The left hind paw of the same animal was used as control, receiving an injection of 30 μL of dialysis buffer. In some experiments the animals were pre-treated with anti-inflammatory drugs given subcutaneously 1 h (esculetin, 50 mg/kg, Sigma) or 4 h (dexamethasone, 0.5 mg/kg, Sigma) before rHPU administration. Increased paw thickness due to edema was measured with a micrometer (Mitutoyo, 0–25 mm,

with 0.002 mm increments) at the AZD1208 in vivo indicated time intervals after the injections. Paw edema was expressed as the difference between the thickness of right and left paws of the same animal. Thus the results represent the net edema (in mm) induced by HPU. Mice paws injected with 45 μg HPU or 30 μL dialysis buffer were fixed in 10% formalin for paraffin block preparation. Sections of 5 μm were stained with hematoxilin–eosin, and studied under light microscopy at the Pathology Service of the Faculty of Veterinary, Universidade Federal Ganetespib manufacturer of Rio Grande do Sul, Porto Alegre, RS, Brazil. All procedures involving animals were conducted in strict accordance to Brazilian legislation (Law no. 6.638/1979) and in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (www.nc3rs.org.uk/ARRIVE),

developed by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Data were analyzed by ANOVA followed by the Tukey–Kramer test using the Instat Graph Pad software and values of p < 0.05 were considered statistically significant. To investigate whether purified rHPU possesses pro-inflammatory activity the model of mouse paw edema was chosen. Fig. 1 shows the time course and dose-dependency curves of paw edema induced by subplantar injection of rHPU in mouse hind paws. As low as 0.5 μg (0.4 pmol)

of injected protein produced an intense paw edema in some animals. At a dose of 45 μg, the rHPU-induced edema peaked at 4–6 h and lasted more than 24 h. Histopathological analysis of the paw edema showed an intense neutrophil infiltration (Fig. 2). Pretreatment of mice with dexamethasone, or with the lipoxygenase inhibitor esculetin, produced significant reduction in the paw edema indicating that eicosanoids, particularly lipoxygenase Carnitine palmitoyltransferase II metabolites, mediate the pro-inflammatory activity of rHPU (Table 1). H. pylori infection induces an acute neutrophil-dominant inflammation and neutrophil density correlates with tissue damage ( Nielsen and Andersen, 1992). H. pylori whole extracts were shown to stimulate chemokine production and activation of neutrophils in vitro ( Shimoyama et al., 2003). Fig. 3A shows that rHPU stimulated human neutrophil migration in a dose-dependent manner. The chemotactic effect of 100 nM rHPU (55.6 ± 6.8 neutrophils/field) was equivalent to that induced by 100 nM fMLP (63 ± 7.2 neutrophils/field). This property of HPU is independent of its ureolytic activity, as rHPU treated with active-site inhibitors promoted the same migration profile ( Fig. 3A).