Use of reference gene expression in rat distal colon after radiation exposure: a caveat.

Research on the effects of ionizing radiation exposure includes transcriptome studies using real-time reverse transcription polymerase chain reaction (RT-PCR). These studies require the use of a reference gene that normalizes for cDNA quantity and corrects for transcription between different samples. In this study, several criteria are reviewed that allow the choice of a reference gene. With the example of five genes selected from the widely used standard housekeeping genes, Gapd (glyceraldehyde-3-phosphate dehydrogenase), Hprt (hypoxanthine-guanine phosphoribosyl transferase), cyclophilin A, AcRP0 (acidic ribosomal protein P0) and 18S, we show that the use of a reference gene without a preliminary study is hazardous. We have shown in rat colon after a hemi-body irradiation that expression of a gene of interest, the serotonin receptor type 1F (5-HT(1F)), was either increased or unchanged, with the result depending on the reference gene used. This work has led us to propose the use of two reference genes, a ribosomal gene, 18S, and another gene with a level of expression closer to that of the gene of interest. The methodology reported here may be applied to other studies of gene expression levels to evaluate the effects of experimental treatment on the expression of potential reference genes.

Increased prokineticin 2 expression in gut inflammation: role in visceral pain and intestinal ion transport.

BACKGROUND: Prokineticin 2 (PROK2) is an inflammatory cytokine-like molecule expressed predominantly by macrophages and neutrophils infiltrating sites of tissue damage. Given the established role of prokineticin signaling on gastrointestinal function, we have explored Prok2 gene expression in inflammatory conditions of the gastrointestinal tract and assessed the possible consequences on gut physiology. METHODS: Prokineticin expression was examined in normal and colitic tissues using qPCR and immunohistochemistry. Functional responses to PROK2 were studied using calcium imaging and a novel antagonist, Compound 3, used to determine the role of PROK2 and prokineticin receptors in inflammatory visceral pain and ion transport. KEY RESULTS: Prok2 gene expression was up-regulated in biopsy samples from ulcerative colitis patients, and similar elevations were observed in rodent models of inflammatory colitis. Prokineticin receptor 1 (PKR1) was localized to the enteric neurons and extrinsic sensory neurons, whereas Pkr2 expression was restricted to sensory ganglia. In rats, PROK2-increased intracellular calcium levels in cultured enteric and dorsal root ganglia neurons, which was blocked by Compound 3. Moreover, PROK2 acting at prokineticin receptors stimulated intrinsic neuronally mediated ion transport in rat ileal mucosa. In vivo, Compound 3 reversed intracolonic mustard oil-induced referred allodynia and TNBS-induced visceral hypersensitivity, but not non-inflammatory, stress-induced visceral pain. CONCLUSIONS & INFERENCES: Elevated Prok2 levels, as a consequence of gastrointestinal tract inflammation, induce visceral pain via prokineticin receptors. This observation, together with the finding that PROK2 can modulate intestinal ion transport, raises the possibility that inhibitors of PROK2 signaling may have clinical utility in gastrointestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.

Direct bacterial killing in vitro by recombinant Nod2 is compromised by Crohn's disease-associated mutations.

BACKGROUND: A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn's disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn's-associated mutations. Wild-type, but not Crohn's-associated, Nod2 LRR domains directly interacted with bacteria in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity. CONCLUSIONS/SIGNIFICANCE: The lack of anti-bacterial activity demonstrated with Crohn's-associated Nod2 mutations in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphisms with the disease.