Of these, BPH medication itself was implicated in the majority of the drug use problems

le; mRNA levels for each target gene were calculated, normalized to glyceraldehyde-3 phosphate dehydrogenase reference gene, according to IL-4 Expression and Effects in Human Osteoarthritic Chondrocytes trend toward a lower percentage of both IL-2Rcc and IL-13Ra1 positive cells in the mid/deep layers, thus suggesting a reduced ability to assemble full functional receptor. No statistical differences were found in the number of cells found to be positive for different IL-4R subunits between control and OA samples. To further investigate which type of IL-4 receptor is present in OA cartilage and controls, co-localization of IL-4R subunits was performed. In control and OA cartilage, the IL-4Ra chain is associated with either IL-2Rcc or IL13-Ra1 to form Type I or Type II IL-4 receptors as indicated by the merged signals. Evaluation of the maintenance of a properly differentiated phenotype and IL-4R subunits expression in high-density chondrocytes Compared to log phase p1 chondrocytes, high-density cultures expressed a higher protein level of SOX-9, the master chondrocytic transcription factor which drives the expression of collagen 2B and aggrecan and is therefore considered to be a marker of differentiated chondrocyte phenotype. The differentiated phenotype was maintained in the cultures because of the high-density seeding. The status of differentiated chondrocytes was also confirmed by evaluating the pattern of IL-4 receptor subunits: real time PCR analysis indicated that highdensity cultures expressed transcripts encoding IL-4Ra, IL-2R common c chain and IL-13Ra1 and therefore these cultures were indeed “primary”according to Guicheux and coworkers. Expression of the three chains was also confirmed at the protein level by flow cytometry, showing a much stronger expression of the chains PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19656604 constituting IL-4R type I, i.e. IL-4Ra and IL-2Rc. Confocal analysis confirmed the presence of a high level of IL-4Ra and the common c chain, with a certain degree of colocalization, thus suggesting the preferential usage of the type I receptor, a feature of differentiated chondrocytes, by chondrocytes cultured in this in vitro model.. IL-4 modulation on IL-1b-stimulated OA chondrocyte production of chemokines and ECM-degrading 481-53-8 biological activity enzymes 1) Chemokines. OA chondrocytes were analyzed for chemokine mRNA expression. Unstimulated and IL-4 treated chondrocytes expressed low levels of chemokine mRNA when analyzed by a sensitive Real Time PCR assay. In response to 24 h stimulation with IL-1b, chemokine mRNA was up-regulated in all the high-density chondrocyte cultures analyzed. Chondrocyte costimulation with IL-1b and IL-4 showed that the presence of IL-4 did not affect GROa/CXCL1 and IL-8/CXCL8 mRNA expression induced by IL-1b. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1a/ CCL3 and MIP-1b/CCL4 mRNA expression induced by IL-1b. ELISA was performed on chondrocyte culture supernatants to investigate the IL-4 modulation of IL-1b-induced chemokine production at protein levels. IL-1b induced secretion of GROa/ CXCL1 and IL-8/CXCL8 protein was unaffected by costimulation with IL-4. Conversely, IL-4 significantly inhibited IL-1b-induced secretion of RANTES/CCL5, in keeping with the effects on mRNA expression, whereas MIP-1a/CCL3 and MIP-1b/CCL4 chemokine production was not significantly modulated by IL-4. 2) Matrix Degrading Enzymes and inhibitors. To understand whether IL-4 is able to modulate ECM remodeling in response to pro-inflammatory stimuli, we evalua