Interfering with interferon-γ signalling in intestinal epithelial cells: selective inhibition of apoptosis-maintained secretion of anti-inflammatory interleukin-18 binding protein.

The intestinal epithelial barrier represents an important component in the pathogenesis of inflammatory bowel diseases. Interferon (IFN)-γ, a T helper type 1 (Th1) cytokine, regulated by the interleukin (IL)-18/IL-18 binding protein (bp) system, modulates the integrity of this barrier. The aim of this work was to study functionally the consequences of IFN-γ on intestinal epithelial cells (IEC) and to interfere selectively with identified adverse IFN-γ effects. IEC lines were stimulated with IFN-γ. IL-18 and IL-18bp were assessed by enzyme-linked immunosorbent assay. Staining of phosphatidylserine, DNA laddering, lactate dehydrogenase (LDH) release, cleavage of poly-adenosine diphosphate-ribose-polymerase (PARP) and activation of caspase-3 were analysed to determine cell death. Inhibitors of tyrosine kinase, caspase-3 or p38 mitogen-activated kinase ((MAP) activity were used. Cytokines were measured in supernatants of colonic biopsies of healthy controls and inflammatory bowel disease (IBD) patients. In IEC lines, IFN-γ up-regulated IL-18bp selectively. Ex vivo, IFN-γ was present in supernatants from cultured biopsies and up-regulated with inflammation. Contrary to previous reports, IFN-γ alone induced apoptosis in IEC lines, as demonstrated by phosphatidylserin staining, DNA cleavage and LDH release. Further, activation of caspase-3, PARP cleavage and expression of pro-apoptotic Bad were induced. Partial inhibition of caspase-3 and of p38 but not JAK tyrosine kinase, preserved up-regulation of IL-18bp expression. Selective inhibition of IFN-γ mediated apoptosis, while preserving its beneficial consequences on the ratio of IL-18/IL-18bp, could contribute to the integrity of the mucosal barrier in intestinal inflammation.

[Toluidin blue in gastrointestinal endoscopy]. / Toluidinblau in der gastrointestinalen Endoskopie.

For decades, methylene blue has been used in gastrointestinal endoscopy as an absorbing dye, it was, however, not approved for that purpose and has now been withdrawn from the market. A possible substitute is toluidine blue, an acidophilic, metachromatic dye that selectively stains cell nuclei; accordingly, since 2007, toluidine blue has been approved as a topical diagnostic agent in chromoendoscopy. Cells with increased DNA synthesis are stained more intensively so that not only malignant cells but also erosions, ulcerations and inflammatory areas are stained with toluidine blue because of the increased reparative cellular processes. Up to now, absolutely no studies have been carried out with regard to the effectiveness of toluidine blue in gastrointestinal endoscopy. We report on a consecutive series of 364 endoscopic applications of toluidine made on the basis of various indications. Besides the known indications (e. g., chromoendoscopy in case of Barrett's oesophagus), we mostly used toluidine blue, diluted in hydroxyethylstarch (HAES), for submucosal injections of flat adenomas prior to endoscopic mucosal resection or endoscopic submucosal dissection, in order to precisely determine the extension of visible lesions. Local and systemic adverse reactions have not been observed. A demarcation of the lesions can be made as effectively as with methylene blue.

Reducing inter-replicate variation in fourier transform infrared spectroscopy by extended multiplicative signal correction.

Fourier transform infrared (FT-IR) spectroscopy is a powerful tool for characterizing biological tissues and organisms, but it is plagued by replicate variation of various sources. Here, a method for estimating and correcting unwanted replicate variation in multivariate measurement signals, based on extended multiplicative signal correction (EMSC), is presented. Systematic patterns of unwanted methodological variations are estimated from replicate spectra, modeled by a linear subspace model, and implemented into EMSC. The method is applied to FT-IR spectra of two different sets of microorganisms (different double gene knockout strains of Saccharomyces cerevisiae and different species of Listeria) and compared to other preprocessing methods used in FT-IR absorption spectroscopy of microorganisms. The EMSC replicate correction turns out to perform best among the compared methods.

Medical prevention and treatment of acute and chronic radiation induced enteritis--is there any proven therapy? a short review.

BACKGROUND: Radiation enteritis is a severe problem in patients receiving irradiation of the abdomen or pelvis in the course of cancer treatment. Nevertheless, there is a lack of standardised strategies for medical prevention and therapy. MATERIALS AND METHODS: A PubMed based literature search was performed to address the available data on the prevention of and therapy for acute and chronic radiation enteritis. RESULTS: Four double-blind and placebo-controlled studies used 5-aminosalycilates in the prevention of acute radiation enteritis. Only for sulphasalzine 2 g/d was a positive effect proven. Prophylactic administration of probiotics reduced the incidence of acute radiation enteritis in a large placebo-controlled trial. If acute radiation enteritis was present octreotide ameliorated radiation-induced diarrhoea in a randomised study. Two investigations, only one of them randomised, described the effectiveness of loperamide in the treatment of acute radiation enteritis. If diarrhoea was also the main symptom of chronic radiation enteritis, loperamide reduced stool frequency in a double-blind and placebo-controlled study. A retrospective analysis of severe cases of chronic radiation enteritis with obstruction and fistula indicated that parenteral nutrition at home was more effective than surgery. CONCLUSION: Reduction of radiation dose and field size are still the most important factors in the prevention of acute and chronic radiation enteritis. Valid data particularly on the treatment of chronic radiation enteritis are lacking. A better understanding of the pathopysiology especially in chronic radiation enteritis might offer new therapeutic perspectives. Inhibition of TGF-beta, for example, might be a new promising therapy approach.

Functional P2X7 receptor polymorphisms (His155Tyr, Arg307Gln, Glu496Ala) in patients with Crohn's disease.

Genetic factors contribute to inflammatory bowel diseases. Recently, the P2X(7) receptor was found to be a key player in caspase-1-mediated processing of the proinflammatory cytokines, interleukin-1beta and interleukin-18. We therefore aimed to determine whether the gain-of-function single nucleotide polymorphism (SNP) His155Tyr and the loss-of-function SNP Arg307Gln and Glu496Ala were associated with susceptibility to Crohn's disease (CD). For association analysis, 681 unrelated CD patients and 736 healthy controls were enrolled. Furthermore, 490 CD trios were included for segregation analysis. Genotyping was performed by the application of the TaqMan(R) MGB biallelic discrimination system. The Arg307Gln polymorphism revealed a borderline significant difference in genotype frequencies between CD patients and controls (P = 0.06) without implying any pathological significance because of low case numbers. Case-control statistics for the variants His155Tyr and Glu496Ala showed no association with CD phenotype (P = 0.19 and 0.99). Subsequent family-based transmission disequilibrium test did not prove an association of the investigated single-nucleotide polymorphisms with CD. In conclusion, the analysed intragenetic variants of the P2X(7) receptor may not be a susceptibility factor for CD.

Reducing psychological distress in patients with inflammatory bowel disease by cognitive-behavioural treatment: exploratory study of effectiveness.

BACKGROUND: This prospective study aimed to determine whether cognitive-behavioural group treatment accompanying medical standard care is effective in reducing psychological distress in patients with inflammatory bowel disease. METHODS: Twenty-eight outpatients with Crohn disease or ulcerative colitis completed the treatment programme. Psychological treatment consisting of 12 weekly sessions was conducted in a group setting. Medical and psychometric assessments were taken at the beginning of the 3-month pretreatment waiting period, at pretreatment, at post-treatment and at the 3, 6 and 9-month follow-ups. RESULTS: During baseline, no change was observed in psychological distress. Disease-related worries and concerns decreased significantly from pretreatment to the follow-ups. The disease groups differed in the decline of concerns between pre- and post-treatment, with a significant reduction of concerns in patients with ulcerative colitis but not Crohn disease. This difference did not occur at the follow-ups, indicating long-term improvement for both disease groups. Depressive coping decreased significantly in women and remained stable at the follow-ups, whereas depressive coping did not change in men. The same gender difference was found for depressive symptoms. CONCLUSIONS: The exploratory findings suggest that psychological group treatment for outpatients is a feasible and effective approach for the short- and long-term reduction of psychological distress for patients with inflammatory bowel disease. However, the revealed gender differences on coping and depression might indicate the necessity to consider gender-specific aspects of inflammatory bowel disease when designing and evaluating psychological interventions.

Butyrate modulates intestinal epithelial cell-mediated neutrophil migration.

Butyrate, a short-chain fatty acid released by colonic bacteria and administered therapeutically in inflammatory bowel diseases, exerts immunomodulatory properties. The aim of the study was to determine the functional consequences of butyrate exposure on the proinflammatory responsiveness of human intestinal epithelial cells (IEC). IL-8 promoter activity in IEC pretreated with butyrate then exposed to proinflammatory stimuli was assayed by transfection of luciferase constructs. IL-8 secretion was determined by ELISA and neutrophil migration by flow cytometry. Receptor mRNA was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Butyrate modulated proinflammatory IL-8 secretion differentially in Caco-2 and HT-29 cells on the transcriptional level. Pointing to the potentially underlying mechanism of increased IL-1 beta-stimulated IL-8 secretion in HT-29 cells, butyrate up-regulated IL-1RI mRNA but not IL-1RII. Butyrate pretreatment of IEC lines stimulated by IL-1 beta modulated neutrophil migration significantly: reduction towards Caco-2 and enhancement towards HT-29/p cells. Pharmacological inhibition of protein tyrosine phosphatases or treatment with mesalamine or sulphasalazine diminished IL-1 beta-stimulated IL-8 secretion by butyrate-exposed HT-29 cells substantially. Immunomodulatory effects of butyrate on IEC are functionally relevant for neutrophil migration. Pharmacological inhibition of enhanced IL-1 beta-mediated IL-8 secretion in a subpopulation of IEC may improve the clinical efficacy of butyrate.

Differential effects of histone deacetylase inhibitors on interleukin-18 gene expression in myeloid cells.

Histone deacetyrase (HDAC) inhibitors induce growth arrest and differentiation of leukemia cell lines and tumor cells derived from a large variety of human tissues. Here we showed that HDAC inhibitors sodium butyrate, TSA, and valproate regulated the expression of Interleukin-18 (IL-18), a cytokine with antitumor and proinflammatory properties, in human acute myeloid leukemia cell lines U937 and HEL. Sodium butyrate increased expression of IL-18 protein and mRNA and activated 1357bp IL-18 gene promoter construct. IL-18 mRNA level was up-regulated by TSA or valproate, which also activated IL-18 full-length promoter. While sodium butyrate or TSA stimulated the 108-bp IL-18 minimal promoter, valproate failed to activate it, indicating that valproate may use a distinct mechanism from sodium butyrate and TSA to activate IL-18 gene expression.

Expression and localization of IL-1beta mRNA is interrelated with cytoskeletal rearrangement in monocytes stimulated by adherence: a light microscopy in situ hybridization study.

Differences in IL-1beta mRNA expression, stability and translation between non-adherent monocytes and those stimulated by adherence suggest that cytokine regulation is coupled to the function and assembly of cytoskeletal structures. In situ hybridization studies were performed to visualize expression and positioning of IL-1beta mRNA in adherently cultivated monocytes. IL-1beta mRNA expression was heterogeneous with high transcript levels found in spread or polarized cells. Transcripts were compartmentalized to the perinuclear region in spread cells, and partially redistributed with polarization. In contrast to mRNA distribution in other motile cell populations, IL-1beta mRNA did not localize to the distal or proximal actin cytoskeleton. Perinuclear confinement of transcripts required intact actin microfilaments. Treatment with cytoskeleton disruption and detergent extraction suggested that most non-translated IL-1beta mRNA was associated with intermediate filaments. In monocytes stimulated by LPS, IL-1beta, but not IL-1Ra transcripts were redistributed and partially associated, yet not bound to actin microfilaments. The present study demonstrates that IL-1beta mRNA expression and localization in adherent monocytes is interrelated with the cytoskeletal rearrangement upon adherence, spreading and polarization.

Gene expression of interleukin 18 in unstimulated peripheral blood mononuclear cells of patients with alcoholic cirrhosis.

BACKGROUND: Most patients with alcohol induced cirrhosis (AC) and chronic endotoxinaemia are not suffering from clinically evident systemic inflammatory reactions. This may be due to altered gene expression of cytokines, possibly related to endotoxin (for example, tolerance and sensitisation). Interleukin 18 (IL-18; interleukin gamma inducing factor) modulates local cytokine production in response to endotoxin (lipopolysaccharide (LPS)). AIM: To investigate the systemic immune response of patients with AC and to see if unstimulated peripheral blood mononuclear cells (PBMC) from patients with AC are activated and contribute to gene expression of IL-18. METHODS: Plasma levels of endotoxin (LPS) and serum levels of IL-18 were measured by enzyme linked immunoassay and the amoebocyte lysate test in 74 abstinent patients with different stages of AC (Child-Pugh stage A, n=18; B, n=22; C, n=34) and compared with healthy controls (n=43). Gene expression of IL-18 was assessed by semiquantitative reverse transcription-polymerase chain reaction in freshly isolated unstimulated PBMC of a subgroup of 14 patients with AC compared with five healthy controls. RESULTS: Gene expression of IL-18 specific mRNA in unstimulated PBMC was significantly enhanced in patients with advanced AC (Child-Pugh stage C) and correlated with plasma LPS and serum CD14 levels (Spearman rank correlation factors r=0.76 and r=0.72). Serum concentrations of IL-18 were also elevated compared with healthy controls (p<0.001) but correlation with serum levels of CD14 and plasma levels of LPS was much weaker compared with mRNA data (Spearman rank correlation factors r=0.47 and r=0.26). CONCLUSIONS: Our in vivo data suggest a presensitisation of "unstimulated" PBMC in the circulation of patients with AC by endotoxin. The term "unstimulated" may be inadequate in patients with AC. Further investigations are needed to define the exact mechanisms and localisation of sensitisation of PBMC in vivo.

Regulation of Staphylococcus aureus-mediated activation of interleukin-18 in peripheral blood mononuclear cells.

Bacteria and bacterial antigens strongly induce cytokine secretion by peripheral blood leukocytes and thereby initiate an inflammatory cascade with potentially deleterious consequences for the host. The present study focussed on receptors and signal transduction pathways involved in activation of interleukin (IL)-18 by heat-inactivated Gram-positive Staphylococcus aureus Cowan strain I (SAC). Similarly to IL-12/IL-12p40, IL-10 and IFN-gamma, SAC dose-dependently activated IL-18. Secretion of IL-18 was independent of functional activity of IL-10, IL-12 or IFN-gamma. Lipoteichoic acid (LTA), a structural component of SAC, was not sufficient for activation of IL-18, while it dose-dependently induced IL-10. In contrast to IL-12, blockade of CD14 only partially diminished secretion of IL-18 and did not affect secretion of IL-10, suggesting involvement of other receptors (e.g., Toll-like receptors) in SAC responses. Further down-stream however, secretion of IL-10, IL-12 and IL-18 was uniformly inhibited by blockade of G-protein-mediated kinase activation by mastoparan. Secretion of IL-18 required phosphatidylinositol-3'-kinase, and secretion of IL-12 phosphotyrosine kinase activity. The data demonstrate that SAC potently activates secretion of IL-18 by peripheral blood mononuclear cells with differential involvement of cell-surface receptors and signal transduction pathways as compared to other natural killer- and T cell-promoting cytokines.

Lipopolysaccharide/endotoxin induces IL-18 via CD14 in human peripheral blood mononuclear cells in vitro.

IL-18 shares activities with IL-12 in generating T-helper 1 cells and cytokine response. It mediates LPS/endotoxin lethality by IL-12 independent interferon-gamma synthesis and it induces bacteria-related organ failure. As peripheral blood mononuclear cells (PBMC) are potent producers of IL-18, we studied the regulation of IL-18 upon exposure to LPS and Staphylococcus aureus (SAC) in vitro. Freshly isolated PBMC constitutively expressed IL-18 mRNA. After unstimulated preincubation for 48 h, however, IL-18 transcripts were nearly not detectable by RT-PCR, but inducible by LPS or SAC (P<0.01). Both LPS and SAC were potent stimuli of IL-18 protein secretion (P<0.01). LPS-mediated IL-18 gene expression and secretion was CD14-dependent and significantly inhibited by co-incubation of PBMC with neutralizing CD14 antibody (P<0.01). We conclude that LPS-driven IL-18 is dependent on the expression of costimulatory factors and that IL-18 inhibition might attenuate IL-18-related toxic effects.

Cellular differentiation causes a selective down-regulation of interleukin (IL)-1beta-mediated NF-kappaB activation and IL-8 gene expression in intestinal epithelial cells.

Interleukin (IL)-1beta signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF-kappaB. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1beta-mediated NF-kappaB activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1beta- and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)-alpha or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1beta exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1beta-mediated IkappaB kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1beta exposure but not after TNF-alpha stimulation. The proximal IL-1 signaling molecule IL-1 receptor-associated kinase was not degraded in IL-1beta-stimulated HT-29 cells, in contrast to Caco-2 cells. kappaB-luciferase reporter gene activity was 16-fold higher following TNF receptor-associated factor-6 transfection after IL-1beta stimulation in HT-29/MTX cells. We conclude that cellular differentiation of HT-29 cells selectively impairs the IL-1beta signaling pathway inhibiting both NF-kappaB and JNK activity in response to IL-1beta. This relative unresponsiveness to IL-1beta may represent an important regulatory mechanism of differentiated intestinal epithelial cells.

Differential expression of interleukin 1 receptor antagonist isoforms in human intestinal epithelial cells.

BACKGROUND & AIMS: Regulatory cytokines mediate intestinal epithelial cell (IEC) participation in mucosal immune responses. The aim of this study was to investigate the expression of secretory and intracellular isoforms of interleukin 1 receptor antagonist (IL-1Ra) in human primary IECs and carcinoma-derived cell lines. METHODS: Primary IECs were isolated from patients with Crohn's disease or ulcerative colitis and from normal controls. Isoform-specific IL-1Ra messenger RNA (mRNA) and protein were assessed by reverse-transcription polymerase chain reaction and Western blot analysis. Expression during cellular differentiation was determined by in situ immunohistochemistry on sequentially released, native IECs and in vitro differentiated cell lines. Intracellular IL-1Ra I function was analyzed by permanent transfection of Caco-2 cells. RESULTS: Intracellular IL-1Ra I protein accumulated in surface IECs with extension to the crypts during inflammation. Secretory IL-1Ra and intracellular IL-1Ra II mRNA, but not the corresponding protein, was detected. Transcription of intracellular IL-1Ra I mRNA was significantly up-regulated with inflammation and in vitro by phorbol myristate acetate and interleukin 1beta. In vitro differentiated cells had higher constitutive intracellular IL-1Ra I protein content. Intracellular IL-1Ra I expression in Caco-2 cells decreased IL-1beta-stimulated interleukin 8 secretion. CONCLUSIONS: Native human IECs and certain cell lines constitutively express intracellular IL-1Ra type I, which is up-regulated by inflammation, inflammatory stimuli, and cellular differentiation. Constitutive expression of this anti-inflammatory cytokine may contribute to mucosal protection.

Subtherapeutic corticosteroids potentiate the ability of interleukin 10 to prevent chronic inflammation in rats.

BACKGROUND & AIMS: Interleukin (IL)-10, which inhibits macrophages and T-helper lymphocyte type 1 (TH1) lymphocytes, attenuates chronic granulomatous inflammation induced by bacterial cell wall polymers. This study determines whether corticosteroids enhance the protective effects of IL-10 in cultured peripheral blood mononuclear cells (PBMNCs) and in vivo when started before or after the onset of experimental chronic granulomatous inflammation. METHODS: Intestines of Lewis rats were injected intramurally with streptococcal peptidoglycan-polysaccharide (PG-APS) polymers. Daily murine recombinant IL-10 and/or dexamethasone (DEX) therapy was started 12 hours before or at several intervals after PG-APS injection. RESULTS: IL-10 plus corticosteroids additively inhibited IL-1beta secretion in human PBMNCs but preserved the beneficial IL-1RA/IL-1beta ratio induced by IL-10. IL-10 started before PG-APS injection significantly attenuated intestinal and extraintestinal inflammation, with even more pronounced effects in combination with subtherapeutic doses of DEX. The combination of DEX decreased the effective dose of IL-10 by at least one half. After onset of systemic inflammation using doses effective for prevention, IL-10 monotherapy had nearly no benefit and DEX plus IL-10 was similar to the mild therapeutic effect of DEX alone. CONCLUSIONS: The combination of IL-10 and corticosteroids allows lower doses of both agents in preventing chronic intestinal and systemic inflammation. However, timing of IL-10 administration is a critical variable in regulating inflammation.

Presence of plasma endotoxin is correlated with tumour necrosis factor receptor levels and disease activity in alcoholic cirrhosis.

Cytokines and plasma endotoxin were measured in a consecutive series of patients with alcoholic cirrhosis (AC). Endotoxaemia was found to be strongly correlated to increased plasma levels of functionally active tumour necrosis factor (TNF) receptors -p55 and -p75, TNF-alpha and the Child-Pugh stage of the disease. Our data support the hypothesis of the pathogenic role of lipopolysaccharide in hepatocellular damage of patients with AC.