pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis.

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD.

A novel approach for reliable microarray analysis of microdissected tumor cells from formalin-fixed and paraffin-embedded colorectal cancer resection specimens.

We present a novel approach for microarray analysis of RNA derived from microdissected cells of routinely formalin-fixed and paraffin-embedded (FFPE) cancer resection specimens. Subsequent to RNA sample preparation and hybridization to standard GeneChips (Affymetrix), RNA samples yielded 36.43 +/- 9.60% (FFPE), 49.90 +/- 4.43% (fresh-frozen), and 53.9% (cell line) present calls. Quality control parameters and Q-RT-PCR validation demonstrated reliability of results. Microarray datasets of FFPE samples were informative and comparable to those of fresh-frozen samples. A systematic measurement difference of differentially processed tissues was eliminated by a correction step for comparative unsupervised data analysis of fresh-frozen and FFPE samples. Within FFPE samples, unsupervised clustering analyses clearly distinguished between normal and malignant tissues as well as to further separate tumor samples according to histological World Health Organization (WHO) subtypes. In summary, our approach represents a major step towards integration of microarrays into retrospective studies and enables further investigation of the relevance of microarray analysis for clinico-pathological diagnostics.