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We have previously identified NOD2 genotype and inflammatory bowel diseases (IBD) phenotype, as associated with shifts in the ileal microbiome (“dysbiosis”) in a patient cohort. Here we report an integrative analysis of an expanded number of Crohn's disease (CD) related genetic defects in innate immune function (NOD2, ATG16L1, IRGM, CARD9, XBP1, ORMDL3) and composition of the ileal microbiome by combining the initial patient cohort (Batch 1, 2005–2010, n = 165) with a second consecutive patient cohort (Batch 2, 2010–2012, n = 118). These combined patient cohorts were composed of three non-overlapping phenotypes: 1.) 106 ileal CD subjects undergoing initial ileocolic resection for diseased ileum, 2.) 88 IBD colitis subjects without ileal disease (predominantly ulcerative colitis but also Crohn’s colitis and indeterminate colitis, and 3.) 89 non-IBD subjects. Significant differences (FDR < 0.05) in microbiota were observed between macroscopically disease unaffected and affected regions of resected ileum in ileal CD patients. Accordingly, analysis of the effects of genetic and clinical factors were restricted to disease unaffected regions of the ileum. Beta-diversity differed across the three disease categories by PERMANOVA (p < 0.001), whereas no significant differences in alpha diversity were noted. Using negative binomial models, we confirmed significant effects of IBD phenotype, C. difficile infection, and NOD2 genotype on ileal dysbiosis in the expanded analysis. The relative abundance of the Proteobacteria phylum was positively associated with ileal CD and colitis phenotypes, but negatively associated with NOD2R genotype. Additional associations with ORMDL3 and XBP1 were detected at the phylum/subphylum level. IBD medications, such as immunomodulators and anti-TNFα agents, may have a beneficial effect on reversing dysbiosis associated with the IBD phenotype. Exploratory analysis comparing microbial composition of the disease unaffected region of the resected ileum between 27 ileal CD patients who subsequently developed endoscopic recurrence within 6–12 months versus 34 patients who did not, suggested that microbial biomarkers in the resected specimen helped stratify patients with respect to risk of post-surgical recurrence. [ABSTRACT FROM AUTHOR]

Background: Incidence and mortality rates of colorectal carcinoma (CRC) are higher in African Americans (AAs) than in Caucasian Americans (CAs). Deficient micronutrient intake due to dietary restrictions in racial/ethnic populations can alter genetic and molecular profiles leading to dysregulated methylation patterns and the inheritance of somatic to germline mutations. Materials and Methods: Total DNA and RNA samples of paired tumor and adjacent normal colon tissues were prepared from AA and CA CRC specimens. Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing were employed to evaluate total genome methylation of 5’-regulatory regions and dysregulation of gene expression, respectively. Robust analysis was conducted using a trimming-and-retrieving scheme for RRBS library mapping in conjunction with the BStool toolkit. Results: DNA from the tumor of AA CRC patients, compared to adjacent normal tissues, contained 1,588 hypermethylated and 100 hypomethylated differentially methylated regions (DMRs). Whereas, 109 hypermethylated and 4 hypomethylated DMRs were observed in DNA from the tumor of CA CRC patients; representing a 14.6-fold and 25-fold change, respectively. Specifically; CHL1, 4 anti-inflammatory genes (i.e., NELL1, GDF1, ARHGEF4, and ITGA4), and 7 miRNAs (of which miR-9-3p and miR-124-3p have been implicated in CRC) were hypermethylated in DNA samples from AA patients with CRC. From the same sample set, RNAseq analysis revealed 108 downregulated genes (including 14 ribosomal proteins) and 34 upregulated genes (including POLR2B and CYP1B1 [targets of miR-124-3p]) in AA patients with CRC versus CA patients. Conclusion: DNA methylation profile and/or products of its downstream targets could serve as biomarker(s) addressing racial health disparity. [ABSTRACT FROM AUTHOR]

Background: Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. Genetic polymorphisms can confer CD risk and influence disease phenotype. Indoleamine 2,3 dioxygenase-1 (IDO1) is one of the most over-expressed genes in CD and mediates potent anti-inflammatory effects via tryptophan metabolism along the kynurenine pathway. We aimed to determine whether non-synonymous polymorphisms in IDO1 or IDO2 (a gene paralog) are important either as CD risk alleles or as modifiers of CD phenotype. Methods: Utilizing a prospectively collected database, clinically phenotyped CD patients (n = 734) and non-IBD controls (n = 354) were genotyped for established IDO1 and IDO2 non-synonymous single nucleotide polymorphisms (SNPs) and novel genetic variants elucidated in the literature. Allelic frequencies between CD and non-IBD controls were compared. Genotype-phenotype analysis was conducted. IDO1 enzyme activity was assessed by calculating the serum kynurenine to tryptophan ratio (K/T). Results: IDO1 SNPs were rare (1.7% non-IBD vs 1.1% CD; p = NS) and not linked to Crohn's disease diagnosis in this population. IDO1 SNPs did however associate with a severe clinical course, presence of perianal disease, extraintestinal manifestations and a reduced serum K/T ratio during active disease suggesting lower IDO1 function. IDO2 minor allele variants were common and one of them, rs45003083, associated with reduced risk of Crohn's disease (p = 0.025). No IDO2 SNPs associated with a particular Crohn's disease clinical phenotype. Conclusions: This work highlights the functional importance of IDO enzymes in human Crohn's disease and establishes relative rates of IDO genetic variants in a US population. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) has the third highest mortality rates among the US population. According to the most recent concept of carcinogenesis, human tumors are organized hierarchically, and the top of it is occupied by malignant stem cells (cancer stem cells, CSCs, or cancer-initiating cells, CICs), which possess unlimited self-renewal and tumor-initiating capacities and high resistance to conventional therapies. To reflect the complexity and diversity of human tumors and to provide clinically and physiologically relevant cancer models, large banks of characterized patient-derived low-passage cell lines, and especially CIC-enriched cell lines, are urgently needed. Principal Findings: Here we report the establishment of a novel CIC-enriched, highly tumorigenic and clonogenic colon cancer cell line, CR4, derived from liver metastasis. This stable cell line was established by combining 3D culturing and 2D culturing in stem cell media, subcloning of cells with particular morphology, co-culture with carcinoma associated fibroblasts (CAFs) and serial transplantation to NOD/SCID mice. Using RNA-Seq complete transcriptome profiling of the tumorigenic fraction of the CR4 cells in comparison to the bulk tumor cells, we have identified about 360 differentially expressed transcripts, many of which represent stemness, pluripotency and resistance to treatment. Majority of the established CR4 cells express common markers of stemness, including CD133, CD44, CD166, EpCAM, CD24 and Lgr5. Using immunocytochemical, FACS and western blot analyses, we have shown that a significant ratio of the CR4 cells express key markers of pluripotency markers, including Sox-2, Oct3/4 and c-Myc. Constitutive overactivation of ABC transporters and NF-kB and absence of tumor suppressors p53 and p21 may partially explain exceptional drug resistance of the CR4 cells. Conclusions: The highly tumorigenic and clonogenic CIC-enriched CR4 cell line may provide an important new tool to support the discovery of novel diagnostic and/or prognostic biomarkers as well as the development of more effective therapeutic strategies. [ABSTRACT FROM AUTHOR]