Gene Expression Profiles of Colonic Mucosa in Healthy Young Adult and Senior Dogs

Background

We have previously reported the effects of age and diet on nutrient digestibility, intestinal morphology, and large intestinal fermentation patterns in healthy young adult and senior dogs. However, a genome-wide molecular analysis of colonic mucosa as a function of age and diet has not yet been performed in dogs.

Methodology/Principal Findings

Colonic mucosa samples were collected from six senior (12-year old) and six young adult (1-year old) female beagles fed one of two diets (animal protein-based vs. plant protein-based) for 12 months. Total RNA in colonic mucosa was extracted and hybridized to Affymetrix GeneChip® Canine Genome Arrays. Results indicated that the majority of gene expression changes were due to age (212 genes) rather than diet (66 genes). In particular, the colonic mucosa of senior dogs had increased expression of genes associated with cell proliferation, inflammation, stress response, and cellular metabolism, whereas the expression of genes associated with apoptosis and defensive mechanisms were decreased in senior vs. young adult dogs. No consistent diet-induced alterations in gene expression existed in both age groups, with the effects of diet being more pronounced in senior dogs than in young adult dogs.

Conclusion

Our results provide molecular insight pertaining to the aged canine colon and its predisposition to dysfunction and disease. Therefore, our data may aid in future research pertaining to age-associated gastrointestinal physiological changes and highlight potential targets for dietary intervention to limit their progression.     

Transcriptomic Meta-Analysis of Multiple Sclerosis and Its Experimental Models

Background

Multiple microarray analyses of multiple sclerosis (MS) and its experimental models have been published in the last years.

Objective

Meta-analyses integrate the information from multiple studies and are suggested to be a powerful approach in detecting highly relevant and commonly affected pathways.

Data sources

ArrayExpress, Gene Expression Omnibus and PubMed databases were screened for microarray gene expression profiling studies of MS and its experimental animal models.

Study eligibility criteria

Studies comparing central nervous system (CNS) samples of diseased versus healthy individuals with n >1 per group and publically available raw data were selected.

Material and Methods

Included conditions for re-analysis of differentially expressed genes (DEGs) were MS, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in rats, proteolipid protein-induced EAE in mice, Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), and a transgenic tumor necrosis factor-overexpressing mouse model (TNFtg). Since solely a single MS raw data set fulfilled the inclusion criteria, a merged list containing the DEGs from two MS-studies was additionally included. Cross-study analysis was performed employing list comparisons of DEGs and alternatively Gene Set Enrichment Analysis (GSEA).

Results

The intersection of DEGs in MS, EAE, TMEV-IDD, and TNFtg contained 12 genes related to macrophage functions. The intersection of EAE, TMEV-IDD and TNFtg comprised 40 DEGs, functionally related to positive regulation of immune response. Over and above, GSEA identified substantially more differentially regulated pathways including coagulation and JAK/STAT-signaling.

Conclusion

A meta-analysis based on a simple comparison of DEGs is over-conservative. In contrast, the more experimental GSEA approach identified both, a priori anticipated as well as promising new candidate pathways.     

Attaching and Effacing Escherichia coli Downregulate DNA Mismatch Repair Protein In Vitro and Are Associated with Colorectal Adenocarcinomas in Humans

Background

Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.

Methodology/Principal Findings

When co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05).

Significance/Conclusions

The ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.     

Effects of Sodium Butyrate and Its Synthetic Amide Derivative on Liver Inflammation and Glucose Tolerance in an Animal Model of Steatosis Induced by High Fat Diet

Background & Aims

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Insulin resistance (IR) appears to be critical in its pathogenesis. We evaluated the effects of sodium butyrate (butyrate) and its synthetic derivative N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) in a rat model of insulin resistance and steatosis induced by high-fat diet (HFD).

Methods

After weaning, young male Sprague-Dawley rats were divided into 4 groups receiving different diets for 6 weeks: 1. control group (standard diet); 2. HFD; 3. HFD plus butyrate (20 mg/kg/die) and 4. HFD plus FBA (42.5 mg/Kg/die, the equimolecular dose of butyrate). Liver tissues of the rats were analyzed by Western blot and real-time PCR. Insulin resistance, liver inflammation and Toll-like pattern modifications were determined.

Results

Evaluation of these two preparations of butyrate showed a reduction of liver steatosis and inflammation in HFD fed animals. The compounds showed a similar potency in the normalisation of several variables, such as transaminases, homeostasis model assessment for insulin resistance index, and glucose tolerance. Both treatments significantly reduced hepatic TNF-α expression and restored GLUTs and PPARs, either in liver or adipose tissue. Finally, FBA showed a higher potency in reducing pro-inflammatory parameters in the liver, via suppression of Toll-like receptors and NF-κB activation.

Conclusions

Our results demonstrated a protective effect of butyrate in limiting molecular events underlying the onset of IR and NAFLD, suggesting a potential clinical relevance for this substance. In particular, its derivative, FBA, could represent an alternative therapeutic option to sodium butyrate, sharing a comparable efficacy, but a better palatability and compliance.     

Differential expression of microRNAs in tumors from chronically inflamed or genetic (APC(Min/+)) models of colon cancer

Background

Chronic inflammation associated with ulcerative colitis predisposes individuals to increased colon cancer risk. The aim of these studies was to identify microRNAs that are aberrantly regulated during inflammation and may participate in transformation of colonic epithelial cells in the inflammatory setting.

Methodology/Principal Findings

We have use quantitative PCR arrays to compare microRNA (miRNA) expression in tumors and control colonic epithelial cells isolated from distal colons of chronically inflamed mice and APC^Min/+ mice. Rank order statistics was utilized to identify differentially regulated miRNAs in tumors that arose due to chronic inflammation and/or to germline APC mutation. Eight high priority miRNAs were identified: miR-215, miR-137, miR-708, miR-31, and miR-135b were differentially expressed in APC tumors and miR-215, miR-133a, miR-467d, miR-218, miR-708, miR-31, and miR-135b in colitis-associated tumors. Four of these (miR-215, miR-708, miR-31, and miR-135b) were common to both tumors types, and dysregulation of these miRNAs was confirmed in an independent sample set. Target prediction and pathway analysis suggests that these microRNAs, in the aggregate, regulate signaling pathways related to MAPK, PI3K, WNT, and TGF-β, all of which are known to be involved in transformation.

Conclusions/Significance

We conclude that these four miRNAs are dysregulated at some very early stage in transformation of colonic epithelial cells. This response is not dependent on the mechanism of initiation of transformation (inflammation versus germline mutation), suggesting that the miRNAs that we have identified are likely to regulate critical signaling pathways that are central to early events in transformation of colonic epithelial cells.