Endothelial cell cultures that had grown confluently were harvested with trypsin-EDTA. Three-dimensional MK-2206 nmr collagen assays and stainings were performed as described [9]. Supernatants were collected for further analyses. For experiments with HUVECs, collagen gels were first cultured for 2 weeks to allow tumour colony

formation, after which RPMI/10% supplemented with 10 ng/mL bFGF and 10 U/mL heparin was added for 24 h. HUVECs were added, and formed a confluent layer in 20 h, after which neutrophils and Ab were added. To measure chemotaxis (specific neutrophil migration) a Boyden Chamber assay was used as described before [34] Fluor-escence was measured in a fluorimeter (excitation wavelength 485 nm/emission wavelength at 520 nm). Lactoferrin ELISA was performed as described [9]. IL-1β, TNF-α and IL-8 ELISA were performed according the manufacture’s instructions (Biosource, Camarillo, CA, USA). Data are shown as mean ± standard deviation (SD) or shown as mean ± standard error of the mean (SEM) as indicated. Statistical differences were determined using two-tailed unpaired Student’s t-tests (two groups) or ANOVA (more than two groups), followed by Bonferroni post hoc tests. *p < 0.05; **p < 0.01. This work was supported by the Dutch Cancer Society (UU2001-2431), Stichting VUmc Cancer Center Amsterdam and the Netherlands Organization for Scientific HCS assay Research

(VENI 916.36.079, M.A Otten and VIDI 016.086.320, J.E. Bakema). The authors declare no financial or commercial conflicts of interest. “
“n-Butyrate deriving from bacterial fermentation in the mammalian intestine is a key determinant in gastrointestinal homeostasis. We examined the effects of this short-chain fatty acid and Toll-like receptor 2 (TLR) and TLR4 engagement on inflammatory/immunity-associated genes, cyclo-oxygenases (COXs), prostaglandins (PGs) and leukotrienes

(LTs) in human monocytes. Before RNA isolation, freshly isolated human monocytes were co-incubated for different time-points with 1 mm n-butyrate alone or in combination with bacterial stimuli. Based on a knowledge-driven approach, a signature of 180 immunity/inflammation-associated genes was picked and real-time PCR analysis was performed. Pathway analysis was carried out Isotretinoin using a web-based database analysing program. Based on these gene expression studies the findings were evaluated at the protein/mediator level by Western blot analysis, FACS and ELISA. Following co-incubation with n-butyrate and lipopolysaccharide, key enzymes of the eicosanoid pathway, like PTGS2 (COX-2), TXS, ALOX5, LTA4H and LTC4S, were significantly up-regulated compared with stimulation with lipopolysaccharide alone. Furthermore, release of the lipid mediators PGE2, 15d-PGJ2, LTB4 and thromboxane B2 was increased by n-butyrate.