Increased numbers of adherent invasive Escherichia coli (AIEC) have been found in Crohn's disease (CD) patients. In this report, we investigate the potential of the probiotic Escherichia coli Nissle 1917 (EcN) to reduce features associated with AIEC pathogenicity in an already established infection with AIEC reference strain LF82.

Background: The era of genome-wide association studies (GWAS) has led to the identification of many inflammatory bowel disease (IBD)-associated single-nucleotide polymorphisms (SNPs) with unknown function. The next step would be to identify the functional consequences of these polymorphisms in order to target them efficiently for therapeutic purposes. One way to study this type of genetic variation is the use of cell line models. However, to characterize the functional effect of a SNP, it is important to know if the selected cell line model itself carries the studied genetic variation. Here. we genotyped 50 IBD markers across 32 susceptibility genes in 9 commonly used gastrointestinal cell lines.Methods: We used Sequenom, TaqMan, and DNA sequencing for the genotyping. To determine the expression profile of the selected genes, we conducted real-time RT-PCR.Results: We found variant SNPs in all analyzed cell lines. Almost every minor allele was carried by at least one of the tested cell lines. We analyzed the effect of 4 SNPs in more detail using quantitative real-time RT-PCR (qRT-PCR) comprising genes ATG16L1, CD14, MDR1, and OCTN2. According to our data, only 2 of the commonly studied SNPs in MDR1 and CD14 have an impact on gene expression.Conclusions: we have identified genotype variants in all analyzed cell lines. Some of them are functional and alter the response to drugs (MDRI) or affect bacterial recognition (TLR4, NOD2). Our results highlight that the genotype should not be neglected in experimental design when using model cell lines.

Inflammatory bowel diseases (IBDs) comprising Crohn disease (CD) and ulcerative colitis (UC) are chronic inflammatory conditions with polygenic susceptibility. Interactions between TNF-alpha and TNF-alpha receptor play a fundamental role in inflammatory response. This study investigates the role that selected single nucleotide polymorphisms (SNPs) and haplotypes in the TNF-alpha receptor (TNSFRSF1B) gene play in the risk of IBD in a New Zealand Caucasian population. DNA samples from 388 CD, 405 UC, 27 indeterminate colitis patients, and 293 randomly selected controls, from Canterbury, New Zealand were screened for 3 common SNPs in TNSFRSF1B: rs1061622 (c.676T > C), rs1061624 (c.*1663A > G), and rs3397 (c.*1690T > C), using TaqMan technologies. Carrying the rs1061624 variant decreased the risk of UC in the left colon (OR 0.73, 95% CI = 0.54-1.00) and of being a smoker at diagnosis (OR 0.62; 95% CI = 0.40-0.96). Carrying the rs3397 variant decreased the risk of penetrating CD (OR 0.62, 95% CI = 0.40-0.95). Three marker haplotype analyses revealed highly significant differences between CD patients and control subjects (chi(2) = 29.9, df = 7, P = .0001) and UC cases and controls (chi(2) = 46.3, df = 7, P < .0001). We conclude that carrying a 3-marker haplotype in the TNSFRSF1B gene may increase (e.g., haplotype of GGC was 2.9-fold more in the CD or UCpatients) or decrease (e.g., TGT was 0.47-fold less in UC patients) the risk of IBD in a New Zealand Caucasian population.

Bacterial sensing is crucial for appropriate response by the innate and adaptive immune system against invading microorganisms. Single nucleotide polymorphisms (SNPs) in genes involved in bacterial recognition, CARD15 and TLR4, increased the risk of inflammatory bowel disease (IBD) in a New Zealand Caucasian case-control cohort. We now consider the effects of SNPs in CD14, TLR9, and BPI, analyzed individually, in association with one another, and with SNPs in CARD15 or TLR4 in this same population group. SNPs in CD14 (c.-159 C>T), TLR9 (c.-1237T>C) and BPI (c.645A>G) showed no significant allele or genotype frequency differences between IBD cases and controls. Genotype-phenotype mapping reveals an association with BPI and ileocolonic Crohn's disease (CD) as well as an association with CD14 and early-onset ulcerative colitis (UC). Genotype interaction analyses using three different statistical approaches provided significant evidence of interaction for the following combinations: CARD15/TLR4 (CD and UC), CARD15/CD14 (CD and UC), CD14/TLR4 (UC only), and CD14/BPI (UC only). A trend for an association between BPI and TLR4 was observed in UC patients, but failed to reach statistical significance. Our findings support the idea of gene-gene interactions for genes involved in closely related pathways (i.e. bacterial detection). There is evidence that carrying two SNPs in genes may lead to statistical significance for genes and SNPs that do not otherwise confirm as risk alleles for disease aetiology when analysed alone.

BACKGROUND:Single nucleotide polymorphisms (SNPs) in the multidrug transporter MDR1 have been associated with inflammatory bowel disease (IBD) in different studies. However, the data are highly controversial. Recently, 6 haplotype tagging SNPs (tSNPs), representing the haplotype variations of the MDR1 gene, were identified. The aims of this study were to genotype these variants and correlate them to disease phenotype in New Zealand IBD patients.MATERIALS AND METHODS:A total of 784 IBD patients and 200 healthy subjects were genotyped for 5 tSNPs and the triallelic MDR1 variant G2677T/A using the Sequenom MassArray platform. Furthermore, the effects of these variants were examined in correlation with phenotypic clinical features.RESULTS:Heterozygous carriers for the variants C1236T, rs2235046 (an SNP in intron 16), and G2677T/A showed a lower risk of developing ulcerative colitis (C1236T: odds ratio [OR] = 0.63, 95% confidence interval [CI] = 0.42-0.93, P = 0.03; G2677T/A: OR = 0.59, CI = 0.39-0.89, P = 0.02; and rs2235046: OR = 0.59, 95% CI = 0.38-0.91, P = 0.009) as compared with homozygotes. None of the analyzed markers were associated with Crohn's disease on a genotypic level. Subgroup analysis revealed an association for 2 variants with IBD when stratified for age of onset (C1236T SNP and rs3789243). The MDR1 variant C3435T was associated with disease behavior in CD (OR = 1.45, 95% CI = 1.01-2.08, P = 0.04), whereas the SNP rs3789243 was found to be associated with pancolitis in UC patients (OR = 1.35, CI = 1.00-1.82, P = 0.05).CONCLUSIONS:The results of our study support the role of MDR1 as a candidate gene for ulcerative colitis. Furthermore, our results suggest the possibility of a heterozygous advantage for certain MDR1 variants for this disease.

Carrying a functional single nucleotide polymorphism (L503F, c. 1672 C>T) in the gene for the Na-dependent organic cation transporter (OCTN1), increases the risk of Crohn's disease (CD) in some, but not all, populations. Case–control data on New Zealand Caucasians show no differences for CD risk between individuals carrying the L503F OCTN1 C-allele when compared with those carrying the variant T-allele. However, more of the New Zealand CD cases report intolerance to maize and mushrooms than those who report beneficial effects or no differences. The OCTN1 gene encodes a transporter for ergothionine, a fungal metabolite at high levels in mushrooms but not widely common in other dietary items. An inability to tolerate mushrooms showed statistically significant associations with the variant OCTN1 genotype. That is, among those individuals reporting adverse effects from mushrooms, there was a higher frequency of the variant T-allele when compared with the general population, or with CD patients overall. We believe that this is a novel gene–diet association, suggesting that individuals carrying the OCTN1 variant single nucleotide polymorphism may have an enhanced risk of adverse symptoms associated with consuming mushrooms. Nutrigenomic approaches to dietary recommendations may be appropriate in this group.

AIM:To investigate the role that single nucleotide polymorphisms (SNPs) in the promoter of the tumour necrosis factor-alpha (TNF-alpha) gene play in the risk of inflammatory bowel diseases (IBDs) in a New Zealand population, in the context of international studies.METHODS:DNA samples from 388 patients with Crohn's disease (CD), 405 ulcerative colitis (UC), 27 indeterminate colitis (IC) and 201 randomly selected controls, from Canterbury, New Zealand were screened for 3 common polymorphisms in the TNF-alpha receptor: -238 G-->A, -308 G-->A and -857C-->T, using a Taqman assay. A meta-analysis was performed on the data obtained on these polymorphisms combined with that from other published studies.RESULTS:Individuals carrying the -308 G/A allele had a significantly (OR = 1.91, c2 = 17.36, P < 0.0001) increased risk of pancolitis, and a 1.57-fold increased risk (OR = 1.57, c2 = 4.34, P = 0.037) of requiring a bowel resection in UC. Carrying the -857 C/T variant decreased the risk of ileocolonic CD (OR = 0.56, c2 = 4.32, P = 0.037), and the need for a bowel resection (OR = 0.59, c2 = 4.85, P = 0.028). The risk of UC was reduced in individuals who were smokers at diagnosis, (OR = 0.48, c2 = 4.86, P = 0.028).CONCLUSION:TNF-alpha is a key cytokine known to play a role in inflammatory response, and the locus for the gene is found in the IBD3 region on chromosome 6p21, known to be associated with an increased risk for IBD. The -308 G/A SNP in the TNF-alpha promoter is functional, and may account in part for the increased UC risk associated with the IBD3 genomic region. The -857 C/T SNP may decrease IBD risk in certain groups. Pharmaco- or nutrigenomic approaches may be desirable for individuals with such affected genotypes.

Background: The IBD5 (MIM# 606348) chromosomal region on 5q31 contains the genes for organic cation transporters, OCTN1 and OCTN2, and has been associated with susceptibility to adult onset CD.While several studies argue that OCTN1 and OCTN2 are the causal genes, others fail to replicate these results.Methods: From the Canterbury Inflammatory Bowel Disease Project, 388 CD, 405 UC, and 27 indeterminate colitis (IC) participants were genotyped for functional SNPs in OCTN1, OCTN2, and IL4 genes, in comparison with 370 controls.TaqMan® technologies were used to genotype IL4 rs2243250, OCTN1 rs1050152, and OCTN2 rs2631367 according to standard methodologies.The frequencies of variant SNPs in the control population were compared with frequencies in cases, where phenotype data for IBD was classed according to the Montreal classification.Results: For those individuals carrying only the variant allele in IL4 or OCTN1, despite trends towards increased risk of developing IBD, there were not statistically significant differences.There were no other specific effects of this allele in the location of CD, behaviour of UC or CD, or any other differences between controls and patient subgroups in this population group.However, carrying the variant OCTN2 rs2631367 allele significantly increased the risk of ileocolonic CD (OR=1.42,95% CI=1.02-1.97,p<0.05).Conclusions: Despite earlier reports suggesting that OCTN1 and OCTN2 variants might provide important risk factors for IBD in New Zealand, this larger population-based study failed to confirm the earlier data.SNPs in the IL4 gene did not affect the analysis.We suggest the possibility that strong effects for these variants in these genes seen in some studies could imply gene-environment interactions.

OBJECTIVES:Published association studies of the TLR4 Asp299Gly polymorphism and inflammatory bowel disease (IBD) in caucasian populations have inconsistent results. We tested two TLR4 variants for association with IBD in the New Zealand caucasian population and assessed the cumulative evidence for association of TLR4 Asp299Gly and IBD. METHODS:The TLR4 Asp299Gly and Thr399Ile polymorphisms were genotyped and tested for case-control frequency differences in a New Zealand white cohort of 389 Crohn's disease (CD) patients, 405 ulcerative colitis (UC) patients, and 416 population controls. Meta-analysis using a random effects model was performed to test whether 299Gly carriage was associated with UC, CD, or phenotypes of CD patients. RESULTS:There were no significant allele or genotype frequency differences between cases and controls or between CD phenotypes in our New Zealand data. Meta-analysis did not identify any significant associations between CD phenotypes and 299Gly carriage. However, meta-analysis demonstrated significantly higher 299Gly carrier frequencies in CD patients (odds ratio 1.45, 95% CI 1.11-1.90) and in IBD patients (odds ratio 1.36, 95% CI 1.01-1.84) compared to controls. CONCLUSIONS:The meta-analysis provides evidence that Asp299Gly is associated with CD and IBD in whites. Only the Asp299Gly polymorphism has been consistently genotyped in previous TLR4 studies with IBD patients, therefore other TLR4 variants with stronger associations with IBD may exist. Additional well-powered studies of Asp299Gly and other TLR4 variants are urgently needed.

Inflammatory bowel disease (IBD) arises in part from a genetic predisposition to develop the disease, through the inheritance of a number of contributory genetic polymorphisms. These variant forms of genes may be associated with an abnormal response to normal luminal bacteria, possibly in combination with an environmental or dietary stimulus, that in turn leads to an excessive immune response. Therefore, central to understanding the etiology of IBD is the identification of the key genetic defects that enhance susceptibility. Characterising the genetic basis for IBD will allow us to understand the pathogenesis of the disease, and provide better targets for future preventative treatments.

Background: Variants in the DLG5 gene have been associated with inflaminatory bowel disease (IFID) in samples from some, but not all populations. In particular, 2 nonsynonymous single-nucleofide polymorphisms (SNPs), R30Q (rsl248696) and P1371Q (rs2289310), have been associated with an increased risk of IBD, and a common haplotype (called haplotype "A") has been associated with reduced risk.Methods: We genotyped R30Q, P1371Q, and a haplotype A tagging SNP (rs228931 1) in a New Zealand Caucasian cohort of 389 Crohn's disease (CD) patients, 406 ulcerative colitis (UC) patients, and 416 population controls. Each SNP was tested for association with disease susceptibility and clinical phenotypes. We also performed a meta-analysis of R30Q data from published association studies.Results: The haplotype A tagging SNP was associated with reduced risk of 1BD at the 0.05 significance level (P = 0.036) with an allefic odds ratio of 0.83 (95% confidence interval [CI]: 0.690.99). Association with haplotype A was strongest (odds ratio similar to 0.57) in UC patients with familial IBD or extraintestinal manifes-tations. The R30Q and P1371Q polymorphisins were not significantly associated with UC, CD, or IBD. Analysis of male and female data did not find any gender-specific associations. Meta-analysis gave no evidence of association of R30Q with 113D.Conclusions: Meta-analysis demonstrates that the minor allele of R30Q is not a risk factor for IBD across populations. This study provides some evidence that DLG5 haplotype A is associated with reduced risk of IBD in the New Zealand Caucasian population, but this association will need to be replicated in an independent sample.

Deficiency of the lysosomal cysteine protease cathepsin L (Ctsl) in mice results in a phenotype affecting multiple tissues, including thymus, epidermis, and hair follicles, and in the heart develops as a progressive dilated cardiomyopathy (DCM). To understand the role of Ctsl in the maintenance of regular heart morphology and function, it is critical to determine whether the DCM in Ctsl-/- mice is primarily because of the lack of Ctsl expression and activity in the cardiomyocytes or is caused by the additional extracardiac pathologies. Cardiomyocyte-specific expression of Ctsl in Ctsl-/- mice, using an alpha-myosin heavy chain promoter-Ctsl transgene, results in improved cardiac contraction, normal mRNA expression of atrionatriuretic peptide, normal heart weight, and regular ultrastructure of cardiomyocytes. Epithelial expression of cathepsin L2 (CTSL2) by a K14 promoter-CTSL2-transgene resulted in rescue of the Ctsl-/- hair loss phenotype. In these mice, cardiac atrionatriuretic peptide expression and end systolic heart dimensions were also significantly attenuated. However, cardiac contraction was not improved, and increased heart weight as well as the typical changes in lysosomal ultrastructure of Ctsl-/- hearts persisted. Myocardial fibrosis was detected in all Ctsl-/- mice irrespective of transgene-mediated cardiac Ctsl expression or extracardiac CTSL2 expression. Expression of collagen 1 was not enhanced in Ctsl-/- hearts, but a reduced collagenolytic activity suggests a role for Ctsl in collagen turnover by cardiac fibroblasts. We conclude that the DCM of Ctsl-/- mice is primarily caused by absence of the protease in cardiomyocytes, whereas the complex gross phenotype of Ctsl-deficient mice, i.e. the fur defect, results in additional stress to the heart.

Accurate measurement of allele frequencies between population groups with differing sensitivities to disease is fundamental to genetic epidemiology. Genotyping errors can markedly influence the biological conclusions of a study. This issue may be especially important now there is increasing recognition of triallelic single nucleotide polymorphisms (SNPs) in the genome and their possible role in diseases like inflammatory bowel disease. For example, the MDR1 (ABCB1) SNP G2677/T/A was, like many other triallelic SNPs, originally described as diallelic. Here, we report a comprehensive analyses of estimated allele frequencies of this SNP in a set of 73 human DNA samples, comparing six commonly used genotyping methods (Applied Biosystems Taqman, Roche LightCycler melting analysis, allelic discrimination PCR, DNA sequencing, Sequenom, and RFLP) from the angle of their error potential. Only Sequenom and DNA sequencing provided accurate measurements, if we had not had prior knowledge of the triallelic nature of this SNP. The other tested methods (with the exception of LightCycler) failed to show any indication of the presence of the rare third A- allele in a diallelic assay. Although most of the errors were due to the inability to detect the third allele, all methods except Sequenom and sequencing produced errors for the detection of the two common alleles G and T (LightCycler, 6 errors; PCR, 4 errors; RFLP, 2 errors; Taqman, 1 error). There is considerable variability in the reported frequencies of the different alleles of the MDR1 G2677/T/A SNP, and the role of this SNP in the etiology of inflammatory bowel disease has been controversial. Our data emphasize the importance of choosing the appropriate method for SNP detection and lead us to suggest that part of the previously reported variation may reflect artifacts associated with the different genotyping methodologies used. The failure to recognize the triallic nature of a SNP may lead to underestimations of real genetic associations.

Inflammatory bowel disease (IBD) arises in part from a genetic predisposition, through the inheritance of a number of contributory genetic polymorphisms. These variant forms of genes may be associated with an abnormal response to normal luminal bacteria. A consistent observation across most populations is that any of three polymorphisms of the Caspase-activated recruitment domain (CARD15) gene are more prevalent in IBD patients as compared with unaffected controls. Similar aberrant responses to bacteria are associated with variants in Autophagy-related 16-like 1 (ATG16L1) and human defensin (HBD-2, -3 and -4) genes. The defective bacterial signal in turn leads to an excessive immune response, presenting as chronic gut inflammation in susceptible individuals. Inconsistent population reports implicate the major histocompatability complex (MHC), that encodes a number of human leukocyte antigens (HLA), MHC class I chain-related gene A (MICA) or cytokines, such as tumour necrosis factor-alpha (TNF-α). Toll-like receptors encoded by the TLR4 or TLR9 genes may also play a role. Recent whole genome scans suggest that a rare variant in the interleukin-23 receptor (IL23R) gene may actually protect against IBD. Other implicated genes may affect mucosal cell polarity (Drosophila discs large homologue 5, DLG5) or mucosal transporter function (sodium dependent organic cation transporters, SLC22A4 and SLC22A5). A variant in ABCB1 (ATP-binding cassette subfamily B member 1) may be especially associated with increased risk of UC. While pharmacogenetics is increasingly being used to predict and optimise clinical response to therapy, nutrigenetics may have even greater potential. In many cases, IBD can be controlled through prescribing an elemental diet, which appears to act through modulating cytokine response and changing the gut microbiota. More generally, no single group of dietary items is beneficial or detrimental to all patients, and elimination diets have been used to individualise dietary requirements. However, recognising the nature of the genes involved may suggest a more strategic approach. Pro- or prebiotics will directly influence the microbial flora, while immunonutrition, including omega-3 fatty acids and certain polyphenols, may reduce the symptoms of gut inflammation. The expression of gut transporters may be modulated through various herbal remedies including green tea polyphenols. Such approaches would require that the gene of interest is functioning normally, other than its expression being up or down-regulated. However, new approaches are being developed to overcome the effects of polymorphisms that affect the function of a gene. A combination of human correlation studies with experimental models could provide a rational strategy for optimising nutrigenetic approaches to IBD.

Although lysosomal proteases are expressed in the heart at considerable levels, their specific functions in this organ remain elusive. Mice deficient for the lysosomal cysteine protease cathepsin L (CTSL) develop a late onset dilated cardiomyopathy (DCM) that is characterized by cardiac chamber dilation, fibrosis, and impaired cardiac contraction at 12 month of age. Investigation of the pathogenic sequence of DCM in ctsl-/- mice revealed numerous dysmorphic lysosome-like structures in heart muscle as early as 3 days after birth, whereas skeletal muscle was not affected. Labeling of the acidic cell compartment of neonatal cardiomyocytes and detection of lysosomal markers after subcellular fractionation confirmed increased lysosome content in CTSL deficient myocardium; however, specific storage materials were not detected. The myocardium of ctsl+/+ and ctsl-/- mice revealed no differences in incidence of cell death, proliferation, and capillary density during DCM progression. However, an observed increase in mRNA expression of natriuretic peptides in young adult mice indicates the activation of the adaptive "fetal" gene program, while proteome analysis revealed decreased levels of the sarcomere-associated proteins alpha-tropomyosin, desmin, and calsarcin 1, as well as considerable changes of metabolic enzymes. Bioinformatic pathway analysis suggested a switch to anaerobic catabolism and impairment of mitochondrial respiration. This interpretation was supported by a 50% reduction in resting state oxygen consumption and impaired respiration capacity in ctsl-/- myocardial homogenates. In summary, the data indicate an essential role of CTSL in maintaining the structure of the endosomal/lysosomal compartment in cardiomyocytes. Lysosomal impairment in ctsl-/- hearts results in metabolic and sarcomeric alterations that promote DCM development.

Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of [Met]enkephalin by cathepsin L occurred by proteolytic processing at dibasic and monobasic prohormone-processing sites. Cellular studies showed the colocalization of cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine chromaffin cells by immunofluorescent confocal and immunoelectron microscopy. Functional localization of cathepsin L to the regulated secretory pathway was demonstrated by its cosecretion with [Met]enkephalin. Finally, in cathepsin L gene knockout mice, [Met]enkephalin levels in brain were reduced significantly; this occurred with an increase in the relative amounts of enkephalin precursor. These findings indicate a previously uncharacterized biological role for secretory vesicle cathepsin L in the production of [Met]enkephalin, an endogenous peptide neurotransmitter.

Dilated cardiomyopathy is a frequent cause of heart failure and is associated with high mortality. Progressive remodeling of the myocardium leads to increased dimensions of heart chambers. The role of intracellular proteolysis in the progressive remodeling that underlies dilated cardiomyopathy has not received much attention yet. Here, we report that the lysosomal cysteine peptidase cathepsin L (CTSL) is critical for cardiac morphology and function. One-year-old CTSL-deficient mice show significant ventricular and atrial enlargement that is associated with a comparatively small increase in relative heart weight. Interstitial fibrosis and pleomorphic nuclei were found in the myocardium of the knockout mice. By electron microscopy, CTSL-deficient cardiomyocytes contained multiple large and apparently fused lysosomes characterized by storage of electron-dense heterogeneous material. Accordingly, the assessment of left ventricular function by echocardiography revealed severely impaired myocardial contraction in the CTSL-deficient mice. In addition, echocardiographic and electrocardiographic findings to some degree point to left ventricular hypertrophy that most likely represents an adaptive response to cardiac impairment. The histomorphological and functional alterations of CTSL-deficient hearts result in valve insufficiencies. Furthermore, abnormal heart rhythms, like supraventricular tachycardia, ventricular extrasystoles, and first-degree atrioventricular block, were detected in the CTSL-deficient mice.