Rasmus Goll

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.

Tissue levels of tumor necrosis factor-alpha correlates with grade of inflammation in untreated ulcerative colitis

Objective. The immune characterization of ulcerative colitis (UC) has been unclear and controversial. One possible explanation for the discrepancies between earlier cytokine studies in UC may be the fact that the patients included were on immunosuppressive therapy. Thus, the aim of this study was to determine the tumor necrosis factor-alpha (TNF-α) level and TH1/TH2 cytokine expression (mRNA) profile in patients with untreated UC. Material and methods. Forty-four untreated UC patients, 10 untreated Crohns disease (CD) patients and 28 healthy controls were included in the study. Colon biopsies were processed for quantitative measurements of TNF-α, interleukin (IL)-10, IL-18, IL-4 and interferon-gamma (IFN-γ) mRNA using real-time polymerase chain reaction (PCR). TNF-α expression in T-cell lymphocytes (CD3) and macrophages (CD68) were further characterized by immunohistochemistry (IHC). Results. Compared with the level in normal controls, the TNF-α mRNA level in UC patients was clearly increased, especially in patients with moderate to severe disease. The levels of TNF-α mRNA increased in proportion to the UC Disease Activity Index (UCDAI) score in UC patients. Differences were also observed between UC and controls for IFN-γ IL-18, IL-4 and IL-10. Only minor quantitative differences in cytokines were observed between UC and CD, and they were more or less similar when comparing moderate to severe UC and CD. CD3+ lymphocytes and macrophages in lamina propria from CD and UC lesions showed increased intracellular staining of TNF-α. Conclusions. TNF-α is highly expressed in UC and correlates to the grade of inflammation. The sources of TNF-α were observed both in CD3+ lymphocytes and in macrophages. Cytokine expression (mRNA) profiles seem to be similar in patients with moderate to severe UC and CD.

Helicobacter pylori Stimulates a Mixed Adaptive Immune Response with a Strong T‐Regulatory Component in Human Gastric Mucosa

Background:  Host factors play an important role in the pathophysiology of Helicobacter pylori infection and development of gastritis and related disease. The established opinion is that the T‐cell‐mediated immune response to H. pylori infection is of Th1 type. Our earlier immune cell phenotype studies indicate a mixed Th1–Th2 profile of the effector cells. Therefore, an extensive adaptive and regulatory cytokine gene expression profile was conducted by quantitative real‐time polymerase chain reaction (qPCR).

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.

Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR

BackgroundIn real-time PCR data analysis, the cycle threshold (CT) method is currently the gold standard. This method is based on an assumption of equal PCR efficiency in all reactions, and precision may suffer if this condition is not met. Nonlinear regression analysis (NLR) or curve fitting has therefore been suggested as an alternative to the cycle threshold method for absolute quantitation. The advantages of NLR are that the individual sample efficiency is simulated by the model and that absolute quantitation is possible without a standard curve, releasing reaction wells for unknown samples. However, the calculation method has not been evaluated systematically and has not previously been applied to a TaqMan platform. Aim: To develop and evaluate an automated NLR algorithm capable of generating batch production regression analysis.ResultsTotal RNA samples extracted from human gastric mucosa were reverse transcribed and analysed for TNFA, IL18 and ACTB by TaqMan real-time PCR. Fluorescence data were analysed by the regular CT method with a standard curve, and by NLR with a positive control for conversion of fluorescence intensity to copy number, and for this purpose an automated algorithm was written in SPSS syntax. Eleven separate regression models were tested, and the output data was subjected to Altman-Bland analysis. The Altman-Bland analysis showed that the best regression model yielded quantitative data with an intra-assay variation of 58% vs. 24% for the CT derived copy numbers, and with a mean inter-method deviation of × 0.8.ConclusionNLR can be automated for batch production analysis, but the CT method is more precise for absolute quantitation in the present setting. The observed inter-method deviation is an indication that assessment of the fluorescence conversion factor used in the regression method can be improved. However, the versatility depends on the level of precision required, and in some settings the increased cost effectiveness of NLR may justify the lower precision.

Intestinal barrier homeostasis in inflammatory bowel disease

Abstract The single-cell thick intestinal epithelial cell (IEC) lining with its protective layer of mucus is the primary barrier protecting the organism from the harsh environment of the intestinal lumen. Today it is clear that the balancing act necessary to maintain intestinal homeostasis is dependent on the coordinated action of all cell types of the IEC, and that there are no passive bystanders to gut immunity solely acting as absorptive or regenerative cells: Mucin and antimicrobial peptides on the epithelial surface are continually being replenished by goblet and Paneth’s cells. Luminal antigens are being sensed by pattern recognition receptors on the enterocytes. The enteroendocrine cells sense the environment and coordinate the intestinal function by releasing neuropeptides acting both on IEC and inflammatory cells. All this while cells are continuously and rapidly being regenerated from a limited number of stem cells close to the intestinal crypt base. This review seeks to describe the cell types and structures of the intestinal epithelial barrier supporting intestinal homeostasis, and how disturbance in these systems might relate to inflammatory bowel disease.

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.

Infliximab therapy decreases the levels of TNF-α and IFN-γ mRNA in colonic mucosa of ulcerative colitis

Objective. The mechanisms of action of infliximab (IFX) in the treatment of ulcerative colitis (UC) are poorly understood. The aim of the study was to investigate the changes in tissue expression of tumor necrosis factor-alpha (TNF-α) and other cytokines in UC patients receiving IFX treatment. Material and methods. The levels of TNF-α, interleukin (IL)-10, IL-4, and interferon-gamma (IFN-γ) mRNA in colonic biopsies from 32 UC patients during IFX treatment were measured by real-time polymerase chain reaction (PCR) and compared with those of 19 controls. Immunohistochemistry was performed to characterize the changes of inflammatory cells during treatment. Results. IFX reduced the expression of TNF-α and IFN-γ mRNA, but not that of IL-10 and IL-4 mRNA. Reductions in TNF-α mRNA were correlated to clinical and endoscopic improvements, and normalization of TNF-α mRNA was obtained in patients with healed mucosa. The numbers of T lymphocytes and macrophages were significantly decreased in patients with healed mucosa after IFX treatment, although compared to normal controls, there were still increased levels of TNF-α-positive cells after treatment. Conclusions. IFX induced down-regulation of the mucosal TNF-α and IFN-γ mRNA expression in UC patients. The numbers of T lymphocytes and macrophages were significantly decreased in patients with endoscopically healed mucosa after IFX treatment.

Reduced expression of microenvironmental Th1 cytokines accompanies adenomas–carcinomas sequence of colorectum

Cytokines have been suggested to be key factors in modulating immune response against tumorigenesis in the microenvironment. Therefore, characterization of cytokine expression along the colorectal adenoma–carcinoma sequence may add important information for understanding the immune-related mechanisms of the development of colorectal carcinoma (CRC). In this study, biopsies from 32 patients with colorectal adenoma (CRA), 20 patients with CRC and 18 healthy controls were examined. Cytokine gene expressions of interleukin-4 (IL-4), IL-10, tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and its upstream inducers (IL-12A and IL-18) were measured at messenger RNA (mRNA) level with quantitative real-time PCR (Q-PCR). Cytokine expressing cells were characterized using immunohistochemistry (IHC). A distinct different cytokine profile between adenoma and CRC was observed: the Th1 cytokines (IFN-gamma, TNF-alpha, IL-12A and IL-18) were increased in local tissues of CRA and decreased in CRC. Consistent with the quantitative cytokine data, IHC examinations revealed slightly increased densities of Th1 cytokine-expressing cells in CRA and a remarkably decreased density of the Th1 cells in CRC. In CRA, the cytokine-expressing cells were highly polarized to the subepithelial stroma while the cells were evenly distributed through the stroma in CRC. In conclusion, distinct changes in the Th1 cytokine profile appear along the colorectal adenoma–carcinoma sequence. This may reflect a change in the host immune regulatory function in the adenoma–carcinoma sequence.

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.