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ABSTRACT: BACKGROUND: Culture-independent phylogenetic analysis of 16S ribosomal RNA (rRNA) gene sequences has emerged as an incisive method of profiling bacteria present in a specimen. Currently, multiple techniques are available to enumerate the abundance of bacterial taxa in specimens, including the Sanger sequencing, the 'next generation' pyrosequencing, microarrays, quantitative PCR, and the rapidly emerging, third generation sequencing, and fourth generation sequencing methods. An efficient statistical tool is in urgent need for the followings tasks: (1) to compare the agreement between these measurement platforms, (2) to select the most reliable platform(s), and (3) to combine different platforms of complementary strengths, for a unified analysis. RESULTS: We present the latent variable structural equation modeling (SEM) as a novel statistical application for the comparative analysis of measurement platforms. The latent variable SEM model treats the true (unknown) relative frequency of a given bacterial taxon in a specimen as the latent (unobserved) variable and estimates the reliabilities of, and similarities between, different measurement platforms, and subsequently weighs those measurements optimally for a unified analysis of the microbiome composition. The latent variable SEM contains the repeated measures ANOVA (both the univariate and the multivariate models) as special cases and, as a more general and realistic modeling approach, yields superior goodness-of-fit and more reliable analysis results, as demonstrated by a microbiome study of the human inflammatory bowel diseases. CONCLUSIONS: Given the rapid evolution of modern biotechnologies, the measurement platform comparison, selection and combination tasks are here to stay and to grow -- and the latent variable SEM method is readily applicable to any other biological settings, aside from the microbiome study presented here.
BMC Bioinformatics 03/2013; 14(1):79. · 2.75 Impact Factor
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ABSTRACT: Previous genome-wide expression studies have highlighted distinct gene expression patterns in inflammatory bowel disease (IBD) compared to control samples, but the interpretation of these studies has been limited by sample heterogeneity with respect to disease phenotype, disease activity, and anatomic sites. To further improve molecular classification of inflammatory bowel disease phenotypes we focused on a single anatomic site, the disease unaffected proximal ileal margin of resected ileum, and three phenotypes that were unlikely to overlap: ileal Crohn's disease (ileal CD), ulcerative colitis (UC), and control patients without IBD. Whole human genome (Agilent) expression profiling was conducted on two independent sets of disease-unaffected ileal samples collected from the proximal margin of resected ileum. Set 1 (47 ileal CD, 27 UC, and 25 Control non-IBD patients) was used as the training set and Set 2 was subsequently collected as an independent test set (10 ileal CD, 10 UC, and 10 control non-IBD patients). We compared the 17 gene signatures selected by four different feature-selection methods to distinguish ileal CD phenotype with non-CD phenotype. The four methods yielded different but overlapping solutions that were highly discriminating. All four of these methods selected FOLH1 as a common feature. This gene is an established biomarker for prostate cancer, but has not previously been associated with Crohn's disease. Immunohistochemical staining confirmed increased expression of FOLH1 in the ileal epithelium. These results provide evidence for convergent molecular abnormalities in the macroscopically disease unaffected proximal margin of resected ileum from ileal CD subjects.
PLoS ONE 01/2012; 7(5):e37139. · 4.09 Impact Factor
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Ellen Li,
Christina M Hamm,
Ajay S Gulati,
R Balfour Sartor,
Hongyan Chen,
Xiao Wu,
Tianyi Zhang,
F James Rohlf,
Wei Zhu,
Chi Gu,
Charles E Robertson,
Norman R Pace,
Edgar C Boedeker,
Noam Harpaz,
Jeffrey Yuan,
George M Weinstock,
Erica Sodergren,
Daniel N Frank
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ABSTRACT: We tested the hypothesis that Crohn's disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum-associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1-V3 and V3-V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides - Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤ 0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides--E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota.
PLoS ONE 01/2012; 7(6):e26284. · 4.09 Impact Factor
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Tianyi Zhang,
Robert A DeSimone,
Xiangmin Jiao,
F James Rohlf,
Wei Zhu,
Qing Qing Gong,
Steven R Hunt,
Themistocles Dassopoulos,
Rodney D Newberry,
Erica Sodergren,
George Weinstock,
Charles E Robertson,
Daniel N Frank, Ellen Li
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ABSTRACT: The aim of this study was to integrate human clinical, genotype, mRNA microarray and 16 S rRNA sequence data collected on 84 subjects with ileal Crohn's disease, ulcerative colitis or control patients without inflammatory bowel diseases in order to interrogate how host-microbial interactions are perturbed in inflammatory bowel diseases (IBD). Ex-vivo ileal mucosal biopsies were collected from the disease unaffected proximal margin of the ileum resected from patients who were undergoing initial intestinal surgery. Both RNA and DNA were extracted from the mucosal biopsy samples. Patients were genotyped for the three major NOD2 variants (Leufs1007, R702W, and G908R) and the ATG16L1T300A variant. Whole human genome mRNA expression profiles were generated using Agilent microarrays. Microbial composition profiles were determined by 454 pyrosequencing of the V3-V5 hypervariable region of the bacterial 16 S rRNA gene. The results of permutation based multivariate analysis of variance and covariance (MANCOVA) support the hypothesis that host mucosal Paneth cell and xenobiotic metabolism genes play an important role in host microbial interactions.
PLoS ONE 01/2012; 7(6):e30044. · 4.09 Impact Factor
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ABSTRACT: : Our objective is to assess the effect of genetic and environmental factors on Crohn's disease location.
: We identified 628 patients with Crohn's disease within the Washington University database (April 2005 through February 2010) that had complete information on 31 Crohn's disease-associated genotypes and clinical information on disease location (L1 to L4), smoking, sex, race, and age at diagnosis. For statistical reasons, the 3 major NOD2 alleles (rs2066844, rs2066845, and rs2066847) were grouped together. Logistic regression incorporating all of the genotypes and clinical covariates, including smoking, was performed with stepwise variable selection and by best subset selection.
: Stepwise variable selection selected 3 major covariates, composite NOD2 genotype, smoking, and TNFSF15 genotype, which are also the 3 covariates selected by the best subset method. Whereas the NOD2 genotype and smoking are positively associated with ileal (L1 + L3) disease, the TNFSF15 genotype is positively associated with isolated colonic (L2) disease.
: The ability to detect disease site associations in this single-center study may be limited by the population size, low allelic frequency, and/or low odds ratio of certain Crohn's disease risk alleles.
: These results indicate that NOD2 genotype, smoking status, and TNFSF15 genotype should be included as covariates in assessing the effect of genetic and environmental factors on Crohn's disease site location.
Diseases of the Colon & Rectum 08/2011; 54(8):1020-5. · 3.13 Impact Factor
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ABSTRACT: Culture-independent microbiological technologies that interrogate complex microbial populations without prior axenic culture, coupled with high-throughput DNA sequencing, have revolutionized the scale, speed and economics of microbial ecological studies. Their application to the medical realm has led to a highly productive merger of clinical, experimental and environmental microbiology. The functional roles played by members of the human microbiota are being actively explored through experimental manipulation of animal model systems and studies of human populations. In concert, these studies have appreciably expanded our understanding of the composition and dynamics of human-associated microbial communities (microbiota). Of note, several human diseases have been linked to alterations in the composition of resident microbial communities, so-called dysbiosis. However, how changes in microbial communities contribute to disease etiology remains poorly defined. Correlation of microbial composition represents integration of only two datasets (phenotype and microbial composition). This article explores strategies for merging the human microbiome data with multiple additional datasets (e.g. host single nucleotide polymorphisms and host gene expression) and for integrating patient-based data with results from experimental animal models to gain deeper understanding of how host-microbe interactions impact disease.
Trends in Microbiology 07/2011; 19(9):427-34. · 7.91 Impact Factor
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Daniel N Frank,
Charles E Robertson,
Christina M Hamm,
Zegbeh Kpadeh,
Tianyi Zhang,
Hongyan Chen,
Wei Zhu,
R Balfour Sartor,
Edgar C Boedeker,
Noam Harpaz,
Norman R Pace, Ellen Li
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ABSTRACT: Abnormal host-microbe interactions are implicated in the pathogenesis of inflammatory bowel diseases. Previous 16S rRNA sequence analysis of intestinal tissues demonstrated that a subset of Crohn's disease (CD) and ulcerative colitis (UC) samples exhibited altered intestinal-associated microbial compositions characterized by depletion of Bacteroidetes and Firmicutes (particularly Clostridium taxa). We hypothesize that NOD2 and ATG16L1 risk alleles may be associated with these alterations.
To test this hypothesis, we genotyped 178 specimens collected from 35 CD, 35 UC, and 54 control patients for the three major NOD2 risk alleles (Leu 1007fs, R702W, and G908R) and the ATG16L1T300A risk allele, that had undergone previous 16S rRNA sequence analysis. Our statistical models incorporated the following independent variables: 1) disease phenotype (CD, UC, non-IBD control); 2) NOD2 composite genotype (NOD2(R) = at least one risk allele, NOD2(NR) = no risk alleles); 3) ATG16L1T300A genotype (ATG16L1(R/R), ATG16L1(R/NR), ATG16L1(NR/NR)); 4) patient age at time of surgery and all first-order interactions. The dependent variable(s) were the relative frequencies of bacterial taxa classified by applying the RDP 2.1 classifier to previously reported 16S rRNA sequence data.
Disease phenotype, NOD2 composite genotype and ATG16L1 genotype were significantly associated with shifts in microbial compositions by nonparametric multivariate analysis of covariance (MANCOVA). Shifts in the relative frequencies of Faecalibacterium and Escherichia taxa were significantly associated with disease phenotype by nonparametric ANCOVA.
These results support the concept that disease phenotype and genotype are associated with compositional changes in intestinal-associated microbiota.
Inflammatory Bowel Diseases 01/2011; 17(1):179-84. · 4.86 Impact Factor
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Tianyi Zhang,
Robert A. DeSimone,
Hongyan Chen,
Christina M. Hamm,
Jeffrey Yuan,
Qing Qing Gong,
Steven R. Hunt,
Themistocles Dassopoulos,
Rodney D. Newberry,
Daniel N. Frank,
Charles E. Robertson,
Norman R. Pace,
Erica Sodergren,
George Weinstock,
Xiangmin Jiao,
Wei Zhu, Ellen Li
IEEE 1st International Conference on Computational Advances in Bio and Medical Sciences, ICCABS 2011, Orlando, FL, USA, February 3-5, 2011; 01/2011
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IEEE 1st International Conference on Computational Advances in Bio and Medical Sciences, ICCABS 2011, Orlando, FL, USA, February 3-5, 2011; 01/2011
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Christina M Hamm,
Melissa A Reimers,
Casey K McCullough,
Elizabeth B Gorbe,
Jianyun Lu,
C Charles Gu, Ellen Li,
Brian K Dieckgraefe,
Qingqing Gong,
Thaddeus S Stappenbeck,
Christian D Stone,
David W Dietz,
Steven R Hunt
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ABSTRACT: NOD2 single nucleotide polymorphisms have been associated with increased risk of ileal Crohn's disease (CD). This exploratory study was conducted to compare ileal mucosal gene expression in CD patients with and without NOD2 risk alleles.
Ileal samples were prospectively collected from 18 nonsmoking CD patients not treated with anti-TNF-α biologics and 9 nonsmoking control patients without inflammatory bowel disease undergoing initial resection and genotyped for the 3 major NOD2 risk alleles (Arg702Trp, Gly908Arg, Leu1007fs). Microarray analysis was performed in samples from 4 NOD2(R) (at least 1 risk allele) CD patients, 4 NOD2(NR) (no risk alleles) CD patients, and 4 NOD2(NR) controls. Candidate genes selected by significance analysis of microarrays (SAM) were confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays of all the samples.
SAM detected upregulation of 18 genes in affected ileum in NOD2(R) compared to NOD2(NR) CD patients, including genes related to lymphocyte activation. SAM also detected altered ileal gene expression in unaffected NOD2(NR) ileal mucosal CD samples compared to NOD2(NR) control samples. qRT-PCR conducted on all the samples confirmed that increased CD3D expression in affected samples was associated with NOD2(R) status, and that increased MUC1, DUOX2, DMBT1 and decreased C4orf7 expression in unaffected samples was associated with CD, independent of NOD2 status.
The results support the concept that NOD2 risk alleles contribute to impaired regulation of inflammation in the ileum. Furthermore, altered ileal gene expression, independent of NOD2 status, is detected in the unaffected proximal margin of resected ileum from CD patients.
Inflammatory Bowel Diseases 02/2010; 16(10):1649-57. · 4.86 Impact Factor
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ABSTRACT: Previous European studies suggest NOD2/CARD15 and interleukin-23 receptor (IL-23R) donor or recipient variants are associated with adverse clinical outcomes in allogeneic hematopoietic stem cell transplantation. We reexamined these findings as well as the role of another inflammatory bowel disease (IBD) susceptibility gene (immunity-related GTPase family, M [IRGM]) on transplantation outcomes in 390 US patients and their matched unrelated donors, accrued between 1995 and 2004. Patients received T-replete grafts with mostly myeloablative conditioning regimens. Multivariate analyses were performed for overall survival, disease-free survival, transplantation-related mortality, relapse, and acute and chronic graft-versus-host disease. Of 390 pairs, NOD2/CARD15 variant single nucleotide polymorphisms (SNPs) were found in 14% of donors and 17% of recipients. In 3% both donor and recipient had a mutant SNP. Thirteen percent of donors and 16% of recipients had variant IL23R SNPs, with 3% having both donor and recipient variants. Twenty-three percent of both donors and recipients had variant IRGM SNPs. None of the 3 IBD-associated alleles showed a statistically significant association with any adverse clinical outcomes. Our results do not support an association between the 3 IBD-associated SNPs and adverse outcomes after matched unrelated donor hematopoietic cell transplantations in US patients.
Blood 02/2010; 115(17):3625-31. · 9.90 Impact Factor
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ABSTRACT: The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand-binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRalpha LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator-activated receptor gamma (PPARgamma) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line-broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo- or hetero-dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor-interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRalpha LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C-terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line-broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRalpha LBD-antagonist-peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRalpha LBD conformations in favor of a compact state, and that a PPARgamma LBD-agonist complex can prevent the antagonist from enhancing the RXRalpha LBD-coactivator binding interaction.
Magnetic Resonance in Chemistry 09/2009; 47(12):1071-80. · 1.44 Impact Factor
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ABSTRACT: Extraintestinal manifestations of Crohn's disease (CD) have not previously included the central nervous system (CNS). Restless legs syndrome (RLS) is a CNS disorder that is either idiopathic or secondary to a number of diseases. The aim of this study was to determine if RLS was associated with CD because both are associated with iron deficiency, inflammation, and bacterial overgrowth.
Consecutive CD outpatients (N = 272) were prospectively surveyed at 4 centers for criteria for RLS. Incidence (having RLS at any point in time), prevalence (having RLS at time of survey), clinical characteristics, risk factors, and potential qualitative relationship between RLS and gastrointestinal symptoms were queried.
The incidence of RLS in patients with CD was 42.7%. Prevalence was 30.2% compared with 9% of spouses. CD patients with and without RLS had a mean age of 46.8 versus 42.6 years, small intestine involvement in 77.9% versus 66.7%, colon involvement in 39.7% versus 63.2%, and prior iron deficiency anemia in 49.3% versus 33.1%. There was no difference between the CD groups with respect to current iron deficiency, RLS family history, or rare prevalence of concomitant RLS disorders. In 91.8% of patients with RLS and CD, RLS started during or after the onset of CD diagnosis. Among 73 patients with RLS, 67 (44.5%) stated there was a relationship between qualitative RLS symptom improvement with overall CD symptom improvement.
These results demonstrate that RLS occurs frequently in CD and appears to be a possible extraintestinal manifestation. The potential relationship of RLS with CD activity warrants further investigation.
Inflammatory Bowel Diseases 08/2009; 16(2):275-9. · 4.86 Impact Factor
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Ken Cadwell,
John Y Liu,
Sarah L Brown,
Hiroyuki Miyoshi,
Joy Loh,
Jochen K Lennerz,
Chieko Kishi,
Wumesh Kc,
Javier A Carrero,
Steven Hunt,
Christian D Stone,
Elizabeth M Brunt,
Ramnik J Xavier,
Barry P Sleckman, Ellen Li,
Noboru Mizushima,
Thaddeus S Stappenbeck,
Herbert W Virgin
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ABSTRACT: Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci. One Crohn's disease risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG16 (ref. 2). It is not known how ATG16L1 or autophagy contributes to intestinal biology or Crohn's disease pathogenesis. To address these questions, we generated and characterized mice that are hypomorphic for ATG16L1 protein expression, and validated conclusions on the basis of studies in these mice by analysing intestinal tissues that we collected from Crohn's disease patients carrying the Crohn's disease risk allele of ATG16L1. Here we show that ATG16L1 is a bona fide autophagy protein. Within the ileal epithelium, both ATG16L1 and a second essential autophagy protein ATG5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell that functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment. ATG16L1- and ATG5-deficient Paneth cells exhibited notable abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to ATG16L1-deficient Paneth cells including increased expression of genes involved in peroxisome proliferator-activated receptor (PPAR) signalling and lipid metabolism, of acute phase reactants and of two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, Crohn's disease patients homozygous for the ATG16L1 Crohn's disease risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy-protein-deficient mice and expressed increased levels of leptin protein. Thus, ATG16L1, and probably the process of autophagy, have a role within the intestinal epithelium of mice and Crohn's disease patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.
Nature 11/2008; 456(7219):259-63. · 36.28 Impact Factor
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Ken Cadwell,
John Y. Liu,
Sarah L. Brown,
Hiroyuki Miyoshi,
Joy Loh,
Jochen K. Lennerz,
Chieko Kishi,
Wumesh Kc,
Javier A. Carrero,
Steven Hunt,
Christian D. Stone,
Elizabeth M. Brunt,
Ramnik J. Xavier,
Barry P. Sleckman, Ellen Li,
Noboru Mizushima,
Thaddeus S. Stappenbeck,
Herbert W. Virgin IV
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ABSTRACT: Susceptibility to Crohn's disease, a complex inflammatory disease involving the small intestine, is controlled by over 30 loci
Nature 10/2008; 456(7219):259-263. · 36.28 Impact Factor
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ABSTRACT: PurposeWe evaluated the effect of potential clinical factors on surgical recurrence of ileal Crohn’s disease after initial ileocolic
resection.
MethodsOne hundred seventy-six patients with ileal Crohn’s disease who underwent an ileocolic resection with anastomosis were identified
from our database. The outcome of interest was time from first to second ileocolic resection. Survival analysis was used to
assess the significance of the Montreal phenotype classification, smoking habit, a family history of inflammatory bowel disease
and other clinical variables.
ResultsIn our final Cox model, a family history of inflammatory bowel disease (hazard ratio 2.24, 95 percent confidence interval
1.16–4.30, P = 0.016), smoking at time of initial ileocolic resection (hazard ratio 2.08, 95 percent confidence interval 1.11–3.91, P = 0.023) was associated with an increased risk of a second ileocolic resection while postoperative prescription of immunomodulators
(hazard ratio 0.40, 95 percent confidence interval 0.18–0.88, P = 0.022) was associated with a decreased risk of a second ileocolic resection.
ConclusionsBoth a family history of inflammatory bowel disease and smoking at the time of the initial ileocolic resection are associated
with an increased risk of a second ileocolic resection. Postoperative prescription of immunomodulators is associated with
a reduced risk of surgical recurrence. This study supports the concept that both genetic and environmental factors influence
the risk of surgical recurrence of ileal Crohn’s disease.
Diseases of the Colon & Rectum 07/2008; 51(8):1211-1216. · 3.13 Impact Factor
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ABSTRACT: We evaluated the effect of potential clinical factors on surgical recurrence of ileal Crohn's disease after initial ileocolic resection.
One hundred seventy-six patients with ileal Crohn's disease who underwent an ileocolic resection with anastomosis were identified from our database. The outcome of interest was time from first to second ileocolic resection. Survival analysis was used to assess the significance of the Montreal phenotype classification, smoking habit, a family history of inflammatory bowel disease and other clinical variables.
In our final Cox model, a family history of inflammatory bowel disease (hazard ratio 2.24, 95 percent confidence interval 1.16-4.30, P = 0.016), smoking at time of initial ileocolic resection (hazard ratio 2.08, 95 percent confidence interval 1.11-3.91, P = 0.023) was associated with an increased risk of a second ileocolic resection while postoperative prescription of immunomodulators (hazard ratio 0.40, 95 percent confidence interval 0.18-0.88, P = 0.022) was associated with a decreased risk of a second ileocolic resection.
Both a family history of inflammatory bowel disease and smoking at the time of the initial ileocolic resection are associated with an increased risk of a second ileocolic resection. Postoperative prescription of immunomodulators is associated with a reduced risk of surgical recurrence. This study supports the concept that both genetic and environmental factors influence the risk of surgical recurrence of ileal Crohn's disease.
Diseases of the Colon & Rectum 07/2008; 51(8):1211-6. · 3.13 Impact Factor
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ABSTRACT: The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acyl-CoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.
Journal of Biological Chemistry 06/2008; 283(20):13510-9. · 4.77 Impact Factor
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ABSTRACT: The C-terminal activation function-2 (AF-2) helix plays a crucial role in retinoid X receptor alpha (RXRalpha)-mediated gene expression. Here, we report a nuclear magnetic resonance (NMR) study of the RXRalpha ligand-binding domain complexed with 9-cis-retinoic acid and a glucocorticoid receptor-interacting protein 1 peptide. The AF-2 helix and most of the C-terminal residues were undetectable due to a severe line-broadening effect. Due to its outstanding signal-to-noise ratio, the C-terminus residue, threonine 462 (T462) exhibited two distinct crosspeaks during peptide titration, suggesting that peptide binding was in a slow exchange regime on the chemical shift timescale. Consistently, the K(d) derived from T462 intensity decay agreed with that derived from isothermal titration calorimetry. Furthermore, the exchange contribution to the (15)N transverse relaxation rate was measurable in either T462 or the bound peptide. These results suggest that T462 is a sensor for coactivator binding and is a potential probe for AF-2 helix mobility.
Biochemical and Biophysical Research Communications 03/2008; 366(4):932-7. · 2.48 Impact Factor
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ABSTRACT: The structural mechanism of allosteric communication between retinoid X receptor (RXR) and its heterodimer partners remains controversial. As a first step towards addressing this question, we report a nuclear magnetic resonance (NMR) study on the GW1929-bound peroxisome proliferator-activated receptor gamma (PPARgamma) ligand-binding domain (LBD) with and without the 9-cis-retinoic acid (9cRA)-bound RXRalpha LBD. Sequence-specific 13C(alpha), 13C(beta), and 13CO resonance assignments have been established for over 95% of the 275 residues in the PPARgamma LBD monomer. The 1HN, 15N, and 13CO chemical shift perturbations induced by the RXRalpha LBD binding are located at not only the heterodimer interface that includes the C-terminal residue Y477 but also residues Y473 and K474 in the activation function-2 (AF-2) helix. This result suggests that 9cRA-bound RXRalpha can affect the PPARgamma AF-2 helix in solution and demonstrates that NMR is a powerful new tool for studying the mechanism of allosteric ligand activation in RXR heterodimers.
Biochemical and Biophysical Research Communications 02/2008; 365(1):42-6. · 2.48 Impact Factor
