected AQPs was assessed by confocal microscopy. Through multiple alignments of the deduced amino acid sequences, the putative three-dimensional structures of AQP1, 3, 7, and 8 were modeled. Results AQP1, 3, 7, and 8 mRNAs were detected in all parts of the intestinal mucosa. Notably, AQP1 and AQP3 mRNA levels were reduced in the ileum of patients with Crohn’s disease, and AQP7 and AQP8 mRNA levels were reduced in the ileum and the colon of patients with ulcerative colitis. Immunofluorescence confocal microscopy showed localization of AQP3, 7, and 8 at the mucosal epithelium, whereas the expression of AQP1 was mainly confined to the endothelial cells and erythrocytes. The reduction in the level of AQP3, 7, and 8 mRNA was confirmed by immunofluorescence, which also indicated a reduction of apical immunolabeling for AQP8 in the colonic surface epithelium and crypts of the IBD samples. This could indicate loss of epithelial polarity in IBD, leading to disrupted barrier function. Conclusion AQPs 1 and 8 and the aquaglyceroporins AQPs 3 and 7 are the AQPs predominantly expressed in the lower intestinal tract of humans. Their expression is significantly reduced in patients with IBD, and they are differentially expressed in specific bowel segments in patients with Crohn’s disease and ulcerative colitis. The data present a link between gut inflammation and water/solute homeostasis, suggesting that AQPs may play a significant role in IBD pathophysiology.

Paucity of mycobacteria in mucosal bowel biopsies from adults and children with early inflammatory bowel disease

BACKGROUND The presence of Mycobacterium avium subspecies paratuberculosis (MAP) has previously been inferred in the genesis of Crohns disease (CD), and a higher incidence of MAP PCR positivity has been demonstrated in the gut and peripheral blood of CD patients than in healthy individuals. The objective of this prospective study was to assess the potential etiological role of MAP in the pathogenesis of CD. METHODS The presence of mycobacteria was assessed in bowel biopsies from newly diagnosed, treatment naïve Norwegian patients with IBD, including CD and ulcerative colitis (UC), as compared to a hospital-based cohort of CD and UC patients. Biopsies were collected from the small and large bowel in 354 individuals with suspected IBD. Detection of mycobacteria was performed by long-term cultivation in combination with direct detection by MAP IS900-specific PCR. RESULTS Among the specimens included from the patients with early IBD, samples from only two of the patients with CD (2.7%) and two of the non-IBD controls (1.5%) exhibited a positive growth signal. None of the CD patients and only one of the non-IBD controls was MAP PCR positive. Only the single PCR positive non-IBD control was also mycobacterial culture positive with Mycobacterium avium subsp. hominissuis. In the referral patients with long-term IBD, the prevalence of growth signal and MAP PCR positivity was higher (52 and 9%, respectively). CONCLUSIONS These findings demonstrate the paucity of MAP in the gut of treatment naïve CD patients. This study does not provide evidence for a role of MAP in early IBD.

Gut bacterial profile in patients newly diagnosed with treatment-naïve Crohn's disease.

Objectives: The aim of this study was to define the composition of the gut bacterial flora in Norwegian patients with early stage Crohn’s disease (CD). Methods: By using a nonselective metagenomics approach, the general bacterial composition in mucosal biopsies from the ileum and the colon of five subjects, four patients with different phenotypes of CD, and one noninflammatory bowel disease control, was characterized. After partial 16S ribosomal RNA (rRNA) gene sequencing, BLAST homology searches for species identification and phylogenetic analysis were performed. Results: An overall biodiversity of 106 different bacterial operational taxonomic units (OTUs) was detected in the cloned libraries. Nearly all OTUs belonged to the phylae Bacteroidetes (42% in CD, 71% in the control) or Firmicutes (42% in CD, 28% in the control), except for some OTUs that belonged to the phylum Proteobacteria (15% in CD, 0% in the control) and a few OTUs that could not be assigned to a phylum (2% in CD, 1% in the control). Conclusion: Based on the high incidence of inflammatory bowel disease (IBD) in Norway, this pilot study represents a relevant determination of the gut microbiota in Norwegian patients compared to previous findings in other countries. The bacterial profile of Norwegian CD patients was found to be similar to that of CD patients in other countries. The findings do not support a particular bacterial composition as a predominant causative factor for the high incidence of IBD that exists in some countries.

Age-Dependent Fecal Bacterial Correlation to Inflammatory Bowel Disease for Newly Diagnosed Untreated Children

The knowledge about correlation patterns between the fecal microbiota and inflammatory bowel diseases (IBD)—comprising the two subforms Crohns disease (CD) and ulcerative colitis (UC)—for newly diagnosed untreated children is limited. To address this knowledge gap, a selection of faecal specimens (CD, n = 27 and UC, n = 16) and non-IBD controls (n = 30) children (age < 18 years) was analysed utilising bacterial small subunit (SSU) rRNA. We found, surprising age dependence for the fecal microbiota correlating to IBD. The most pronounced patterns were that E. coli was positively (R 2 = 0.16, P = 0.05) and Bacteroidetes, negatively (R 2 = 0.15, P = 0.05) correlated to age for CD patients. For UC, we found an apparent opposite age-related disease correlation for both Bacteroides and Escherichia. In addition, there was an overrepresentation of Haemophilus for the UC children. From our, results we propose a model where the aetiology of IBD is related to an on-going immunological development in children requiring different age-dependent bacterial stimuli. The impact of our findings could be a better age stratification for understanding and treating IBD in children.

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

BackgroundNucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample.ResultsA further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample.ConclusionsThe present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.

Determinants of optimal bowel function in ileal pouch-anal anastomosis – physiological differences contributing to pouch function

Abstract Background: Variability in functional outcome after ileal-pouch anal anastomosis (IPAA) is to a large extent unexplained. The aim of this study was to perform multiple physiological and biochemical tests including an endoscopic examination with histology on IPAA patients with well and poorly functioning pouches to determine factors, or combinations thereof, contributing to functional outcome. Methods: All patients with ulcerative colitis undergoing restorative proctocolectomy between 2000 and 2013 (N = 108) were interviewed using a pouch functioning score. The best and worst functioning quartiles were invited to undergo examination with a barostat measuring pouch volume at preset variable distension pressures, and a pouch endoscopy. Results: Forty five of 58 eligible patients agreed to participate. The most significant physiological parameter differing between the well and poorly functioning pouches was pouch volume at first sensation, urge and discomfort (p value <.001). Urge volumes were 213 (CI 171–256) ml for poorly and 352 (CI 305–401) ml for well functioning pouches. Pouchitis episodes were negatively correlated to function. The poorly functioning patients had a higher prevalence of histological signs of inflammation and hand-sewn anastomosis, and a longer remaining rectal cuff, however, nonsignificant. The pouch pressure at sensation thresholds did not differ between the groups. Conclusions: Pouch volume is the most dominant predictor of pouch function in this study. The present comprehensive study of a multitude of different factors that possibly could be contributing to functional outcome, failed to shed much further light on the functional variability among pouch patients. The pouch physiology remains to a large extent unexplained.

P866 Faecal microbiota in newly diagnosed Crohn’s disease and its relation to treatment escalation

Faecal microbiota in newly diagnosed Crohns disease and its relation to treatment escalation