Ingrid Christiansen

TH1 and TH17 interactions in untreated inflamed mucosa of inflammatory bowel disease, and their potential to mediate the inflammation.

BACKGROUND Crohns disease (CD) and ulcerative colitis (UC) have been associated with a T helper1 (TH1) and a TH2 cytokine profile, respectively. Recently, a TH17 lineage has been introduced, but their role in the inflammation of CD and UC is not fully understood. AIM To characterize the cytokines directing the TH17 cells and their interactions with TH1 cells in the mucosa of untreated patients with CD and UC. METHOD Seventy-nine patients with untreated UC, 32 patients with untreated CD and 23 controls with no signs of colon disease were included in the study. Clinical indices for ulcerative colitis (UCDAI) and Crohns disease (CDAI) were assessed. Biopsies for measurements of interleukin (IL)-17A, IL-23, IL-6, transforming growth factor-beta (TGF-β), interferon-gamma (IFN-γ), mRNA levels as well as immunohistochemical (IHC) analyses were performed. RESULTS The gene expression for all cytokines in UC and for all cytokines except for TGF-β in CD were significantly increased compared with the controls. The immunohistochemical analysis showed significantly increased number of IL-17A positive cells in lamina propria and epithelium of both UC and CD compared to controls. The levels of IL-17A and IL-23 mRNA were significantly higher in UC than in CD while the levels of IL-6 were significantly higher in CD compared with UC. The levels of IL-17A, IL-6 and IL-23 mRNA were associated with the disease activity score in both UC and CD. IFN-γ was associated with the disease activity in UC, but did not reach significant level in CD. CONCLUSION Increased levels of IL-17A and IL-23 were found in both UC and CD compared to controls. Association to the grade of inflammation and clinical activity was also observed. IL-17A and IL-23 were significantly higher in UC than in CD. TH1 and TH17 cytokines seem to act synergistically in inflammatory bowel disease (IBD) with no apparent polarization between UC and CD.

TNF-alpha gene expression in colorectal mucosa as a predictor of remission after induction therapy with infliximab in ulcerative colitis

BACKGROUND It has been documented that treatment with infliximab (IFX) induces remission in 1/3 of patients with moderate to severe ulcerative colitis (UC). Predictors of response could improve selection of patients with a higher probability of favorable outcome. AIM To determine predictor factors for the clinical outcome of IFX induction therapy in UC. METHODS UC patients with moderate to severe disease who received 5mg/kg IFX at weeks 0, 2 and 6weeks were included. Ulcerative colitis disease activity index (UCDAI) score including endoscopic sub-scores were assessed before and after treatment. Several predictors, including TNF-alpha mRNA expression, were tested in a regression model. RESULTS Fifty-nine patients completed the study. Age, gender, steroid therapy, immunosuppressive, pancolitis, endoscopic sub-score, disease duration, C-reactive protein, interleukin-(IL)-4, IL-10 or interferon-gamma (IFN-gamma) did not predict mucosal or clinical remission. There was an inverse and independent association between pre-treatment TNF-alpha expression levels and clinical and endoscopic remission of IFX treatment (logistic regression, p=0.01 and p=0.003, odds ratio 2.5 and 4.8, respectively). CONCLUSION The clinical outcome of an induction therapy with IFX in UC is inversely associated with the pre-treatment gene expression levels of TNF-alpha in colorectal mucosa.

Mucosal cytokine gene expression profiles as biomarkers of response to infliximab in ulcerative colitis

Abstract Objective. Mucosal cytokine profile determines T cell differentiation and may play an important role in the clinical course of inflammatory bowel disease (IBD). Cytokines from different T helper (Th) cell subsets are elevated in inflamed mucosa of patients with ulcerative colitis (UC), contributing to the inflammation. The aim of this study was to determine the predictive value of pre-treatment mucosal cytokine profile in response to therapy with the anti-TNF agent infliximab (IFX). Material and methods. The expression of Th1, Th17, Th2 and T-regulatory (Treg)-related cytokines was quantified by real-time PCR in mucosal biopsies from 74 UC patients before initiation of IFX induction therapy. Clinical and endoscopic effects were assessed after three infusions. Remission was defined as ulcerative colitis disease activity index (UCDAI) below 3. Results. Higher gene expression levels of IL-17A and IFN-γ were significantly associated with remission after three IFX infusions (OR = 5.4, p = 0.013 and OR = 5.5, p = 0.011, respectively). IL-17A and IFN-γ mRNA expression showed positive correlation. Th2 and Treg-related mediators were not significantly associated with clinical outcome, but were expressed at higher levels in UC patients compared with the controls. Immunohistochemistry (IHC) confirmed the presence of cells expressing both IL-17A and IFN-γ. Conclusions. High expression of Th1- and Th17-related cytokines in the mucosa of UC patients can potentially predict a favorable outcome of IFX induction therapy. Th2 and Treg-related mediators do not appear useful as predictive markers.

Improvement of real‐time polymerase chain reaction for quantifying TNF‐α mRNA expression in inflamed colorectal mucosa: An approach to optimize procedures for clinical use

Objective. The precise measurement of local tumor necrosis factor alpha (TNF‐α) expression in tissue is important in understanding the pathogenesis of inflammatory bowel diseases (IBD). Real‐time polymerase chain reaction (PCR) is a sensitive, versatile method and is becoming a commonly used tool for the quantification of gene expression. The aim of this study was to optimize the laboratory procedure for biopsy sampling, storage and calibration of result for TNF‐α mRNA quantification with real‐time PCR of colorectal biopsies. Material and methods. Endoscopic biopsies from the colorectum were obtained from 18 patients with ulcerative colitis (UC), 11 patients with Crohns disease (CD) and 18 normal controls. Optimization of procedures for real‐time PCR performance was carried out. Results. The transport medium, RNAlater, exhibited a high preservation effect against RNA degradation even after 8 days of storage at room temperature; one biopsy from each patient was sufficient for RNA extraction, cDNA synthesis and TNF‐mRNA quantification. An assay was established with a technical reproducible sensitivity of 100 copies/µL. The observed interassay variations were 7.4 % coefficient of variation (CV) and 7.2 % CV in low and high TNF‐α mRNA expression biopsies, respectively. TNF‐α mRNA levels in colorectal biopsies from patients with either CD or moderate to severe UC were markedly increased, and 8∼9‐fold higher than those in healthy controls. Conclusions. This optimization improves the clinical use of real‐time PCR for quantification of TNF‐α gene expression in colorectal biopsies and provides a sensitive reproducible assay.

Normalization of mucosal cytokine gene expression levels predicts long-term remission after discontinuation of anti-TNF therapy in Crohn's disease

Abstract Objective. To investigate mucosal cytokine gene expression levels in healed mucosa after anti-tumor necrosis factor (TNF) therapy in patients with Crohns disease (CD) as possible risk factors for relapse after discontinuation of therapy. Design. Thirty-seven CD patients treated with anti-TNF agents until complete mucosal healing, documented by endoscopy, discontinued anti-TNF treatment and entered a follow-up study. Levels of mRNA expression of interleukin (IL)17A (IL17A), IL23, interferon-gamma (IFNG), TNF-alpha (TNF), IL10 and Forkhead Box P3 (FOXP3) were measured in biopsies from healed mucosa and analyzed as possible risk factors of relapse. Mucosal cytokine transcript levels from patients without CD served as controls. Results. Patients were followed after therapy withdrawal until relapse. Median time to relapse was 20 and 68 weeks for patients with elevated and normalized IL17A and TNF expression levels, respectively (p = 0.02 for IL17A and p = 0.003 for TNF, log-rank). Expression levels of TNF, IL17A and FOXP3 were significantly higher in patients who relapsed before 26 weeks than in those who did not relapse, and also higher in patients with relapse before week 52 versus non-relapsers. Elevated expression levels of TNF and IL17A in healed mucosa significantly increased the risk of relapse (HR = 3.4, p = 0.03, sensitivity 80%, specificity 38% and HR = 4.1, p = 0.008, sensitivity 81%, specificity 61%, respectively). Conclusions. Normalization of mucosal gene expression of cytokines after anti-TNF therapy does not occur in all patients with healed mucosa as judged by endoscopy. Normalization of TNF and/or IL17A expression predicts long-term remission.

The effect of adalimumab for induction of endoscopic healing and normalization of mucosal cytokine gene expression in Crohn's disease

Abstract Objective. To investigate the effects of adalimumab on the induction of complete endoscopic healing and normalization of mucosal cytokine gene expression in patients with active Crohns disease. Material and methods. A prospective, single-center study including 77 patients. All were examined by endoscopy before initiation of adalimumab induction therapy with a minimum of six adalimumab injections. Patients were treated until documentation of complete endoscopic healing. Biopsies for measurements of mRNA expression levels of interleukin(IL)-17A (IL17A), IL23, interferon-gamma (IFNG), tumor necrosis factor-alpha (TNF), IL10 and Forkhead Box P3 (FOXP3), as well as for immunohistochemistry (IHC) were sampled at pre- and post-treatment endoscopy, and from 17 control patients. Results. Complete endoscopic healing was achieved in 27.3% after 10 weeks of treatment, documented by endoscopy at week 12. Cumulative endoscopic healing after 52 weeks was 44.2%. Complete endoscopic healing led to a significant reduction in mRNA expression levels for all cytokines except IL10. Elevated expression of TNF and IL-17A persisted in 52% and 76%, respectively, of patients with complete endoscopic remission. Pre-treatment cytokine gene expression levels did not predict response to adalimumab therapy. Conclusions: Adalimumab induces accumulated complete endoscopic healing in 44% of patients after 52 weeks of therapy. Normalization of mucosal gene expression of cytokines does not occur in all patients with endoscopy-verified healed mucosa. Inclusion of normalized mucosal cytokine expression into the concept of mucosal healing could have an impact on long-term clinical outcome.