Mikael Lördal

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 × 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 × 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 Crohns disease loci showed that roughly half of the known Crohns disease associations are shared with ulcerative colitis. These data implicate approximately 30 loci in ulcerative colitis, thereby providing insight into disease pathogenesis.

A novel tachykinin NK2 receptor antagonist prevents motility-stimulating effects of neurokinin A in small intestine

MEN 11420 (nepadutant) is a potent, selective and competitive antagonist of tachykinin NK2 receptors. The objective of the present study was to assess the capability of the drug to antagonize the stimulatory effects of neurokinin A (NKA) on gastrointestinal motility, as well as to change the fasting migrating motor complex (MMC). Thirty‐four male volunteers were randomized to treatment with either placebo or MEN 11420 in a double‐blinded manner. Effects of MEN 11420 (8u2003mg intravenously) were evaluated as changes in phases I, II and III of MMC, as well as contraction frequency, amplitude and motility index during baseline conditions and during stimulation of motility using NKA (25u2003pmolu2003kg−1u2003min−1 intravenously). NKA preceded by placebo increased the fraction of time occupied by phase II, increased contraction frequency, amplitude and motility index. MEN 11420 effectively antagonized the motility‐stimulating effects of NKA. MEN 11420 reduced the phase II‐stimulating effect of NKA. In addition, the stimulatory effect of NKA on contraction frequency and amplitude, as well as motility index were inhibited by MEN 11420. MEN 11420 did not affect the characteristics of MMC during saline infusion. Plasma levels of MEN 11420 peaked during the first hour after infusion and decreased to less than half during the first 2u2003h. In conclusion, intravenous MEN 11420 effectively inhibited NKA‐stimulated, but not basal gastrointestinal motility, and was well tolerated by all subjects.

Tachykinins influence interdigestive rhythm and contractile strength of human small intestine

The effect of the putative entericneurotransmitters neurokinin A and substance P wereinvestigated on human small intestinal motility. Eitherneurokinin A, at doses of 6-25 pmol/kg/min, or substanceP at doses of 1- 6 pmol/kg/min were administeredintravenously to healthy volunteers over 4 hr.Neurokinin A dose-dependently increased the fraction ofphase II of the migrating motor complex, contraction frequency, motility index, and amplitude ofcontractions. At the highest dose, neurokinin A induceda phase II-like pattern, disrupting the migratingmyoelectric complex. Substance P dose-dependentlyincreased phase II of the migrating motor complex. Thecontraction frequency increased slightly at the highestdose, but neither motility index nor contractionamplitude changed. It is concluded that neurokinin A and substance P stimulate small intestinalmotility in man, and it can be speculated that they playa role in the control of human small intestinalmotility.

Multiple Polymorphisms Affect Expression and Function of the Neuropeptide S Receptor (NPSR1)

Background neuropeptide S (NPS) and its receptor NPSR1 act along the hypothalamic-pituitary-adrenal axis to modulate anxiety, fear responses, nociception and inflammation. The importance of the NPS-NPSR1 signaling pathway is highlighted by the observation that, in humans, NPSR1 polymorphism associates with asthma, inflammatory bowel disease, rheumatoid arthritis, panic disorders, and intermediate phenotypes of functional gastrointestinal disorders. Because of the genetic complexity at the NPSR1 locus, however, true causative variations remain to be identified, together with their specific effects on receptor expression or function. To gain insight into the mechanisms leading to NPSR1 disease-predisposing effects, we performed a thorough functional characterization of all NPSR1 promoter and coding SNPs commonly occurring in Caucasians (minor allele frequency >0.02). Principal Findings we identified one promoter SNP (rs2530547 [−103]) that significantly affects luciferase expression in gene reporter assays and NPSR1 mRNA levels in human leukocytes. We also detected quantitative differences in NPS-induced genome-wide transcriptional profiles and CRE-dependent luciferase activities associated with three NPSR1 non-synonymous SNPs (rs324981 [Ile107Asn], rs34705969 [Cys197Phe], rs727162 [Arg241Ser]), with a coding variant exhibiting a loss-of-function phenotype (197Phe). Potential mechanistic explanations were sought with molecular modelling and bioinformatics, and a pilot study of 2230 IBD cases and controls provided initial support to the hypothesis that different cis-combinations of these functional SNPs variably affect disease risk. Significance these findings represent a first step to decipher NPSR1 locus complexity and its impact on several human conditions NPS antagonists have been recently described, and our results are of potential pharmacogenetic relevance.

Analysis of 39 Crohn's disease risk loci in Swedish inflammatory bowel disease patients

To the Editor: Whether Crohn’s disease (CD) and ulcerative colitis (UC) represent distinct disorders or comprise a continuum is a key question in inflammatory bowel disease (IBD). Recently, genome-wide association studies (GWAS) have identified several susceptibility loci, particularly for CD. This progress has enabled attempts to define the molecular similarity of IBD subtypes by specifically testing CD loci in UC. Here we extend this approach by genotyping a Swedish sample of 736 CD patients (age 49.4 15.9 SD, 50.9% males), 935 UC patients (age 51.6 13.1 SD, 54.2% males) and 1460 common controls (age 49.8 15.6 SD, 35.6% males) for 39 of 40 loci nominally associated with CD in a recent GWAS meta-analysis. IBD patients were recruited at the Karolinska University Hospital, Stockholm, and in other participating centers across Sweden. Diagnosis of IBD (CD or UC) was based on standard clinical, endoscopic, radiologic, and histologic criteria. Control individuals were Swedish healthy blood donors, and subjects free of inflammatory disease enrolled in the Swedish EIRA study of rheumatoid arthritis. Informed consent was obtained from all participants and local ethics committees approved the study. One single nucleotide polymorphism (SNP) was selected at each CD locus based on previous associations and genotyped with iPlex chemistry (www.sequenom.com). The average genotyping success rate was 96.3%, and no marker deviated significantly (P < 0.001) from Hardy–Weinberg equilibrium in controls. SNPs were tested for association with CD and UC by trend tests implemented in PLINK (pngu.mgh.harvard.edu/ purcell/plink), with >80% power to detect odds ratios >1.3 for 90% of the markers (risk allele frequency >0.10). A Bonferroni correction for the number of tested loci (39) was applied, which set P < 0.0013 as the level of significance in our experiment (Table 1). With the exception of IL23R and NOD2, few of the CD loci studied here have been investigated in Swedish CD patients. We replicate associations withstanding correction for multiple testing for SNPs in or near IL23R, IRGM, ZNF365, LRRK2, and C13orf31. Loci showing best nominal significance (P < 0.05) include regions harboring TNFSF18, IL18RAP, JAK2, CUL2, and NKX2-3, whereas other loci consistently replicated across different populations appeared only of marginal (ATG16L1, NOD2, and TNFSF15) or no relevance (C5orf56 and PTPN2) in our sample. In particular, the minor role of NOD2 in Swedish CD has been reported previously, and stems from the low frequency of its risk variants in Scandinavia. Three CD loci were associated with UC in our study after correction for multiple testing: those containing IL23R, MST1, and GSDMB. Interestingly, the latter 2 appeared to be relevant primarily to UC in our sample, an observation that diverges from previous reports in UK patients, where MST1 was found to be associated with both CD and UC, while the region harboring GSDMB did not show evidence of association with UC. One locus, CUL2, showed P-values nearing the Bonferroni significance level, and had similar effects on both CD and UC susceptibility. Finally, although type 2 errors cannot be excluded, another 8 CD loci also recently implicated in UC in different studies only showed nominal significance (C1orf81, BTNL2, JAK2) or were not associated in our sample (ILRAP18, IL12B, LYRM4, CDKAL1, STAT3), despite Swedish control frequencies similar to those of other European populations. Our findings are only partially concordant with previous results, and highlight the importance of assessing the relevance of genetic risk variants in different populations, even within Europe. Several GWA screens have been performed, and others are imminent. These rapidly increasing data promise to more conclusively define the genetic architecture of IBD, and the magnitude of the effects that specific risk alleles have in its various forms, and in different populations.

Nitric oxide pathway-related gene alterations in inflammatory bowel disease

Abstract Objective. To reveal specific gene activation in nitric oxide (NO)-related inflammation we studied differential gene expression in inflammatory bowel disease (IBD). Methods. Total RNA was isolated from 20 biopsies of inflamed mucosa from Crohns disease (CD) and ulcerative colitis (UC) patients each as well as from six controls, labeled with 32P-dCTP and hybridized to a human NO gene array. Significant genes were analyzed for functional gene interactions and heatmaps generated by hierarchical clustering. A selection of differentially expressed genes was further evaluated with immunohistochemical staining. Results. Significant gene expression differences were found for 19 genes in CD and 23 genes in UC compared to controls, both diseases with high expression of ICAM1 and IL-8. Correlation between microarray expression and corresponding protein expression was significant (r = 0.47, p = 0.002). Clustering analysis together with functional gene interaction analysis revealed clusters of coregulation and coexpression in CD and UC: transcripts involved in angiogenesis, inflammatory response mediated by the transcription factor hypoxia-inducible factor 1, and tissue fibrosis. Also, a fourth cluster with transcripts regulated by the transcription factor Sp1 was found in UC. Conclusions. Expression analysis in CD and UC revealed disease-specific regulation of NO-related genes, which might be involved in perpetuating inflammatory disease activity in IBD.

Neurokinin A increases duodenal mucosal permeability, bicarbonate secretion, and fluid output in the rat

The aim of this study was to examine the integrative response to neurokinin A (NKA) on duodenal mucosal permeability, bicarbonate secretion, fluid flux, and motility in an in situ perfusion model in anesthetized rats. Intravenous infusion of NKA (100, 200, and 400 pmol.kg-1.min-1) induced duodenal motility. Furthermore, duodenal mucosal bicarbonate secretion, fluid output, and mucosal permeability increased in response to NKA. Pretreatment with the nicotinic antagonist hexamethonium did not change the response in any of the parameters investigated, whereas the NK2-receptor antagonist MEN 10,627 effectively inhibited all responses to NKA. Indomethacin induced duodenal motility and stimulated bicarbonate secretion. In indomethacin-treated rats, NKA further increased motility but decreased indomethacin-stimulated bicarbonate secretion by 70%. The NKA-induced increase in mucosal permeability was unaltered by indomethacin. It is concluded that NKA not only induces motility but also increases mucosal permeability and fluid output. Furthermore, the neuropeptide may have both stimulative and inhibitory effects on bicarbonate secretion. All responses to NKA are dependent on NK-2 receptor activation but are not mediated through nicotinic receptors.The aim of this study was to examine the integrative response to neurokinin A (NKA) on duodenal mucosal permeability, bicarbonate secretion, fluid flux, and motility in an in situ perfusion model in anesthetized rats. Intravenous infusion of NKA (100, 200, and 400 pmol ⋅ kg-1 ⋅ min-1) induced duodenal motility. Furthermore, duodenal mucosal bicarbonate secretion, fluid output, and mucosal permeability increased in response to NKA. Pretreatment with the nicotinic antagonist hexamethonium did not change the response in any of the parameters investigated, whereas the NK2-receptor antagonist MEN 10,627 effectively inhibited all responses to NKA. Indomethacin induced duodenal motility and stimulated bicarbonate secretion. In indomethacin-treated rats, NKA further increased motility but decreased indomethacin-stimulated bicarbonate secretion by 70%. The NKA-induced increase in mucosal permeability was unaltered by indomethacin. It is concluded that NKA not only induces motility but also increases mucosal permeability and fluid output. Furthermore, the neuropeptide may have both stimulative and inhibitory effects on bicarbonate secretion. All responses to NKA are dependent on NK-2 receptor activation but are not mediated through nicotinic receptors.

Dental caries, prevalence and risk factors in patients with Crohn's disease.

Objective The present study tested the hypothesis that patients with Crohn’s disease (CD) have a higher prevalence and risk for caries compared to people without CD. Material and Methods Patients with CD were divided into groups; 71 patients (50.7±13.9 years) who had gone through resective intestinal surgery and 79 patients (42.0±14.4 years) who had not. The patients were compared to 75 controls (48.6±13.4 years) regarding DMF-T and DMF-S, Lactobacilli (LB), Streptococcus mutans (SM), salivary flow and dental plaque. Statistical methods including ANOVA or Chi-square test for calculation of demographic differences between groups, analysis of covariance (ANCOVA) to compare the clinical variable and Post hoc analyses were done with Fischers Least Significant Difference test or Chi-square. Non-parametric Spearman’s correlation matrix coefficient was estimated between clinical variables and disease duration. Results CD patients who had been subjected to resective surgery had a higher DMF-S score (50.7 versus 36.5; pu200a=u200a0.01) compared to the control group after adjusting for age, gender and smoking. These patients had higher counts of SM (1.5 versus 0.9; pu200a=u200a0.04) and LB (10000.0 versus 1000.0; pu200a=u200a0.01), and more dental plaque (53.7 versus 22.6; pu200a=u200a0.001). CD patients reported a more frequent consumption of sweetened drinks between meals compared to controls (pu200a=u200a0.001). Conclusions The present study shows that patients with CD who had undergone resective surgery had a higher DMFs score, and higher salivary counts of Lactobacilli and Streptococcus mutans compared to the control group.

Eosinophil associated genes in the inflammatory bowel disease 4 region: Correlation to inflammatory bowel disease revealed

AIMnTo study the association between inflammatory bowel disease (IBD) and genetic variations in eosinophil protein X (EPX) and eosinophil cationic protein (ECP).nnnMETHODSnDNA was extracted from ethylene diamine tetraacetic acid blood of 587 patients with Crohns disease (CD), 592 with ulcerative colitis (UC) and 300 healthy subjects. The EPX405 (G > C, rs2013109), ECP434 (G > C, rs2073342) and ECP562 (G > C, rs2233860) gene polymorphisms were analysed, by the 5-nuclease allelic discrimination assay. For determination of intracellular content of EPX and ECP in granulocytes, 39 blood samples was collected and extracted with a buffer containing cetyltrimethylammonium bromide. The intracellular content of EPX was analysed using an enzyme-linked immunosorbent assay. The intracellular content of ECP was analysed with the UniCAP(®) system as described by the manufacturer. Statistical tests for calculations of results were χ(2) test, Fishers exact test, ANOVA, Student-Newman-Keuls test, and Kaplan-Meier survival curve with Log-rank test for trend, the probability values of P < 0.05 were considered statistically significant.nnnRESULTSnThe genotype frequency for males with UC and with an age of disease onset of ≥ 45 years (n = 57) was for ECP434 and ECP562, GG = 37%, GC = 60%, CC = 4% and GG = 51%, GC = 49%, CC = 0% respectively. This was significantly different from the healthy subjects genotype frequencies of ECP434 (GG = 57%, GC = 38%, CC = 5%; P = 0.010) and ECP562 (GG = 68%, GC = 29%,CC = 3%; P = 0.009). The genotype frequencies for females, with an age of disease onset of ≥ 45 years with CD (n = 62), was for the ECP434 and ECP562 genotypes GG = 37%, GC = 52%, CC = 11% and GG = 48%, GC = 47% and CC = 5% respectively. This was also statistically different from healthy controls for both ECP434 (P = 0.010) and ECP562 (P = 0.013). The intracellular protein concentration of EPX and ECP was calculated in μg/10(6) eosinophils and then correlated to the EPX 405 genotypes. The protein content of EPX was highest in the patients with the CC genotype of EPX405 (GG = 4.65, GC = 5.93, and CC = 6.57) and for ECP in the patients with the GG genotype of EPX405 (GG = 2.70, GC = 2.47 and CC = 1.90). ANOVA test demonstrated a difference in intracellular protein content for EPX (P = 0.009) and ECP (P = 0.022). The age of disease onset was linked to haplotypes of the EPX405, ECP434 and ECP562 genotypes. Kaplan Maier curve showed a difference between haplotype distributions for the females with CD (P = 0.003). The highest age of disease onset was seen in females with the EPX405CC, ECP434GC, ECP562CC haplotype (34 years) and the lowest in females with the EPX405GC, ECP434GC, ECP562GG haplotype (21 years). For males with UC there was also a difference between the highest and lowest age of the disease onset (EPX405CC, ECP434CC, ECP562CC, mean 24 years vs EPX405GC, ECP434GC, ECP562GG, mean 34 years, P = 0.0009). The relative risk for UC patients with ECP434 or ECP562-GC/CC genotypes to develop dysplasia/cancer was 2.5 (95%CI: 1.2-5.4, P = 0.01) and 2.5 (95%CI: 1.1-5.4, P = 0.02) respectively, compared to patients carrying the GG-genotypes.nnnCONCLUSIONnPolymorphisms of EPX and ECP are associated to IBD in an age and gender dependent manner, suggesting an essential role of eosinophils in the pathophysiology of IBD.

Mediation of irregular spiking activity by multiple neurokinin‐receptors in the small intestine of the rat

We have studied the small intestinal myoelectric response to the natural tachykinins substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and the neurokinin‐receptor selective agonists substance P methyl esther (SPME), [β‐Ala8]neurokinin A 4‐10, and senktide in conscious rats. The effects of the agonists were studied before and after administration of the selective neurokinin 2 (NK2)‐receptor antagonist MEN 10,627. Under basal conditions SP, NKA, NKB, as well as the selective NK1‐receptor agonist SPME, the NK2‐receptor agonist [β‐Ala8]NKA 4–10, and the NK3‐receptor agonist senktide, disrupted the interdigestive rhythm with regularly recycling migrating myoelectric complexes and induced a phase II‐like irregular spiking activity. MEN 10,627 given alone did not affect the interdigestive rhythm. MEN 10,627 inhibited the response to [β‐Ala8]NKA 4–10 but not to SP, SPME, NKA, NKB or senktide. It is concluded that not only NK2 receptors, but also other receptors, such as NK1 and NK3 receptors, may mediate the motility‐stimulating action of different tachykinins in vivo. It is further concluded that MEN 10,627 exerts a selective NK2‐receptor antagonism, and may be a valuable tool for assessing the functional role of NK2‐receptors in gastrointestinal physiology.