Jacob Tveiten Bjerrum

Metabonomics in Ulcerative Colitis: Diagnostics, Biomarker Identification, And Insight into the Pathophysiology

Nuclear magnetic resonance (NMR) spectroscopy and appropriate multivariate statistical analyses have been employed on mucosal colonic biopsies, colonocytes, lymphocytes, and urine from patients with ulcerative colitis (UC) and controls in order to explore the diagnostic possibilities, define new potential biomarkers, and generate a better understanding of the pathophysiology. Samples were collected from patients with active UC (n = 41), quiescent UC (n = 33), and from controls (n = 25) and analyzed by NMR spectroscopy. Data analysis was carried out by principal component analysis and orthogonal-projection to latent structure-discriminant analysis using the SIMCA P+11 software package (Umetrics, Umea, Sweden) and Matlab environment. Significant differences between controls and active UC were discovered in the metabolic profiles of biopsies and colonocytes. In the biopsies from patients with active UC higher levels of antioxidants and of a range of amino acids, but lower levels of lipid, glycerophosphocholine (GPC), myo-inositol, and betaine were found, whereas the colonocytes only displayed low levels of GPC, myo-inositol and choline. Interestingly, 20% of inactive UC patients had similar profiles to those who were in an active state. This study demonstrates the possibilities of metabonomics as a diagnostic tool in active and quiescent UC and provides new insight into pathophysiologic mechanisms.

IL-33 is upregulated in colonocytes of ulcerative colitis.

Interleukin-33 (IL-33) is a novel member of the IL-1 cytokine family. It has been shown to elicit a Th2-like cytokine response in immunocompetent cells through binding and activation of the T1/ST2 receptor. IL-33 has recently been associated with immune responses to helminthic intestinal infections, airway inflammation and arthritis in animal models. We now report IL-33 to be produced by colonic epithelial cells in humans and it is highly upregulated in ulcerative colitis (UC). Little mRNA expression was found in control subjects (N=9), whereas patients with UC in remission (N=7) and active UC (N=9) had a 3-fold (p<0.006) and 13-fold (p<0.0002) increased expression, respectively. On the protein level, IL-33 in its uncleaved form was overexpressed in active UC compared to controls (p<0.006) and inactive UC (p<0.03). Immunohistochemistry of IL-33 confirmed expression in active UC in colonic epithelial cells, whereas no detectable epithelial expression was seen in control specimens. Caspase 1, which is known to activate IL-33, was expressed in colonocytes, albeit at just detectable levels when the activated p20 caspase 1 was measured. Since IL-33 recently has been shown to be biologically active in its pro-form, and cleavage seems to inactivate IL-33, IL-33 is suggested to be active in UC. We found no IL-33 expression in Caco2 cells, regardless of stimulation by pro-inflammatory cytokines. In contrast to the IL-33 expression data, we could not show any difference in the production of another member of the IL-1 cytokine family, IL-1beta. This is the first study to describe that IL-33 is upregulated in UC. If IL-33 is driving a Th2-like cytokine response in UC, inhibition of the IL-33 T1/ST2 receptor pathway could be a future therapeutic option in UC.

Diagnosis of ulcerative colitis before onset of inflammation by multivariate modeling of genome-wide gene expression data

Background: Endoscopically obtained mucosal biopsies play an important role in the differential diagnosis between ulcerative colitis (UC) and Crohns disease (CD), but in some cases where neither macroscopic nor microscopic signs of inflammation are present the biopsies provide only inconclusive information. Previous studies indicate that CD cannot be diagnosed by molecular and histological diagnostic tools using colonic biopsies without microscopic signs of inflammation, but it is unknown if this is also the case for UC. Methods: The aim of the present study was to apply multivariate modeling of genome‐wide gene expression to investigate if a diagnosable preinflammatory state exists in biopsies of noninflamed UC colon, and to exploit such information to build a diagnostic tool. Results: Genome‐wide gene expression data were obtained from control subjects and UC and CD patients. In total, 89 biopsies from 78 patients were included. A diagnostic model was derived with the random forest method based on 71 biopsies from 60 patients. The model‐internal out‐of‐bag performance measure yielded perfect classification. Furthermore, the model was validated in independent 18 noninflamed biopsies from 18 patients (7 UC, 7 CD, 4 control) where the model achieved 100% sensitivity (95% confidence limits: 60.0–100) and 100% specificity (95% confidence limits: 71.5–100). Conclusions: The present study demonstrates a preinflammatory state in patients diagnosed with UC. In addition, we demonstrate the usefulness of random forest modeling of genome‐wide gene expression data for distinguishing quiescent and active UC colonic mucosa versus control and CD colonic mucosa.

miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic biomarkers in ulcerative colitis

AIM To use microarray-based miRNA profiling of colonic mucosal biopsies from patients with ulcerative colitis (UC), Crohns disease (CD), and controls in order to identify new potential miRNA biomarkers in inflammatory bowel disease. METHODS Colonic mucosal pinch biopsies from the descending part were obtained endoscopically from patients with active UC or CD, quiescent UC or CD, as well as healthy controls. Total RNA was isolated and miRNA expression assessed using the miRNA microarray Geniom Biochip miRNA Homo sapiens (Febit GmbH, Heidelberg, Germany). Data analysis was carried out by principal component analysis and projection to latent structure-discriminant analysis using the SIMCA-P+12 software package (Umetrics, Umea, Sweden). The microarray data were subsequently validated by quantitative real-time polymerase chain reaction (qPCR) performed on colonic tissue samples from active UC patients (n = 20), patients with quiescent UC (n = 19), and healthy controls (n = 20). The qPCR results were analyzed with Mann-Whitney U test. In silico prediction analysis were performed to identify potential miRNA target genes and the predicted miRNA targets were then compared with all UC associated susceptibility genes reported in the literature. RESULTS The colonic mucosal miRNA transcriptome differs significantly between UC and controls, UC and CD, as well as between UC patients with mucosal inflammation and those without. However, no clear differences in the transcriptome of patients with CD and controls were found. The miRNAs with the strongest differential power were identified (miR-20b, miR-99a, miR-203, miR-26b, and miR-98) and found to be up-regulated more than a 10-fold in active UC as compared to quiescent UC, CD, and controls. Two miRNAs, miR-125b-1* and let-7e*, were up-regulated more than 5-fold in quiescent UC compared to active UC, CD, and controls. Four of the seven miRNAs (miR-20b, miR-98, miR-125b-1*, and let-7e*) were validated by qPCR and found to be specifically upregulated in patients with UC. Using in silico analysis we found several predicted pro-inflammatory target genes involved in various pathways, such as mitogen-activated protein kinase and cytokine signaling, which are both key signaling pathways in UC. CONCLUSION The present study provides the first evidence that miR-20b, miR-98, miR-125b-1*, and let-7e* are deregulated in patients with UC. The level of these miRNAs may serve as new potential biomarkers for this chronic disease.

Influence of Smoking on Colonic Gene Expression Profile in Crohn's Disease

Background The development and course of Crohns disease (CD) is related to both genetic and environmental factors. Smoking has been found to exacerbate the course of CD by increasing the risk of developing fistulas and strictures as well as the need for surgery, possibly because of an interaction between smoking or nicotine on macrophage function and the intestinal microvasculature. Several genes are involved in the pathogenesis of CD, and in this study the gene expression differences of the descending colonic mucosa were investigated in CD (smokers or never smokers) and controls (smokers or never smokers). Aim To identify any difference in gene expression of the descending colonic mucosa between smoking and never-smoking CD patients (and controls) by determining genetic expression profiles from microarray analysis. Methods Fifty-seven specimens were obtained by routine colonoscopy from the included material: CD smokers (n = 28) or never-smokers (n = 14) as compared to fifteen healthy controls (8 smokers and 7 never-smokers). RNA was isolated and gene expression assessed with Affymetrix GeneChip Human Genome U133 Plus 2.0. Data were analyzed by principal component analysis (PCA), Wilcoxon rank sum test and multiple linear regressions. Real-time (RT) PCR was subsequently applied to verify microarray results. Results The PCA analysis showed no intrinsic clustering of smokers versus never-smokers. However, when Wilcoxon rank sum test corrected with Q values were performed, six known genes were significantly expressed differently in the inflamed CD smokers as compared to the inflamed CD never-smokers: ring finger protein 138 (RNF138), metalothionein 2A (MT2A) and six transmembrane epithelial antigen of the prostate 3 (STEAP3), SA hypertension-associated homolog, PGM2L1 and KCNJ2. The subsequent RT-PCR-analyses verified, however, that only RNF138, MT2A and STEAP3 were significantly up-regulated in CD smokers in specimens with inflammatory activity of the descending colon. Conclusions The present study demonstrates that the genes, RNF138, MT2A, and STEAP3 are differently expressed in the inflamed descending colon of smoking versus never-smoking CD patients, which might be of relevance for the poorer clinical course among CD smokers. Many gastroenterologists are still not totally aware of the benefits of smoking cessation in relation to CD, and do not put much effort into getting the patients to quit, therefore more information on the negative effects of smoking, seems warranted.

Genome-wide gene expression analysis of mucosal colonic biopsies and isolated colonocytes suggests a continuous inflammatory state in the lamina propria of patients with quiescent ulcerative colitis

Background: Genome‐wide gene expression (GWGE) profiles of mucosal colonic biopsies have suggested the existence of a continuous inflammatory state in quiescent ulcerative colitis (UC). The aim of this study was to use DNA microarray‐based GWGE profiling of mucosal colonic biopsies and isolated colonocytes from UC patients and controls in order to identify the cell types responsible for the continuous inflammatory state. Methods: Adjacent mucosal colonic biopsies were obtained endoscopically from the descending colon in patients with active UC (n = 8), quiescent UC (n = 9), and with irritable bowel syndrome (controls, n = 10). After isolation of colonocytes and subsequent extraction of total RNA, GWGE data were acquired using Human Genome U133 Plus 2.0 GeneChip Array (Affymetrix, Santa Clara, CA). Data analysis was carried out by principal component analysis and projection to latent structure‐discriminant analysis using the SIMCA‐P 11 software (Umetrics, Umeå, Sweden). Results: A clear separation between active UC, quiescent UC, and control biopsies were found, whereas the model for the colonocytes was unable to distinguish between quiescent UC and controls. The differentiation between quiescent UC and control biopsies was governed by unique profiles containing gene expressions with significant fold changes. These primarily belonged to the family of homeostatic chemokines, revealing a plausible explanation for the abnormal regulated innate immune response seen in patients with UC. Conclusions: This study has demonstrated the presence of a continuous inflammatory state in quiescent UC, which seems to reflect an altered gene expression profile of lamina propria cells. Inflamm Bowel Dis 2009

Recent Advances Using Immunomodulators for Inflammatory Bowel Disease

Use of the immunomodulators thiopurines and methotrexate (MTX) in the treatment of inflammatory bowel disease (IBD), i.e., Crohns disease and ulcerative colitis (UC), is considered to be good clinical practice. However, despite being administered to a considerable number of IBD patients over the years, questions remain about the most rational treatment regimens of azathioprine (AZA), 6‐mercaptopurine (6‐MP), and MTX, and results from a range of recent studies necessitate increased attention to how to optimize the use of these immunomodulators. First and foremost, it is of utmost importance to define the subgroup of IBD patients in need of immunomodulators, including those in need of combination therapy with biologic agents, especially because some side effects may be rather severe. Second, colorectal cancer is observed more often in IBD patients than in the background population. However, a recent nationwide Dutch study pointed to a preventive effect of thiopurines. Finally, the need for an appropriate approach to the discontinuation of immunomodulators is emphasized. Since controversy continues regarding the most appropriate use of immunomodulators, this paper is focusing on pharmacokinetics, pharmacogenetics, and therapeutic blood testing, as well as the occurrence of adverse events, when using AZA, 6‐MP, and MTX in an attempt to determine a more up‐to‐date and rational treatment regimen in IBD.

Assessment of the validity of a multigene analysis in the diagnostics of inflammatory bowel disease.

The findings of a previous multigene study indicated that the expression of a panel of seven specific genes had strong differential power regarding inflammatory bowel disease (IBD) versus non‐IBD, as well as ulcerative colitis (UC) versus Crohns disease (CD). This prospective confirmatory study based on an independent patient cohort from a national Danish IBD centre was conducted in an attempt to verify these earlier observations.

Transcriptional analysis of left-sided colitis, pancolitis, and ulcerative colitis-associated dysplasia.

Background:It is unknown why patients with extensive ulcerative colitis (UC) have a higher risk of colorectal cancer compared with patients with left-sided UC. This study characterizes the inflammatory processes in left-sided UC, pancolitis, and UC-associated dysplasia at the transcriptional level to identify potential biomarkers and transcripts of importance for the carcinogenic behavior of chronic inflammation. Methods:The Affymetrix GeneChip Human Genome U133 Plus 2.0 was applied on colonic biopsies from UC patients with left-sided UC, pancolitis, dysplasia, and controls. Reverse transcription polymerase chain reaction and immunohistochemistry were performed for validating selected transcripts in the initial cohort and in 2 independent cohorts of patients with UC. Microarray data were analyzed by principal component analysis, and reverse transcription polymerase chain reaction and immunohistochemistry data by the Wilcoxons rank-sum test. Results:The principal component analysis results revealed separate clusters for left-sided UC, pancolitis, dysplasia, and controls. Close clustering of dysplastic and pancolitic samples indicated similarities in gene expression. Indeed, 101 and 656 parallel upregulated and downregulated transcripts, respectively, were identified in specimens from dysplasia and pancolitis. Validation of selected transcripts hereof identified insulin receptor alpha (INSRA) and MAP kinase interacting serine/threonine kinase 2 (MKNK2) with an enhanced expression in dysplasia compared with left-sided UC and controls, whereas laminin &ggr;2 (LAMC2) was found with a lower expression in dysplasia compared with the remaining 3 groups. Conclusions:This study demonstrates pancolitis and left-sided UC as distinct inflammatory processes at the transcriptional level, and identifies INSRA, MKNK2, and LAMC2 as potential critical transcripts in the inflammation-driven preneoplastic process of UC.

Biological treatment of Crohn's disease.

Introduction of biological agents for the treatment of Crohn’s disease (CD) has led to a transformation of the treatment paradigm. Several biological compounds have been approved for patients with CD refractory to conventional treatment: infliximab, adalimumab and certolizumab pegol (and natalizumab in several countries outside the European Union). However, despite the use of biologics for more than a decade, questions still remain about the true efficacy and the best treatment regimens – especially about when to discontinue treatment. Furthermore, a need for optimizing treatment with biologics still exists, as 20–40% of patients with CD (depending on selection criteria) do not have any relevant response to the current biological agents (i.e. primary failures). A better patient selection might maximize the clinical outcome while minimizing the complications associated with this type of therapy. However, the clinical tools capable of identifying such patients are still unavailable, and the trough level strategy may help the clinician to optimize therapy and to avoid loss of response and/or immunogenicity (i.e. a low but measurable antibody level exists just before the periodic administration of the biological agent). On the other hand, peak levels and average levels should not exceed concentrations associated with increased toxicity. Randomized, controlled studies focusing on trough levels and antibodies towards the biological agent in routine clinical situations may add important pieces to the puzzle for a more rational treatment algorithm of CD patients. In some situations, the risks (i.e. immunogenicity, serious infections and the promotion of neoplasia) may, however, not outweigh the benefits of biological treatment.