Cohort profile: design and first results of the Dutch IBD Biobank: a prospective, nationwide biobank of patients with inflammatory bowel disease.

PURPOSE: The Dutch IBD Biobank aims to facilitate the discovery of predictors for individual disease course and treatment response in patients with inflammatory bowel disease (IBD). In this paper, we aim to describe the establishment of the Dutch IBD Biobank, including the facilitators and barriers to establishment. Moreover, we aim to provide a complete overview of the content of the Dutch IBD Biobank. PARTICIPANTS: Since 2007, every patient with IBD treated in one of the eight Dutch university medical centres is asked to participate in the Dutch IBD Biobank in which 225 standardised IBD-related data items and biomaterials, such as serum, DNA, biopsies and a stool sample, are collected. FINDINGS TO DATE: As of June 2014, the Dutch IBD Biobank had enrolled 3388 patients with IBD: 2118 Crohn's disease (62.5%), 1190 ulcerative colitis (35.1%), 74 IBD-unclassified (2.2%) and 6 IBD-indeterminate (0.2%). The inclusion of patients with IBD is ongoing. The quality of the biomaterials is good and serum, DNA and biopsies have been used in newly published studies. FUTURE PLANS: The genotyping (750 000 genetic variants) of all participants of the Dutch IBD Biobank is currently ongoing, enabling more genetic research. In addition, all participants will start reporting disease activity and outcome measures using an online platform and mobile app.

Back/joint pain, illness perceptions and coping are important predictors of quality of life and work productivity in patients with inflammatory bowel disease: a 12-month longitudinal study.

BACKGROUND AND AIMS: Back and joint pain are the most common extraintestinal symptoms reported by patients with inflammatory bowel disease (IBD). We assessed the impact of back/joint pain, illness perceptions, and coping on quality of life (QOL) and work productivity in patients with IBD. METHODS: Our cohort included 155 IBD patients with and 100 without arthropathy. Arthropathy was defined as daily back pain for ≥3 months and/or peripheral joint pain and/or joint swelling over the last year. At baseline and at 12 months, patients completed questionnaires on the extent of back/joint pain, IBD disease activity, illness perceptions, coping, QOL, and work productivity. The impact of back/joint pain, illness perceptions and coping on QOL and work productivity was determined, using linear mixed models. RESULTS: In total, 204 IBD patients (72% Crohn's disease, 40% male, mean age 44 ± 14 years) completed questionnaires at both time points. At both time points, IBD patients with back/joint pain reported a significantly lower QOL and work productivity compared with IBD patients without back/joint pain. Predictors of low QOL were back/joint pain (ß = -1.04, 95% confidence interval [CI] -1.40, -0.68), stronger beliefs about the illness consequences (ß = -0.39, 95% CI -0.59, -0.18) and emotional impact of IBD (ß = -0.47, 95% CI -0.66, -0.28), and the coping strategy 'decreasing activity' (ß = -0.26, 95% CI -0.48, -0.03). Predictors of work productivity were back/joint pain (ß = 0.22, 95% CI 0.07, 0.37) and illness consequences (ß = 0.14, 95% CI 0.06, 0.22). CONCLUSION: Back/joint pain, illness perceptions, and coping are significant predictors of QOL and work productivity, after controlling for disease activity.

Value-based healthcare in Lynch syndrome.

Lynch syndrome (LS), one of the most frequent forms of hereditary colorectal cancer (CRC), is caused by a defect in one of the mismatch repair (MMR) genes. Carriers of MMR defects have a strongly increased risk of developing CRC and endometrial cancer. Over the last few years, value-based healthcare has been introduced as an approach to the cost-effective delivery of measurable patient value over complete cycles of care. This requires all involved stakeholders to formulate and validate 'patient value' for Lynch syndrome, as well as to identify targets and associated costs. The aim of this study was to develop a value-based care model for Lynch syndrome that can determine patient value and associated costs, and to design a coordinated care pathway from existing guidelines. All specialists in our hospital involved in the management of LS patients evaluated the care delivered to these patients at their department and formulated outcome measures relevant to patient value. Patients were then invited to complete a questionnaire that assessed the importance of these measures on a scale of 1-10. Six high-value outcomes were identified: (1) prevention of cancer or detection of early stage cancer (2) rapid results from MMR gene mutation testing (3) rapid investigation of the colon and uterus (4) no/little pain during colonoscopy and gynaecologic examination/biopsy (5) the offer of psychological help and (6) registration with the Dutch Lynch syndrome registry. A total of 38 (59 %) out of 62 patients completed the questionnaire. The relevance of all outcomes was confirmed by the patients and mean scores varied from 7.2 to 9.9. Patients underscored the relevance of both proper patient education and the efficiency of surveillance during their care cycle. Value-based care delivery for Lynch syndrome includes the implementation of six parameters related to prevention and early detection of cancer, a short cycle time and registration to ensure continuation of care. Estimated costs are 3320 for the first cycle of care ( 3550 including gynaecologic surveillance) and approximately 720 per subsequent annual cycle ( 950 including gynaecologic surveillance).

A randomised phase I study of etrolizumab (rhuMAb ß7) in moderate to severe ulcerative colitis.

OBJECTIVE: Etrolizumab (rhuMAb ß7, anti-ß7, PRO145223) is a humanised monoclonal antibody targeting the ß7 subunit of the heterodimeric integrins α4ß7 and αEß7, which are implicated in leucocyte migration and retention in ulcerative colitis (UC). This randomised phase I study evaluated the safety and pharmacology of etrolizumab in patients with moderate to severe UC. DESIGN: In the single ascending dose (SAD) stage, etrolizumab (0.3, 1.0, 3.0, 10 mg/kg intravenous, 3.0 mg/kg subcutaneous (SC) or placebo) was administered 4:1 (n=25) in each cohort. In the multiple dose (MD) stage, new patients received monthly etrolizumab (0.5 mg/kg SC (n=4), 1.5 mg/kg SC (n=5), 3.0 mg/kg SC (n=4), 4.0 mg/kg intravenous (n=5)) or placebo (n=5). The pharmacokinetics was studied and Mayo Clinic Score evaluated at baseline, day 29 (SAD), and days 43 and 71 (MD). RESULTS: In the SAD stage, there were no dose limiting toxicities, infusion or injection site reactions. Two impaired wound healing serious adverse events occurred in two patients receiving etrolizumab. In the MD stage, there were no dose limiting toxicities, and no infusion or injection site reactions. Headache was the most common adverse event, occurring more often in etrolizumab patients. Antietrolizumab antibodies were detected in two subjects. The duration of ß7 receptor full occupancy was dose related. A clinical response was observed in 12/18 patients, and clinical remission in 3/18 patients treated with etrolizumab in the MD stage, compared with 4/5 and 1/5 placebo patients, respectively. CONCLUSION: Etrolizumab is well tolerated in moderate to severe UC. Further investigation is warranted.

Hedgehog signalling stimulates precursor cell accumulation and impairs epithelial maturation in the murine oesophagus.

OBJECTIVE: In the intestine Hedgehog (Hh) signalling is directed from epithelium to mesenchyme and negatively regulates epithelial precursor cell fate. The role of Hh signalling in the oesophagus has not been studied in vivo. Here the authors examined the role of Hh signalling in epithelial homeostasis of oesophagus. DESIGN: The authors used transgenic mice in which the Hh receptor Patched1 (Ptch1) could be conditionally inactivated in a body-wide manner and mice in which Gli1 could be induced specifically in the epithelium of the skin and oesophagus. Effects on epithelial homeostasis of the oesophagus were examined using immunohistochemistry, in situ hybridisation, transmission electron microscopy and real-time PCR. Hh signalling was examined in patients with oesophageal squamous cell carcinoma (SCC) by quantitative real-time PCR. RESULTS: Sonic Hh is signalled in an autocrine manner in the basal layer of the oesophagus. Activation of Hh signalling resulted in an expansion of the epithelial precursor cell compartment and failure of epithelial maturation and migration. Levels of Hh targets GLI1, HHIP and PTCH1 were increased in SCC compared with normal tissue from the same patients. CONCLUSION: Here the authors find that Hh signalling positively regulates the precursor cell compartment in the oesophageal epithelium in an autocrine manner. Since Hh signalling targets precursor cells in the oesophageal epithelium and signalling is increased in SCCs, Hh signalling may be involved in oesophageal SCC formation.

Farnesoid X receptor (FXR) activation and FXR genetic variation in inflammatory bowel disease.

BACKGROUND: We previously showed that activation of the bile salt nuclear receptor Farnesoid X Receptor (FXR) protects against intestinal inflammation in mice. Reciprocally, these inflammatory mediators may decrease FXR activation. We investigated whether FXR activation is repressed in the ileum and colon of inflammatory bowel disease (IBD) patients in remission. Additionally, we evaluated whether genetic variation in FXR is associated with IBD. METHODS: mRNA expression of FXR and FXR target gene SHP was determined in ileal and colonic biopsies of patients with Crohn's colitis (n = 15) and ulcerative colitis (UC; n = 12), all in clinical remission, and healthy controls (n = 17). Seven common tagging SNPs and two functional SNPs in FXR were genotyped in 2355 Dutch IBD patients (1162 Crohn's disease (CD) and 1193 UC) and in 853 healthy controls. RESULTS: mRNA expression of SHP in the ileum is reduced in patients with Crohn's colitis but not in patients with UC compared to controls. mRNA expression of villus marker Villin was correlated with FXR and SHP in healthy controls, a correlation that was weaker in UC patients and absent in CD patients. None of the SNPs was associated with IBD, UC or CD, nor with clinical subgroups of CD. CONCLUSIONS: FXR activation in the ileum is decreased in patients with Crohn's colitis. This may be secondary to altered enterohepatic circulation of bile salts or transrepression by inflammatory signals but does not seem to be caused by the studied SNPs in FXR. Increasing FXR activity by synthetic FXR agonists may have benefit in CD patients.

A meta-analysis of genome-wide association scans identifies IL18RAP, PTPN2, TAGAP, and PUS10 as shared risk loci for Crohn's disease and celiac disease.

Crohn's disease (CD) and celiac disease (CelD) are chronic intestinal inflammatory diseases, involving genetic and environmental factors in their pathogenesis. The two diseases can co-occur within families, and studies suggest that CelD patients have a higher risk to develop CD than the general population. These observations suggest that CD and CelD may share common genetic risk loci. Two such shared loci, IL18RAP and PTPN2, have already been identified independently in these two diseases. The aim of our study was to explicitly identify shared risk loci for these diseases by combining results from genome-wide association study (GWAS) datasets of CD and CelD. Specifically, GWAS results from CelD (768 cases, 1,422 controls) and CD (3,230 cases, 4,829 controls) were combined in a meta-analysis. Nine independent regions had nominal association p-value <1.0 x 10⁻5 in this meta-analysis and showed evidence of association to the individual diseases in the original scans (p-value < 1 x 10⁻² in CelD and < 1 x 10⁻³ in CD). These include the two previously reported shared loci, IL18RAP and PTPN2, with p-values of 3.37 x 10⁻8 and 6.39 x 10⁻9, respectively, in the meta-analysis. The other seven had not been reported as shared loci and thus were tested in additional CelD (3,149 cases and 4,714 controls) and CD (1,835 cases and 1,669 controls) cohorts. Two of these loci, TAGAP and PUS10, showed significant evidence of replication (Bonferroni corrected p-values <0.0071) in the combined CelD and CD replication cohorts and were firmly established as shared risk loci of genome-wide significance, with overall combined p-values of 1.55 x 10⁻¹° and 1.38 x 10⁻¹¹ respectively. Through a meta-analysis of GWAS data from CD and CelD, we have identified four shared risk loci: PTPN2, IL18RAP, TAGAP, and PUS10. The combined analysis of the two datasets provided the power, lacking in the individual GWAS for single diseases, to detect shared loci with a relatively small effect.

Misclassification of dysplasia in patients with inflammatory bowel disease: consequences for progression rates to advanced neoplasia.

BACKGROUND: The natural behavior of flat low-grade (LGD) and indefinite dysplasia (IND) in patients with inflammatory bowel disease (IBD) remains uncertain and seems to be dependent on the interpretation of the pathologist. We studied the progression rate of flat LGD and IND to advanced neoplasia (high-grade dysplasia [HGD] or colorectal cancer [CRC]) before and after histopathological review by a panel of gastrointestinal expert pathologists. METHODS: A nationwide pathology database was used to identify IBD patients with dysplasia in six Dutch university medical centers between 1990 and 2006. Medical charts of patients with recorded flat LGD or IND were reviewed. Histological slides from three university medical centers were reviewed by a panel of three expert gastrointestinal pathologists. RESULTS: We identified 113 flat LGD patients and 26 flat IND patients. Advanced neoplasia was found in 18 flat LGD patients (16%) after a median follow-up of 48 months, resulting in a 5-year progression rate of 12%. Five IND patients (19%) developed advanced neoplasia after a median follow-up of 24 months, resulting in a 5-year progression rate of 21%. Review of 1547 histological slides from 87 patients resulted in an increase of the 5-year progression rate of flat LGD to advanced neoplasia to 37%, whereas the progression rate of IND decreased to 5%. CONCLUSIONS: A diagnosis of flat LGD that is confirmed by a panel of expert gastrointestinal pathologists is associated with a substantial risk of progression to advanced neoplasia, while confirmed IND is associated with a low risk of progression.

The London Position Statement of the World Congress of Gastroenterology on Biological Therapy for IBD with the European Crohn's and Colitis Organization: when to start, when to stop, which drug to choose, and how to predict response?

The advent of biological therapy has revolutionized inflammatory bowel disease (IBD) care. Nonetheless, not all patients require biological therapy. Selection of patients depends on clinical characteristics, previous response to other medical therapy, and comorbid conditions. Availability, reimbursement guidelines, and patient preferences guide the choice of first-line biological therapy for luminal Crohn's disease (CD). Infliximab (IFX) has the most extensive clinical trial data, but other biological agents (adalimumab (ADA), certolizumab pegol (CZP), and natalizumab (NAT)) appear to have similar benefits in CD. Steroid-refractory, steroid-dependent, or complex fistulizing CD are indications for starting biological therapy, after surgical drainage of any sepsis. For fistulizing CD, the efficacy of IFX for inducing fistula closure is best documented. Unique risks of NAT account for its labeling as a second-line biological agent in some countries. Patients who respond to induction therapy benefit from systematic re-treatment. The combination of IFX with azathioprine is better than monotherapy for induction of remission and mucosal healing up to 1 year in patients who are naïve to both agents. Whether this applies to other agents remains unknown. IFX is also effective for treatment-refractory, moderate, or severely active ulcerative colitis. Patients who have a diminished or loss of response to anti-tumor necrosis factor (TNF) therapy may respond to dose adjustment of the same agent or switching to another agent. Careful consideration should be given to the reasons for loss of response. There are insufficient data to make recommendations on when to stop anti-TNF therapy. Preliminary evidence suggests that a substantial proportion of patients in clinical remission for >1 year, without signs of active inflammation can remain in remission after stopping treatment.

[Guideline 'Diagnosis and treatment of inflammatory bowel disease in adults'. I. Diagnosis and treatment]. / Richtlijn 'Diagnostiek en behandeling van inflammatoire darmziekten bij volwassenen'. I. Diagnostiek en behandeling.

The Dutch national practice guideline 'Diagnosis and treatment of inflammatory bowel diseases (IBD) in adults' describes the multidisciplinary approach for adult patients with (suspected) IBD, recommended following analysis of the literature according to the principles of evidence based guideline development. The symptoms on first presentation of a patient with IBD are mainly connected with the localisation and severity of the disease and less with the resulting diagnosis 'Crohn's disease' or 'ulcerative colitis'. There is no test by which the diseases can be distinguished with certainty. Clinical course, ileocolonoscopy and histopathological investigation following biopsy form the 'gold standard' for diagnosis of IBD. The final diagnostic step is disease assessment according to the Montreal classification in order to enable unambiguous communication with medical professionals. The first aim of treatment is to treat and stabilise active disease (induction therapy); at the same time maintenance therapy is initiated. A step-up approach is recommended for both treatment aims. Surgical intervention is indicated if the medical treatment is ineffective, in case of intractable gastrointestinal bleeding, in clinically significant gastrointestinal stenosis due to fibrotic scar tissue, or if complications of the inflammation occur such as abscess, peritonitis, or complicated fistula formation. Nutrition and diet do not play a primary therapeutic role in treatment of adult patients with IBD. However, supportive nutritional care is warranted. Probiotics have a demonstrable effect in preventing pouchitis, but not in the treatment of IBD. Alternative medicine has no role to play in the treatment of IBD. The risk of developing colorectal carcinoma is slightly elevated in IBD patients. Therefore, endoscopic surveillance strategies, aimed at early detection of dysplasia, is indicated according to a schedule in which the frequency increases according to the time elapsed since first clinical signs of IBD.

Loss of Indian Hedgehog activates multiple aspects of a wound healing response in the mouse intestine.

BACKGROUND & AIMS: Indian Hedgehog (Ihh) is expressed by the differentiated epithelial cells of the small intestine and signals to the mesenchyme where it induces unidentified factors that negatively regulate intestinal epithelial precursor cell fate. Recently, genetic variants in the Hh pathway have been linked to the development of inflammatory bowel disease. METHODS: We deleted Ihh from the small intestinal epithelium in adult mice using Cyp1a1-CreIhh(fl/fl) conditional Ihh mutant mice. Intestines were examined by immunohistochemistry, in situ hybridization, and real-time polymerase chain reaction. RESULTS: Deletion of Ihh from the intestinal epithelium initially resulted in a proliferative response of the intestinal epithelium with lengthening and fissioning of crypts and increased Wnt signaling. The epithelial proliferative response was associated with loss of bone morphogenetic protein and Activin signaling from the epithelium of the villus and crypts, respectively. At the same stage we observed a substantial influx of fibroblasts and macrophages into the villus core with increased mesenchymal transforming growth factor-ß signaling and deposition of extracellular matrix proteins. Prolonged loss of Ihh resulted in progressive leukocyte infiltration of the crypt area, blunting and loss of villi, and the development of intestinal fibrosis. CONCLUSIONS: Loss of Ihh initiates several events that are characteristic of an intestinal wound repair response. Prolonged loss resulted in progressive inflammation, mucosal damage, and the development of intestinal fibrosis. Ihh is a signal derived from the superficial epithelial cells that may act as a critical indicator of epithelial integrity.

Genome-wide association identifies multiple ulcerative colitis susceptibility loci.

Ulcerative colitis is a chronic, relapsing inflammatory condition of the gastrointestinal tract with a complex genetic and environmental etiology. In an effort to identify genetic variation underlying ulcerative colitis risk, we present two distinct genome-wide association studies of ulcerative colitis and their joint analysis with a previously published scan, comprising, in aggregate, 2,693 individuals with ulcerative colitis and 6,791 control subjects. Fifty-nine SNPs from 14 independent loci attained an association significance of P < 10(-5). Seven of these loci exceeded genome-wide significance (P < 5 x 10(-8)). After testing an independent cohort of 2,009 cases of ulcerative colitis and 1,580 controls, we identified 13 loci that were significantly associated with ulcerative colitis (P < 5 x 10(-8)), including the immunoglobulin receptor gene FCGR2A, 5p15, 2p16 and ORMDL3 (orosomucoid1-like 3). We confirmed association with 14 previously identified ulcerative colitis susceptibility loci, and an analysis of acknowledged Crohn's disease loci showed that roughly half of the known Crohn's disease associations are shared with ulcerative colitis. These data implicate approximately 30 loci in ulcerative colitis, thereby providing insight into disease pathogenesis.

The absence of functional PI3Kgamma prevents leukocyte recruitment and ameliorates DSS-induced colitis in mice.

Phosphatidylinositol-3-kinase gamma (PI3Kgamma) is the major PI3K that is activated in response to chemoattractants. It is responsible for the migration of leukocytes from the bloodstream to sites of injury or infection. Constant migration of new leukocytes to the intestinal mucosa may be an important factor in maintenance of inflammation and tissue damage in inflammatory bowel disease (IBD). Reducing this influx, for example by inhibition of PI3Kgamma, might therefore be a potential goal for therapy. Here we investigated the role of PI3Kgamma in the migration of leukocytes to sites of intestinal inflammation. We induced colitis in mice with a point mutation that inactivates PI3Kgamma enzymatic activity ('kinase-dead') by oral administration of dextran sodium sulphate (DSS). Mice were treated with 1.5% DSS for 1 week and effects on cytokine production, leukocyte recruitment and disease severity were examined. Both clinical and histological parameters showed that the severity of colitis was significantly reduced in PI3Kgamma-kinase-dead mice compared to controls. Although mutant mice had a less severe colitis than controls they produced significantly more pro-inflammatory Th1 cytokines such as Il-12, Tnfalpha and Ifngamma and more Il-10. PI3Kgamma mutant mice showed increased numbers of resident macrophages and T cells in the colonic lamina propria in an unstressed condition but failed to recruit new leukocytes to the mucosa upon treatment with DSS despite the increased cytokine levels. These results suggest that PI3Kgamma plays a critical role in lamina propria leukocyte trafficking and that loss of PI3Kgamma-activity ameliorates DSS-induced colitis in mice.

Prospective randomized open-label multicenter phase I/II dose escalation trial of visilizumab (HuM291) in severe steroid-refractory ulcerative colitis.

BACKGROUND: Visilizumab is a humanized IgG(2) monoclonal anti-CD3 antibody. We evaluated its safety and dose response in severe intravenous steroid-refractory ulcerative colitis (UC). METHODS: In all, 104 patients were treated. In Stage I, 73 patients were randomly assigned to receive intravenous visilizumab 5, 7.5, 10, or 12.5 microg/kg/day for 2 consecutive days. In Stage II, 33 patients received visilizumab at the optimal clinical dose (OCD) of 5 microg/kg/day for 2 days. Symptomatic response and remission were defined by the modified Truelove-Witts severity index. Clinical response and remission were defined by the Mayo score. RESULTS: The rates of symptomatic response at day 15 in the 5, 7.5, 10, or 12.5 microg/kg dose groups were 71%, 70%, 50%, and 61%, respectively, in Stage I and in 54% in Stage II. The symptomatic remission rates were 35%, 5%, 22%, and 11% in Stage I and 18% in Stage II. The rates of clinical response at day 30 in the 5, 7.5, 10, or 12.5 microg/kg dose groups were 71%, 65%, 50%, and 67%, respectively, in Stage I and 55% in Stage II. The clinical remission rates were 6%, 5%, 0%, and 11% in Stage I and 6% in Stage II. All patients experienced adverse events. Serious adverse events included abdominal abscess, cytomegalovirus infection, atrial fibrillation, herpes zoster, and esophageal candidiasis. CONCLUSIONS: Treatment with visilizumab induced symptomatic response and clinical response. Results with 5 microg/kg/day were similar to those observed with higher doses.

5-Aminosalicylic acid inhibits TGF-beta1 signalling in colorectal cancer cells.

The transforming growth factor-beta (TGF-beta) pathway is an important pathway in the initiation and progression of colorectal cancer. We aimed to determine the effects of 5-aminosalicylic acid (5-ASA) on TGF-beta signalling in colorectal cancer cells in vitro. 5-ASA inhibited TGF-beta1 signalling in HCT116 cells and colonic fibroblasts, as judged by a TGF-beta-specific reporter gene assay, plasminogen activator inhibitor-1 mRNA and protein levels, fibroblast trans-differentiation, Smad3 phosphorylation and nuclear translocation. We conclude that 5-ASA inhibits TGF-beta1 signalling in colorectal cancer cells, and might be a potent adjuvant therapeutic drug, interfering with aberrant TGF-beta signalling in colorectal cancer.

Significant differences between Crohn's disease and ulcerative colitis regarding the impact of body mass index and initial disease activity on responsiveness to azathioprine: results from a European multicenter study in 1,176 patients.

In a survey comprising 1,176 patients with inflammatory bowel disease (IBD) we recently showed that azathioprine (AZA) beyond 4 years is beneficial in ulcerative colitis (UC) patients and in a subset of Crohn's disease (CD) patients. Here, we show for the first time that azathioprine responsiveness depends on body mass index (BMI). The relationship is reciprocal in UC and CD, with a better outcome in UC patients with a BMI<25 and in CD patients with a BMI>25. These observations are particularly interesting considering the evolving concept of a relationship between fatty metabolism and immune regulation. Additionally, we show that CD patients, but not UC patients, respond better to AZA when it is started in clinical remission. This observation may support data favouring a "hit hard and early" regime in CD. Finally, we were able to demonstrate a decrease in the incidence of CD-related complications requiring surgery through treatment with AZA.

Comparison of peptide array substrate phosphorylation of c-Raf and mitogen activated protein kinase kinase kinase 8.

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.

Regulation of p110delta PI 3-kinase gene expression.

BACKGROUND: Despite an intense interest in the biological functions of the phosphoinositide 3-kinase (PI3K) signalling enzymes, little is known about the regulation of PI3K gene expression. This also applies to the leukocyte-enriched p110delta catalytic subunit of PI3K, an enzyme that has attracted widespread interest because of its role in immunity and allergy. PRINCIPAL FINDINGS: We show that p110delta expression is mainly regulated at the transcriptional level. In fibroblasts, lymphocytes and myeloid cells, p110delta gene transcription appears to be constitutive and not subject to acute stimulation. 5'RACE experiments revealed that p110delta mRNA transcripts contain distinct upstream untranslated exons (named exon -1, -2a, -2b, -2c and -2d), which are located up to 81 kb upstream of the translational start codon in exon 1. The levels of all the different p110delta transcripts are higher in leukocytes compared to non-leukocytes, with the p110delta transcript containing exon -2a most abundantly expressed. We have identified a highly conserved transcription factor (TF) binding cluster in the p110delta gene which has enhanced promoter activity in leukocytes compared to non-leukocytes. In human, this TF cluster is located immediately upstream of exon -2a whilst in mouse, it is located within exon -2a. CONCLUSION: This study identifies a conserved PIK3CD promoter region that may account for the predominant leukocyte expression of p110delta.

Depletion of the colonic epithelial precursor cell compartment upon conditional activation of the hedgehog pathway.

BACKGROUND & AIMS: The intestinal epithelium is a homeostatic system in which differentiated cells are in dynamic equilibrium with rapidly cycling precursor cells. Wnt signaling regulates intestinal epithelial precursor cell fate and proliferation. Homeostatic systems exist by virtue of negative feedback loops, and we have previously identified the Hedgehog (Hh) pathway as a potential negative feedback signal in the colonic epithelium. Indian hedgehog (Ihh) is produced by the differentiated enterocytes and negatively regulates Wnt signaling in intestinal precursor cells. We studied the role of members of the Hh signaling family in the intestine using a conditional genetic approach. METHODS: We inactivated the Hh receptor Patched1 (Ptch1) in adult mice, resulting in constitutive activation of the Hh signaling pathway. Effects on colonic mucosal homeostasis were examined. Colon tissues were examined by immunohistochemistry, in situ hybridization, transmission electron microscopy, and real-time polymerase chain reaction. RESULTS: Ihh but not Sonic hedgehog (Shh) was expressed in colonic epithelium. Expression of Ptch1 and Gli1 was restricted to the mesenchyme. Constitutive activation of Hh signaling resulted in accumulation of myofibroblasts and colonic crypt hypoplasia. A reduction in the number of epithelial precursor cells was observed with premature development into the enterocyte lineage and inhibition of Wnt signaling. Activation of Hh signaling resulted in induction of the expression of bone morphogenetic proteins (Bmp) and increased Bmp signaling in the epithelium. CONCLUSIONS: Hh signaling acts in a negative feedback loop from differentiated cells via the mesenchyme to the colonic epithelial precursor cell compartment in the adult mouse.