Dietary inclusion of fermentable carbohydrates (fCHO) is reported to reduce large intestinal formation of putatively toxic metabolites derived from fermentable proteins (fCP). However, the influence of diets high in fCP concentration on epithelial response and interaction with fCHO is still unclear. Thirty-two weaned piglets were fed 4 diets in a 2 × 2 factorial design with low fCP/low fCHO [14.5% crude protein (CP)/14.5% total dietary fiber (TDF)]; low fCP/high fCHO (14.8% CP/16.6% TDF); high fCP low fCHO (19.8% CP/14.5% TDF); and high fCP/high fCHO (20.1% CP/18.0% TDF) as dietary treatments. After 21-23 d, pigs were killed and colon digesta and tissue samples analyzed for indices of microbial ecology, tissue expression of genes for cell turnover, cytokines, mucus genes (MUC), and oxidative stress indices. Pig performance was unaffected by diet. fCP increased (P < 0.05) cell counts of clostridia in the Clostridium leptum group and total short and branched chain fatty acids, ammonia, putrescine, histamine, and spermidine concentrations, whereas high fCHO increased (P < 0.05) cell counts of clostridia in the C. leptum and C. coccoides groups, shifted the acetate to propionate ratio toward acetate (P < 0.05), and reduced ammonia and putrescine (P < 0.05). High dietary fCP increased (P < 0.05) expression of PCNA, IL1β, IL10, TGFβ, MUC1, MUC2, and MUC20, irrespective of fCHO concentration. The ratio of glutathione:glutathione disulfide was reduced (P < 0.05) by fCP and the expression of glutathione transferase was reduced by fCHO (P < 0.05). In conclusion, fermentable fiber ameliorates fermentable protein-induced changes in most measures of luminal microbial ecology but not the mucosal response in the large intestine of pigs.

Regulatory T cells (Treg) are supportive to cancer development in most tissues, but their role in colitisassociated colon cancer (CAC) remains unclear. In this study, we investigated the role of CD4

As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the anti-inflammatory function of the AhR in the context of systemic endotoxin shock, AhR and AhRR act in concert to dampen intestinal inflammation. Specifically, AhRR contributes to the maintenance of colonic intraepithelial lymphocytes and prevents excessive IL-1β production and Th17/Tc17 differentiation. In contrast, the AhRR enhances IFN-γ-production by effector T cells in the inflamed gut. Our findings highlight the physiologic importance of cell-type specific balancing of AhR/AhRR expression in response to microbial, nutritional and other environmental stimuli.

Several studies have shown that specific mRNA sequences can be successfully detected in formalin-fixed, paraffin-embedded tissues using reverse transcriptase-polymerase chain reaction (RT-PCR). Here, we test the hypothesis that gene expression levels can be accurately quantified in formalin-fixed, paraffin-embedded tissues by determining the ratio between the copy number of the mRNA molecule of interest and the mRNA copy number of a so-called housekeeping gene. The mRNA copy numbers of the variably expressed multiple drug resistance gene (MDR)-1 and four housekeeping genes (hypoxanthine phosphoribosyl-transferase-1, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, and elongation factor-1a) were quantified by real-time-quantitative RT-PCR before and after formalin-fixation and paraffin-embedding of 576 tissue samples (heart, kidney, spleen, liver) from three beagle dogs. The results indicate that fixation and embedding drastically altered the ratios between the different mRNA copy numbers and that the relative expression levels of MDR-1 per any of the housekeeping genes were artificially increased or decreased up to more than tenfold. It would thus appear questionable to normalize quantitative expression data from fixed and embedded tissues by using housekeeping genes as reference. In contrast, tissue autolysis of up to 24 h and long-term storage of embedded tissues of up to 20 years had no additional effects.

Rapid contact- and contamination-free procurement of histologic material for proteomic and genomic analysis can be achieved by laser microdissection of the sample of interest followed by laser-induced transport (laser pressure catapulting). The dynamics of laser microdissection and laser pressure catapulting of histologic samples of 80 mum diameter was investigated by means of time-resolved photography. The working mechanism of microdissection was found to be plasma-mediated ablation initiated by linear absorption. Catapulting was driven by plasma formation when tightly focused pulses were used, and by photothermal ablation at the bottom of the sample when defocused pulses producing laser spot diameters larger than 35 microm were used. With focused pulses, driving pressures of several hundred MPa accelerated the specimen to initial velocities of 100-300 m/s before they were rapidly slowed down by air friction. When the laser spot was increased to a size comparable to or larger than the sample diameter, both driving pressure and flight velocity decreased considerably. Based on a characterization of the thermal and optical properties of the histologic specimens and supporting materials used, we calculated the evolution of the heat distribution in the sample. Selected catapulted samples were examined by scanning electron microscopy or analyzed by real-time reverse-transcriptase polymerase chain reaction. We found that catapulting of dissected samples results in little collateral damage when the laser pulses are either tightly focused or when the laser spot size is comparable to the specimen size. By contrast, moderate defocusing with spot sizes up to one-third of the specimen diameter may involve significant heat and ultraviolet exposure. Potential side effects are maximal when samples are catapulted directly from a glass slide without a supporting polymer foil.