2 mM of

dNTP, 0.1–0.2 μM of each primer, 2–3.75 mM of MgCl2, 1.5 units of Taq Platinum DNA polymerase (Invitrogen), 0.04 μL of Sybr Green 100×, 2 μL of buffer 10× 50 mM of KCl, 10 mM of Tris–HCl pH 9.0 (Invitrogen), and 0.5 μL of dimethyl sulfoxide (DMSO; Sigma). For each sample, the cycle threshold (Ct) mean was obtained and normalized to a reference gene. Three reference genes were evaluated: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), HPRT-1 (hypoxanthine phosphoribosyltransferase 1) and RPL-19 (ribosomal protein L19). The relative quantification was evaluated by mathematic modeling based on JQ1 the PCR efficiencies (E) of the target and endogenous genes and on Ct variation of samples from the experimental groups, according to Pfaffl et al. (2002). For this analysis, the Relative Expression Software Tool (REST©) was used, which applies a nonparametric significance test called the Pair Wise Fixed Reallocation Randomisation Test©. LBH589 Histological data (eosinophils, mast cells and globule leukocytes) were analyzed by the GLM procedure using the SAS program (SAS, 2002/2003). The average eosinophil and mast cell counts were 30.96 (±S.D. 5.55) and 10.31 (±S.D. 9) in the non-infected group and 28.41 (±S.D. 2.35) and 17.12 (±S.D. 1.95) in the infected group, respectively. No significant

differences were found between groups for the eosinophil counts (p = 0.30) and mast cell counts (p = 0.32) in the mucous membrane of the abomasum ( Fig. 1). No globular leukocyte was observed in the slides in any group. Among the three reference genes tested to be used in the relative quantification, the RPL-19 gene was chosen because it presented more constant Ct values (19 ± S.D. 2.3 in the abomasum and 20.7 ± S.D. 1.2

in lymph node) than the other two genes analyzed. Relative quantification of target genes showed that IL-4 (14×; p = 0.002), IL-13 (26×; p = 0.003) and TNF-α (10×; p = 0.03) were up-regulated in the abomasal lymph nodes of the infected group in comparison with control group ( Fig. 2). In the abomasum tissue, IL-13 was up-regulated (4.8×; p = 0.03) in the infected group and TNF-α was down-regulated (4.0×; p = 0.032) in the same group ( Fig. 3). The mRNA levels of the other genes were not influenced by H. placei Rebamipide larval exposure in the abomasal lymph node, as well as in the abomasum tissue (p > 0.05). In this study, we compared cytokine gene expression of Nellore calves in primary infection with H. placei infections caused by helminths have been studied in many species and have usually been associated with Th2 response in infected animals. In cattle, these infections are not characterized by a severe immune response and most become chronic. Reduction in the number of adult parasites begins after exposure to the parasite and at the same time there is a significant increase in eosinophil, globular leukocyte and mast cell counts in the sites of the infection ( Grencis, 2001 and Bricarello et al., 2004).