Bovicin HC5 stock solutions (1 mg ml-1 in PBS (10 mM, pH 7.2)) were stored at −20°C until use. Protein concentration was determined using a bicinchoninic acid protein assay (Pierce Chemical Corp., Bonn, Germany), with bovine serum albumin as the standard. Experimental animals The BALB/c mice used in this study were housed in an animal facility at the Universidade Federal de Viçosa, according to standards and guidelines as set forth in the Animal #AZD1390 mw randurls[1|1|,|CHEM1|]# Welfare Legislation, the Guide for the Care and Use of Laboratory
Animals, the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the National Council for Animal Experimentation Control (CONCEA), following approval by the Institutional Animal Care and Use Committee
(IACUC) of the Universidade Federal de Viçosa under the protocol number BLZ945 cell line CEUA/UFV 97/2011. Five-week-old female BALB/c mice were randomly divided into three experimental groups: Group 1, untreated mice (negative control, NC group); Group 2, mice given purified bovicin HC5 (Bov group); Group 3, mice given ovalbumin (Sigma Chemicals Co., St. Louis, MO, 99% of purity) (positive control, PC group). Two independent experiments were performed and a total number of eight animals were used per experimental group. The sensitization procedure was developed based on previously established protocols [18]. Mice from the Bov group were subcutaneously sensitized with bovicin HC5 (4 μg/g animal weight/day or approximately 70 μg/animal [35]), while animals from the PC group were sensitized with ovalbumin (100 μl of a 1 mg ml-1 stock solution in sterile ultrapure water, or 100 μg OVA/animal). Aluminum hydroxide was used as adjuvant (50 μl; 20 mg ml-1 stock solution in sterile saline) at the first sensitization (day 0). After three weeks, each mouse group was subcutaneously boosted (without the use of adjuvant)
with the respective substances RANTES (second sensitization, day 21). The NC group was sensitized with sterile PBS (10 mM, pH 7.2), using the same procedure described above. PBS, bovicin HC5 or ovalbumin (100 μl) were administered without adjuvant to the NC, Bov and PC groups, respectively, by daily gavages (18-gauge stainless steel feeding needles). Oral administration started one week after the second sensitization (day 28) and continued for 30 days uninterruptedly (day 58). The mice were weekly weighted and behavior, general appearance and adverse reactions were monitored daily. Gut permeability The gut permeability was determined by the uptake of β-lactoglobulin (β-LG) following challenge ([36], with modifications]). At the end of the trial period (day 58), the animals of the NC, Bov and PC groups, were orally challenged with 200 μl of the respective samples (PBS, bovicin HC5 or ovalbumin).