Increased expression of HIF2α during iron deficiency-associated megakaryocytic differentiation.

BACKGROUND: Iron deficiency is associated with reactive thrombocytosis; however, the mechanisms driving this phenomenon remain unclear. We previously demonstrated that this occurs alongside enhanced megakaryopoiesis in iron-deficient rats, without alterations in the megakaryopoietic growth factors thrombopoietin, interleukin-6, or interleukin-11. OBJECTIVES: The aim of this study was to evaluate megakaryocyte differentiation under iron deficiency in an in vitro model and to investigate potential genes involved in this process. METHODS: Human erythroleukemia and megakaryoblastic leukemia cell lines, as well as cord-blood derived hematopoietic stem cells were cultured under iron deficiency. Cell morphology, ploidy, expression of CD41, CD61, and CD42b, and proplatelet formation were assessed in iron-deficient cultures. Polymerase chain reaction arrays were used to identify candidate genes that were verified using real-time polymerase chain reaction. Hypoxia-inducible factor 1, α subunit (HIF2α) protein expression was assessed in bone marrow sections from iron-deficient rats and vascular endothelial growth factor (VEGF)-A in culture supernatants. RESULTS AND CONCLUSIONS: Iron deficiency enhanced megakaryoid features in cell lines, increasing ploidy and initiating formation of proplatelet-like structures. In cord blood cell cultures, iron deficiency increased the percentage of cells expressing megakaryopoietic markers and enhanced proplatelet formation. HIF2α and VEGF were identified as potential pathways involved in this process. HIF2α protein expression was increased in megakaryocytes from iron-deficient rats, and VEGF-A concentration was higher in iron-deficient culture supernatants. Addition of VEGF-A to cell cultures increased percentage expression of megakaryocyte CD41. In conclusion, the data demonstrate that iron deficiency augments megakaryocytic differentiation and proplatelet formation and a potential role of HIF2α in megakaryopoiesis.

[Colorectal cancer: screening and surveillance in inflammatory bowel diseases - consensus of the working group for inflammatory bowel diseases of the Austrian Society of Gastroenterology and Hepatology]. / Kolorektales Karzinom: Screening und Surveillance bei chronisch entzündlichen Darmerkrankungen - Konsensus der Arbeitsgruppe für chronisch entzündliche Darmerkrankungen der ÖGGH.

Patients with ulcerative colitis and Crohn's colitis are at increased risk of colorectal cancer (CRC). This risk is dependent on the duration and extent of disease, inflammatory activity and possible additional risk factors. Thus, the aim is to reduce this risk and to detect dysplastic and malignant lesions at an early stage. The working group for Inflammatory Bowel Diseases (IBD) of the Austrian Society of Gastroenterology and Hepatology (ÖGGH) has developed consensus statements on the following topics: risk of colorectal cancer, screening and surveillance, procedure of surveillance colonoscopy, dysplasia and its management, and chemoprevention. This consensus is intended to increase awareness of the increased risk of CRC in IBD and to support a standardised approach in cancer prevention.

A randomized head-to-head study of small-bowel imaging comparing MiroCam and EndoCapsule.

BACKGROUND AND STUDY AIMS: The MiroCam is a new video capsule device offering a higher frame rate and a longer battery life-expectancy. We aimed to quantify its clinical impact and performed a randomized head-to-head comparison with the EndoCapsule device with respect to the rate of complete small-bowel examinations, diagnostic yield in the small bowel, and capsule transit time. PATIENTS AND METHODS: Patients referred for video capsule endoscopy because of obscure gastrointestinal bleeding, chronic diarrhea, and anemia of unknown origin were randomly assigned to swallow either the MiroCam first, followed by the EndoCapsule 2 hours later, or vice versa. All videos were analyzed by two independent investigators. RESULTS: A total of 50 patients (median age 61, range 21-84) were included. Complete small-bowel examination was achieved in 48/50 patients using the MiroCam and 45/50 using the EndoCapsule (96% vs. 90%, odds ratio [OR] 2.67, 95% confidence interval [CI] 0.49-14.45; P=0.38). There was diagnostic yield in the small bowel for 25/50 patients using the MiroCam and 24/50 using the EndoCapsule (50% vs. 48%, OR 1.08, 95%CI 0.49-2.37; P>0.99). However, the findings were concordant in 68% only (kappa = 0.50). The combined diagnostic yield was 58%. Even solitary findings had a relevant clinical impact during a 6-month follow-up. CONCLUSION: In this direct comparison the MiroCam and EndoCapsule devices were not statistically different with regard to their rates of complete small-bowel examinations or diagnostic yield. Their moderate concordance, mainly caused by missed pathological findings, which affected both devices, needs consideration in clinical practice.

An intracytoplasmic IL-10 receptor variant permits rapid reduction in STAT3 activation.

Within the interleukin-10 receptor 1 (IL10R1) gene, two common variants are associated with certain diseases: single-nucleotide polymorphism 3 (SNP3), a serine-138 to glycine mutation is in linkage disequilibrium with SNP4, a glycine-330 to arginine mutation, both of which are considered loss-of-function alleles. However, the molecular consequence of G330R is unknown. We investigated possible roles of G330R on the dynamics of IL10R1 surface expression and signal transducer and activator of transduction (STAT) phosphorylation. HeLa cells expressing the respective IL10R1 haplotype were stimulated with IL-10. Significant reduction of IL10R1 surface expression was observed after ligand binding. Receptor expression remained low on continuous incubation with IL-10. In contrast, when treated with an IL-10 pulse, IL10R1 surface expression returned to its resting state within 3-9 h irrespective of the haplotype. STAT3 was rapidly phosphorylated both in cells with wild-type (WT) or variant IL10R1, and maintained phosphorylated when cells were cultured with IL-10. On IL-10 pulse, however, STAT3 phosphorylation declined rapidly in cells expressing IL10R1-G330R but not IL10R1-WT or S138G. Similar dynamics were observed with STAT1 phosphorylation at Tyr701. No differences in janus kinase 1 (JAK1) activation were observed in cells with WT or variant IL10R1. Our results indicate that IL10R1-G330R does not alter surface expression but duration of STAT phosphorylation, indicating that the position of G330 is important in stabilizing the STAT signal.

[Diagnosis and treatment of iron deficiency and anaemia in inflammatory bowel diseases. Consensus of the Austrian IBD Working Party]. / Diagnose und Behandlung von Eisenmangel und Anämie bei chronisch entzündlichen Darmerkrankungen. Konsensus der österreichischen Arbeitsgruppe für CED.

Iron deficiency with and without anaemia is a common burden of patients with inflammatory bowel diseases (IBD) and has considerable impact on their quality of life and the ability to perform. The IBD working group of the Austrian Society of Gastroenterology and Hepatology developed five consensus statements on the following topics: (i) diagnosis of iron deficiency and (ii) anaemia, (iii) screening of iron deficiency, (iv) treatment of iron deficiency and (v) therapeutic goals. The clinical importance of intravenous iron replacement therapy in IBD with regard to effectiveness and compliance was discussed.

Systematic review: molecular chemoprevention of colorectal malignancy by mesalazine.

BACKGROUND: Mesalazine (mesalamine) (5-ASA) is considered an anti-inflammatory drug for the treatment of inflammatory bowel disease. It is well tolerated by most patients and induces mucosal healing specifically in ulcerative colitis. Besides its anti-inflammatory properties, 5-ASA has been studied for cancer inhibitory activities as it seems to reduce colorectal cancer incidence in patients using this drug for long periods of time. However, detailed molecular mechanisms of drug action are vague. AIMS: To evaluate known molecular mechanisms of 5-ASA on chemoprevention of colorectal malignancy. METHODS: Systematic review with search terms '5 aminosalicylic acid, mesalazine, 5-ASA, mesalazine, molecular mechanisms, chemoprevention' between 2006 and August 2009. RESULTS: A total of 48 studies were retrieved that link 5-ASA chemopreventive properties to five distinct pathways. These include interference with cell cycle progression (12 references), scavenging of reactive oxygen- or nitrogen-derived metabolites (16 references), TNF-alpha/TGF-ss signalling (11 references), WNT/beta-catenin signalling (5 references) and anti-bacterial properties (4 references). CONCLUSIONS: In the recent years, a large amount of molecular data has accumulated supporting the notion that 5-ASA biological effects interfere with colorectal cancer development. These molecular pathways are of special interest in the search for 5-ASA's molecular target(s) and development of novel chemopreventive compounds.

IL10R1 loss-of-function alleles in rheumatoid arthritis and systemic lupus erythematosus.

OBJECTIVE: IL-10 is a pleiotropic cytokine involved in the regulation of innate and cell-mediated immunity and a key mediator within the disturbed SLE immune system. IL-10 binds to IL10R1, which is expressed on a variety of immune cells and activates the JAK-STAT pathway. Two (out of several known) genetic IL10R1 variants may alter IL-10 binding or signal transduction. Here we investigate the differential activity of these IL10R1 variants and their possible association with RA or SLE susceptibility. METHODS: IL10R1-wt, IL10R1-S138G, IL10R1-G330R, or IL10R1- S138G +G330R were cloned into pIRESpuro3 and transfected into HeLa cells. Single cell clones were tested for IL-10-induced SOCS3- and SLAM gene expression by real-time PCR. DNA from 182 RA patients, 222 SLE patients, and 250 healthy controls was genotyped by allele-specific PCR. RESULTS: A biphasic increase of SOCS3 mRNA was observed that peaked at 15 minutes and 4 hours after IL-10 stimulation. The presence of IL10R1 S138G and G330R showed a weaker induction of both SOCS3 and SLAM upon stimulation with IL-10. In RA a homozygous G330R genotype was more commonly present than in controls (15.4% vs. 7.6%; p<0.05). In SLE the G330R allele frequency was also increased (36.3% vs. 30.0%; p<0.05) without showing a gene-dose relationship at the genotype level. CONCLUSIONS: Based on these results, both variants of the IL10R1 gene are loss-of-function alleles. IL10R1 G330R may possibly contribute to RA or SLE disease susceptibility in Caucasian populations.

In vivo effects of mesalazine or E. coli Nissle 1917 on microsatellite instability in ulcerative colitis.

BACKGROUND: Microsatellite instability (MSI) occurs in chronically inflamed colorectal tissue and may evolve to colitis-associated cancer. In vitro data suggest that mesalazine (5-ASA) improves MSI. AIM: To analyse the changes in MSI in 156 distal colonic biopsies of 39 patients with ulcerative colitis that had been treated within a randomized, double-blind trial comparing 5-ASA with E. coli Nissle (EcN). METHODS: Two biopsies had been collected before and after 1 year of treatment. MSI testing was performed using a panel of eight markers, including 3 dinucleotide and 5 mononucleotide repeats. RESULTS: No MSI was observed with any of the mono-repeats, and among dinucleotide repeats, only D5S346 (maps to APC) and D17S250 (p53) were consistently informative. Overall, 31/156 (20%) biopsies displayed MSI. After 1 year, 3/11 patients displayed MSI improvement [change to microsatellite stability (MSS); 1 on 5-ASA, 2 on EcN] at D5S346 and 4/11 showed MSI worsening (change from MSS to MSI; all 5-ASA). For D17S250, the corresponding data were for 3/9 patients (2 on 5-ASA, 1 on EcN) and 2/9 (both on 5-ASA), respectively. CONCLUSIONS: In the set of biopsies taken from patients treated with 1.5 g 5-ASA for 1 year, there was no improvement in the prevalence of MSI in the distal colon.

The IL-10R1 S138G loss-of-function allele and ulcerative colitis.

Genetic predisposition is a risk factor for the development of inflammatory bowel diseases (IBDs). Disruption of the interleukin (IL)-10 pathway in mice causes intestinal inflammation similar to human IBD. Two common non-synonymous IL-10R1 variants, S138G and G330R, were cloned and expressed in HeLa and Ba/F3. A reduction in IL-10-induced STAT1 and STAT3 activation was seen for IL-10R1-S138G (but not IL-10R1-G330R) by phosphospecific western blotting in both cell types. When analyzing 52 world populations for the presence of IL-10R1 variants, a strong dissimilarity was found between major geographical regions. In addition, when 182 IBD-parent trios were genotyped for both variants, a reduced transmission of haplotype -7 (carrying the S138G variant allele) to offspring with ulcerative colitis (UC) was observed. This UC-protective effect of S138G was confirmed in a Hungarian cohort (n=185, allele frequency 11.6 versus 17.5%; P=0.017) but not in an independent Belgian cohort (n=666, allele frequency 15.9 versus 15.5%; P=0.8). In conclusion, the IL-10R1 S138G variant is a loss-of-function allele for IL-10-induced STAT1 and STAT3 activation but does not protect from UC susceptibility.

Systematic review: does concurrent therapy with 5-ASA and immunomodulators in inflammatory bowel disease improve outcomes?

BACKGROUND: With greater use of immunomodulators in inflammatory bowel disease (IBD), it is uncertain whether concurrent therapy with both 5-aminosalicylic acid [5-ASA, mesalazine (mesalamine)] and an immunomodulator is necessary. AIM: To determine whether concurrent therapy with both 5-ASA and immunomodulator(s) improves outcomes in IBD. METHODS: Systematic review with search terms 'azathioprine, 6-mercaptopurine, thiopurine(s), 5 aminosalicylic acid, mesalazine, inflammatory bowel disease, ulcerative colitis, Crohn's disease, immunosuppressant(s), immunomodulator and methotrexate' in November 2007 to identify clinical trials on concurrent 5-ASA and immunomodulator therapy. RESULTS: Two small controlled studies were found. Neither showed a benefit on disease control beyond immunomodulator monotherapy. Potential pharmacological interactions exist between 5-ASA and thiopurines. Whilst circumstantial, epidemiological and laboratory evidence suggests that 5-ASA may assist colorectal cancer (CRC) chemoprevention, it may simply be via anti-inflammatory effects. With changes in practice, ethical issues and the long lead-time needed to demonstrate or disprove an effect, no clinical studies can/will directly answer this. The costs of avoiding one CRC in IBD may be as low as 153 times the annual cost of 5-ASA therapy. CONCLUSIONS: It is unclear whether concurrent 5-ASA and immunomodulator therapy improves outcomes of disease control, drug toxicity or compliance. Concurrent therapy of 5-ASA and immunomodulators may decrease CRC risk at 'acceptable' cost.

Activated neutrophils induce an hMSH2-dependent G2/M checkpoint arrest and replication errors at a (CA)13-repeat in colon epithelial cells.

OBJECTIVE: Chronic inflammation in ulcerative colitis is associated with increased risk for colorectal cancer. Its molecular pathway of cancer development is poorly understood. We investigated the role of neutrophil-derived cellular stress in an in vitro model of neutrophils as effectors and colon epithelial cells as targets, and tested for changes in cell cycle distribution and the appearance of replication errors. DESIGN: Colon epithelial cells with different mismatch repair phenotypes were co-cultured with activated neutrophils. Target cells were analysed for cell cycle distribution and replication errors by flow cytometry. Changes in nuclear and DNA-bound levels of mismatch repair- and checkpoint-related proteins were analysed by western blotting. RESULTS: Activated neutrophils cause an accumulation of target cells in G2/M, consistent with the installation of a DNA-damage checkpoint. Cells that do not express hMSH2, p53 or p21(waf1/cip1) failed to undergo the G2/M arrest. Phosphorylation of p53 at site Ser15 and Chk1 at Ser317, as well as accumulation of p21(waf1/cip1), was observed within 8-24 h. Superoxide dismutase and catalase were unable to overcome this G2/M arrest, possibly indicating that neutrophil products other than superoxide or H(2)O(2) are involved in this cellular response. Finally, exposure to activated neutrophils increased the number of replication errors. CONCLUSIONS: By using an in vitro co-culture model that mimics intestinal inflammation in ulcerative colitis, we provide molecular evidence for an hMSH2-dependent G2/M checkpoint arrest and for the presence of replication errors.

Systematic review: managing anaemia in Crohn's disease.

BACKGROUND: Anaemia is a serious complication of Crohn's disease that triggers hospitalization and, if not interfered with, may lead to death. AIMS: To systematically summarize and compare the literature on anaemia in Crohn's disease. METHODS: For this systematic review the literature was searched for English-language articles using anaemia, Crohn* and IBD as key words. 144 articles were identified and sorted according to the following topics: prevalence, aetiology, diagnostic tests and therapy. RESULTS: The reported prevalence of anaemia varied between 6.2% and 73.7%, with higher reported frequencies in older studies and in in-patients. Iron deficiency is the most common underlying condition. Vitamin B12 deficiency is related to the extent of ileal resection but has rarely impact on anaemia. Diagnostic criteria are not established and treatment guidelines are missing. Oral iron supplementation seems effective for short periods but intolerance leads to discontinuation in up to 21%. Eleven of 11 studies show that oral iron enhances intestinal inflammation and colon carcinogenesis in animal models of colitis. Intravenous iron supplementation with iron sucrose has been tested in over 250 Crohn's disease patients, is safe, effective and does not carry such hazards. CONCLUSIONS: As disease activity is determining the degree of anaemia in Crohn's disease, implementation of more effective therapy for Crohn's disease will lower its incidence. However, further studies regarding the safety and effectiveness of iron supplementation are needed.

Bi-allelic presence of the interleukin-10 receptor 1 G330R allele is associated with cirrhosis in chronic HCV-1 infection.

Immune response to viral infection is an important determinant of liver injury in chronic hepatitis C (CHC). Experimental and clinical data suggest a protective role of interleukin-10 (IL-10) in hepatic fibrogenesis. The significance of two SNPs of the interleukin-10 receptor 1 (IL-10R1), S138G (SNP3) and G330R (SNP4) was investigated on (i) susceptibility to CHC, (ii) progression of hepatic fibrosis and (iii) response to interferon/ribavirin therapy. DNA and liver biopsies were obtained from 212 patients with HCV (hepatitis C virus)-genotype-1 infection. The allele frequencies were 0.17 for SNP3 and 0.33 for SNP4, both of which were indifferent from healthy controls (0.17 and 0.32, respectively). Stage 1 liver fibrosis was found in 22 cases (10.4%), stage 2 in 108 (50.9%), stage 3 in 27 (12.8%), and stage 4 (cirrhosis) in 55 (25.9%). An association was found between the SNP4 allele and the presence of cirrhosis (P=0.01). Homozygous SNP4 individual variants segregated within the cirrhosis group (P=0.03). We found neither an association with SNP3 nor with the necroinflammatory disease activity (as measured by ALT levels) nor with the response to antiviral therapy. Our work implies that IL-10R1 SNP4 is a recessively inherited risk factor for hepatic cirrhosis in HCV genotype-1 infection.

Review article: the chemoprevention of colorectal carcinoma.

This review aims to provide an insight into some of the common pathways involved in the development of colorectal cancer--so-called chromosomal instability and microsatellite instability. There is both clinical and molecular evidence to show that colorectal cancer development occurs over an extended period of time. Together with a knowledge of the molecular pathogenesis of colorectal cancer, this time window allows physicians to counteract cancer development, a strategy known as chemoprevention. In familial and sporadic colorectal cancer, aspirin, other nonsteroidal anti-inflammatory drugs and cyclo-oxygenase-2 inhibitors have been tested for their ability to reduce polyp and cancer development in clinical studies. In inflammatory bowel disease-related colorectal cancer, evidence from retrospective case-control studies shows that mesalazine (mesalamine) can reduce the development of dysplastic lesions or cancer. Although the mechanisms behind this are incompletely understood, general anti-inflammatory, oxygen scavenger and some pro-apoptotic and cyclo-oxygenase inhibitory activities of mesalazine are thought to be involved. New molecular evidence points to a direct DNA stabilizing effect of mesalazine, resulting in a significant reduction of spontaneous microsatellite mutations.

Iron, anaemia, and inflammatory bowel diseases.

Iron deficiency anaemia is one of the most common disorders in the world. Also, one third of inflammatory bowel disease (IBD) patients suffer from recurrent anaemia. Anaemia has significant impact on the quality of life of affected patients. Chronic fatigue, a frequent IBD symptom itself, is commonly caused by anaemia and may debilitate patients as much as abdominal pain or diarrhoea. Common therapeutic targets are the mechanisms behind anaemia of chronic disease and iron deficiency. It is our experience that virtually all patients with IBD associated anaemia can be successfully treated with a combination of iron sucrose and erythropoietin, which then may positively affect the misled immune response in IBD.

A randomised placebo controlled trial of pegylated interferon alpha in active ulcerative colitis.

BACKGROUND: Pilot studies of interferon alpha (IFN-alpha) suggest a high remission rate in the treatment of active ulcerative colitis. We evaluated the safety of pegylated interferon alpha (PegIFN) and its role in induction of remission in patients with active ulcerative colitis, in a multicentre placebo controlled trial. METHODS: Sixty patients with a clinical activity score (CAI) of >6 were randomised to receive placebo (n=20), PegIFN 0.5 microg/kg (n=19), or PegIFN 1.0 microg/kg body weight (n=21) once weekly (PegIntron; Schering-Plough, USA) over 12 weeks. Patients receiving 5-aminosalicylates, steroids, and/or azathioprine in stable dosages were included. RESULTS: Serious adverse events were seen in none of the placebo patients, in 3/19 patients in the PegIFN 0.5 microg/kg group (hospitalisation due to disease flare up n=3), and in 3/21 in the PegIFN 1.0 microg/kg group (hospitalisation due to disease flare up n=1; thrombosis n=1; grand mal seizure n=1). Otherwise, we observed only minor IFN-alpha side effects. Clinical remission rates at week 12 (CAI < or =4) were 7/20 (35%) in the placebo, 9/19 (47%) in the PegIFN 0.5 microg/kg group, and 7/21 (33%) in the PegIFN 1.0 microg/kg group (NS). Early withdrawal from the study was observed in 11/20 placebo patients, in 6/19 in the PegIFN 0.5 microg/kg group, and in 10/21 in the PegIFN 1.0 microg/kg group, mainly due to lack of efficacy. The higher PegIFN dose was associated with a significant decrease in levels of C reactive protein (p=0.003, day 0 v 85). CONCLUSIONS: PegIFN is safe but not effective, at the dosages used, in patients with ulcerative colitis.