Karyometry of the colonic mucosa

Objective: The study summarizes results of karyometric measurements in epithelial cells of the colorectal mucosa to document evidence of a field effect of preneoplastic development among patients with colorectal adenocarcinoma or adenoma.

The topography of DNA methylation in the non-neoplastic colonic mucosa surrounding colorectal cancers

The degree of gene hypermethylation in non-neoplastic colonic mucosa (NNCM) is a potentially important event in the development of colorectal cancer (CRC), particularly for the subgroup with a CpG island methylator phenotype (CIMP). In this study, we aimed to use an unbiased and high-throughput approach to evaluate the topography of DNA methylation in the non-neoplastic colonic mucosa (NNCM) surrounding colorectal cancer (CRC). A total of 61 tissue samples comprising 53 NNCM and 8 tumor samples were obtained from hemicolectomy specimens of two CRC patients (Cases 1 and 2). NNCM was stripped from the underlying colonic wall and samples taken at varying distances from the tumor. The level of DNA methylation in NNCM and tumor tissues was assessed at 1,505 CpG sites in 807 cancer-related genes using Illumina GoldenGate® methylation arrays. Case 1 tumor showed significantly higher levels of methylation compared to surrounding NNCM samples (P?

Microbiota of De-Novo Pediatric IBD: Increased Faecalibacterium Prausnitzii and Reduced Bacterial Diversity in Crohn's But Not in Ulcerative Colitis

OBJECTIVES: The gastrointestinal microbiota is considered important in inflammatory bowel disease (IBD) pathogenesis. Discoveries from established disease cohorts report reduced bacterial diversity, changes in bacterial composition, and a protective role for Faecalibacterium prausnitzii in Crohn's disease (CD). The majority of studies to date are however potentially confounded by the effect of treatment and a reliance on established rather than de-novo disease.

METHODS: Microbial changes at diagnosis were examined by biopsying the colonic mucosa of 37 children: 25 with newly presenting, untreated IBD with active colitis (13 CD and 12 ulcerative colitis (UC)), and 12 pediatric controls with a macroscopically and microscopically normal colon. We utilized a dual-methodology approach with pyrosequencing (threshold >10,000 reads) and confirmatory real-time PCR (RT-PCR).

RESULTS: Threshold pyrosequencing output was obtained on 34 subjects (11 CD, 11 UC, 12 controls). No significant changes were noted at phylum level among the Bacteroidetes, Firmicutes, or Proteobacteria. A significant reduction in bacterial alpha-diversity was noted in CD vs. controls by three methods (Shannon, Simpson, and phylogenetic diversity) but not in UC vs. controls. An increase in Faecalibacterium was observed in CD compared with controls by pyrosequencing (mean 16.7% vs. 9.1% of reads, P = 0.02) and replicated by specific F. prausnitzii RT-PCR (36.0% vs. 19.0% of total bacteria, P = 0.02). No disease-specific clustering was evident on principal components analysis.

CONCLUSIONS: Our results offer a comprehensive examination of the IBD mucosal microbiota at diagnosis, unaffected by therapeutic confounders or changes over time. Our results challenge the current model of a protective role for F. prausnitzii in CD, suggesting a more dynamic role for this organism than previously described.

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.

Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

Role of NleH, a type III secreted effector from attaching and effacing pathogens, in colonization of the bovine, ovine, and murine gut

The human pathogen enterohemorrhagic Escherichia coli (EHEC) O157:H7 colonizes human and animal gut via formation of attaching and effacing lesions. EHEC strains use a type III secretion system to translocate a battery of effector proteins into the mammalian host cell, which subvert diverse signal transduction pathways implicated in actin dynamics, phagocytosis, and innate immunity. The genomes of sequenced EHEC O157:H7 strains contain two copies of the effector protein gene nleH, which share 49% sequence similarity with the gene for the Shigella effector OspG, recently implicated in inhibition of migration of the transcriptional regulator NF-kappaB to the nucleus. In this study we investigated the role of NleH during EHEC O157:H7 infection of calves and lambs. We found that while EHEC DeltanleH colonized the bovine gut more efficiently than the wild-type strain, in lambs the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. Using the mouse pathogen Citrobacter rodentium, which shares many virulence factors with EHEC O157:H7, including NleH, we observed that the wild-type strain exhibited a competitive advantage over the mutant during mixed infection. We found no measurable differences in T-cell infiltration or hyperplasia in colons of mice inoculated with the wild-type or the nleH mutant strain. Using NF-kappaB reporter mice carrying a transgene containing a luciferase reporter driven by three NF-kappaB response elements, we found that NleH causes an increase in NF-kappaB activity in the colonic mucosa. Consistent with this, we found that the nleH mutant triggered a significantly lower tumor necrosis factor alpha response than the wild-type strain.

RUNX3 Inactivation in Colorectal Polyps Arising Through Different Pathways of Colonic Carcinogenesis

OBJECTIVES: We hypothesized that RUNX3 inactivation by promoter hypermethylation in colorectal polyps is an early molecular event in colorectal carcinogenesis.
METHODS: RUNX3 protein expression was analyzed immunohistochemically in 50 sporadic colorectal polyps comprising 19 hyperplastic polyps (HPs), 14 traditional serrated adenomas (TSAs), and 17 sporadic traditional adenomas (sTAs) as well as in 19 familial adenomatous polyposis (FAP) samples from 10 patients showing aberrant crypt foci (ACF) (n=91), small adenomas (SmAds) (n=40), and large adenomas (LAds) (n=13). In addition, we assessed the frequency of promoter hypermethylation of RUNX3 by methylation-specific PCR (MSP) in all the 50 sporadic polyps as well as 38 microdissected FAP polyps comprising ACF, SmAds, and LAds obtained from 7 FAP samples. A total of 12 normal colon samples were also included for RUNX3 MSP analysis.
RESULTS: Compared to normal colon (2 of 12, 16%) and sTAs (3 of 17, 18%), HPs (15 of 19, 79%) and TSAs (8 of 14, 57%) displayed significant inactivation of RUNX3 (P<0.05). In FAP, RUNX3 inactivation was more frequently seen in ACF (78 of 91, 86%), SmAds (25 of 40, 62%), and LAds (6 of 13, 46%) compared to normal mucosa (0 of 19, 0%) in the same samples (all P<0.05). Promoter hypermethylation of RUNX3 was significantly higher in colorectal polyps (64 of 87, 74%) compared to normal colon (2 of 12, 16%) (P=0.001). Serrated polyps such as HPs (17 of 19, 89%) and TSAs (12 of 14, 86%) were significantly more methylated than sTAs (7 of 17, 44%) (P=0.004). RUNX3 hypermethylation was observed in 28 of the total 38 (74%) FAP polyps. Overall, RUNX3 promoter methylation correlated with inactivation of RUNX3 expression in sporadic (27 of 36, 75%) (P=0.022) and FAP (21 of 28, 75%) (P=0.021) polyps.
CONCLUSIONS: Our data suggest that RUNX3 inactivation due to promoter hypermethylation in colorectal polyps represents an early event in colorectal cancer (CRC) progression. In addition, epigenetic RUNX3 inactivation is a frequent event in the serrated colonic polyps as well as in the ACF of FAP polyps.

Electrical activity induced by nitric oxide in canine colonic circular muscle.

Nitric oxide generates slow electrical oscillations (SEOs) in cells near the myenteric edge of the circular muscle layer, which resemble slow waves generated by interstitial cells of Cajal (ICCs) at the submucosal edge of this muscle. The properties of SEOs were studied to determine whether these events are similar to slow waves. Rapid frequency membrane potential oscillations (MPOs; 16 +/- 1 cycles/min and 9.6 +/- 0.2 mV) were recorded from control muscles near the myenteric edge. Sodium nitroprusside (0.3 microM) reduced MPOs and initiated SEOs (1.3 +/- 0.3 cycles/min and 13.4 +/- 1.4 mV amplitude). SEOs were abolished by the guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (10 microM). MPOs were abolished by nifedipine (1 microM), whereas SEO frequency increased and the amount of depolarization decreased. BAY K 8644 (1 microM) prolonged SEOs and reduced their frequency. SEOs were abolished by Ni(2+) (0.5 mM), low Ca(2+) solution (0.1 mM Ca(2+)), cyclopiazonic acid (10 microM), and the mitochondrial uncouplers antimycin (10 microM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (1 microM). Oligomycin (10 microM) was without effect. These effects are similar to those described for colonic slow waves. Our results suggest that nitric oxide-induced SEOs are similar in mechanism to slow waves, an activity not previously thought to be generated by myenteric pacemakers.