Azucena Salas

Sa1874 Transcriptional Analysis of the Intestinal Mucosa of Patients With Ulcerative Colitis in Remission Reveals Lasting Epithelial Cell Alterations

Objective Ulcerative colitis (UC) is a chronic condition characterised by the relapsing inflammation despite previous endoscopic and histological healing. Our objective was to identify the molecular signature associated with UC remission. Design We performed whole-genome transcriptional analysis of colonic biopsies from patients with histologically active and inactive UC, and non-inflammatory bowel disease (non-IBD) controls. Real-time reverse transcriptase-PCR and immunostaining were used for validating selected genes in independent cohorts of patients. Results Microarray analysis (n=43) demonstrates that UC patients in remission present an intestinal transcriptional signature that significantly differs from that of non-IBD controls and active patients. Fifty-four selected genes were validated in an independent cohort of patients (n=30). Twenty-nine of these genes were significantly regulated in UC-in-remission subjects compared with non-IBD controls, including a large number of epithelial cell-expressed genes such as REG4, S100P, SERPINB5, SLC16A1, DEFB1, AQP3 and AQP8, which modulate epithelial cell growth, sensitivity to apoptosis and immune function. Expression of inflammation-related genes such as REG1A and IL8 returned to control levels during remission. REG4, S100P, SERPINB5 and REG1A protein expression was confirmed by immunohistochemistry (n=23). Conclusions Analysis of the gene signature associated with remission allowed us to unravel pathways permanently deregulated in UC despite histological recovery. Given the strong link between the regulation of some of these genes and the growth and dissemination of gastrointestinal cancers, we believe their aberrant expression in UC may provide a mechanism for epithelial hyper-proliferation and, in the context of malignant transformation, for tumour growth.

Late Crohn’s disease patients present an increase in peripheral Th17 cells and cytokine production compared with early patients

Aliment Pharmacol Ther 31, 561–572

Defective IL-10 production in severe phenotypes of Crohn's disease

Loss of tolerance toward commensal bacteria has been invoked as a mechanism for Crohns disease. IL‐10 is a key anti‐inflammatory cytokine that plays a role in induction and maintenance of tolerance. The aim of this study is to determine IL‐10 production in response to bacterial components in Crohns disease patients, who were classified according to their phenotypes as stricturing, penetrating, or inflammatory. Peripheral blood was obtained from Crohns disease patients and healthy controls. Cytokine production was measured in whole blood cultures, isolated CD4+ cells, and monocyte‐derived dendritic cells (MDDCs). Under unstimulated conditions, IL‐10, but not IL‐12, was down‐regulated significantly in blood cultures of patients with severe phenotypes, compared with inflammatory, nonpenetrating, nonstricturing Crohns disease patients. In response to LPS, IL‐10 was up‐regulated more significantly in patients with no fistulae or fibrosis. Study of IL‐10 production by isolated cell subsets showed that DCs, but not CD4+ T cells, from penetrating Crohns disease produced significantly less IL‐10 in response to LPS. Differences were not associated with the 1082A/G polymorphism in the IL‐10 gene promoter. We show a defect in IL‐10 production in whole blood cell cultures and MDDCs in patients with severe forms of Crohns disease. This defect in IL‐10 production by a group of Crohns disease patients may represent a mechanism mediating more severe manifestations of the disease. We propose that treatment with IL‐10 or IL‐10‐inducing therapies could be of particular benefit to these group of patients.

Reperfusion-induced oxidative stress in diabetes: cellular and enzymatic sources

Reactive oxygen metabolites (ROMs) have been implicated in the pathogenesis of the inflammatory response to ischemia/reperfusion (I/R), which is exacerbated in diabetes. This study revealed an increased (P < 0.01) ROMs production in mesenteric tissue (measured using the oxidant‐sensitive fluorochrome dihydrorhodamine 123) after I/R in control and diabetic rats, with larger increments (P < 0.0001) observed in the latter group, that was associated with an increased inflammatory response measured by intravital microscopy. Either xanthine oxidase inhibition, superoxide scavenging, ICAM‐1 immunoneutralization, or blockade of platelet‐activating factor or leukotrienes effectively reduced leukocyte recruitment and ROMs production in control and diabetic rats. Moreover, neutrophils from diabetic rats showed an enhanced production of ROMs in vitro in basal and stimulated conditions. We conclude that the oxidative stress during reperfusion is markedly enhanced in diabetes and this appears to result from increased leukocyte recruitment and a higher capacity of diabetic leukocytes to generate ROMs in response to stimulation. J. Leukoc. Biol. 66: 59–66; 1999.

Evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis

Background:Clinical trials in ulcerative colitis (UC) rely on certain parameters to evaluate responses that are highly subjective or of low sensitivity. Here, using a select group of genes, we tested the accuracy of gene expression analysis as a biomarker of clinical, endoscopic, and histologic improvements. Methods:Intestinal biopsies were obtained from UC patients included in two cohorts. Cohort 1 was used to select for genes whose expression was modulated in active (vs. inactive) UC. Cohort 2 included patients recruited in a phase II study receiving placebo, mesalazine, or dersalazine sodium for 4 weeks. The expression of 44 genes identified in Cohort 1 was assessed at weeks 0 and 4, and was then correlated with biomarkers, as well as with clinical, endoscopic, and histologic scores. Results:Significant changes in the expression of 31 of the 44 genes tested were detected in Cohort 2 at week 4. Gene expression (&Dgr;Ct) significantly correlated with the total Mayo score, C-reactive protein (CRP), and fecal calprotectin. The number of genes significantly regulated at week 4 was highly associated with histologic and endoscopic responses. Logistic regression analysis identified four separate genes (IFITM1, ITGB2, IL1R2, IL2RA) whose relative change was independently associated with endoscopic remission with high specificity and sensitivity. Conclusions:Change in the expression of a select set of genes can serve as an early biomarker, one with high specificity and sensitivity to clinical, endoscopic, and histologic responses. This could represent a new tool for identifying early response to treatment in mild to moderately active UC patients.

Usefulness of Transcriptional Blood Biomarkers as a Non-invasive Surrogate Marker of Mucosal Healing and Endoscopic Response in Ulcerative Colitis

Abstract Background and Aims Ulcerative colitis [UC] is a chronic inflammatory disease of the colon. Colonoscopy remains the gold standard for evaluating disease activity, as clinical symptoms are not sufficiently accurate. The aim of this study is to identify new accurate non-invasive biomarkers based on whole-blood transcriptomics that can predict mucosal lesions and response to treatment in UC patients. Methods Whole-blood samples were collected for a total of 152 UC patients at endoscopy. Blood RNA from 25 UC individuals and 20 controls was analysed using microarrays. Genes that correlated with endoscopic activity were validated using real-time polymerase chain reaction in an independent group of 111 UC patients, and a prediction model for mucosal lesions was evaluated. Responsiveness to treatment was assessed in a longitudinal cohort of 16 UC patients who started anti-tumour necrosis factor [TNF] therapy and were followed up for 14 weeks. Results Microarray analysis identified 122 genes significantly altered in the blood of endoscopically active UC patients. A significant correlation with the degree of endoscopic activity was observed in several genes, including HP, CD177, GPR84, and S100A12. Using HP as a predictor of endoscopic disease activity, an accuracy of 67.3% was observed, compared with 52.4%, 45.2%, and 30.3% for C-reactive protein, erythrocyte sedimentation rate, and platelet count, respectively. Finally, at 14 weeks of treatment, response to anti-TNF therapy induced alterations in blood HP, CD177, GPR84, and S100A12 transcripts that correlated with changes in endoscopic activity. Conclusions Transcriptional changes in UC patients are sensitive to endoscopic improvement and appear to be an effective tool to monitor patients over time.

Letter: pathogenicity of Th17 cells may differ in ulcerative colitis compared with Crohn's disease - authors' reply

SIRS, Thank you for giving us the opportunity to respond to the letter from Raza and Shata concerning our article. After careful consideration, we believe that the data discussed in the letter and the conclusions made have some important caveats. First, the authors use the Harvey-Bradshaw index, an abbreviated form of the Crohn’s disease activity index, to evaluate both Crohn’s disease (CD) and ulcerative colitis (UC) clinical symptoms. The Harvey-Bradshaw index includes parameters, such as the presence of fistulae or abdominal mass that apply to CD, but not UC, whereas it does not account for rectal bleeding – a good indicator of disease severity in UC. More importantly, Raza and Shata make the assumption that anti-IL-17 (or anti-IL-17R) therapy may have failed in CD due to, according to their data, a lack of production of IL-17 in these patients, whereas they hypothesise that it may work in UC. It is important to emphasise that increased production of IL-17 in active UC and CD patients has been documented by several groups (apart from us) and it is well-known that clinical and endoscopic activity in both UC (measured using the Mayo score) and CD patients is accompanied by increased production of IL-17. Moreover, anti-IL-17 therapy appears to worsen disease in some patients suggesting that, as it has been seen in different animal models, lack of IL-17 may have deleterious effects to intestinal homeostasis. Given the evidence, we believe that the assumption that anti-IL-17 may be beneficial in UC, based on the data the authors provide in the letter, is misleading.