5 μCi/well of [methyl-3H] thymidine (1 Ci/mmol; China Institute of Atomic Energy, China) for the last 16 hrs of cultivation. The cultured cells were collected and put on the glass fiber membrane for dry at 70 °C in the oven. The radioactivity was counted by a liquid scintillation counter (Beckman Coulter, USA). [Methyl-3H] thymidine incorporation was calculated in cpm. Stimulatory index: Cpm of experimental 1 well − cpm of blank control well/cmp of blank control well. Level of total IgA in the supernatant of homogenized small

intestine was analyzed using sIgA radioimmunoassay kit (China Institute of Atomic Energy, China) according manufacture’s instruction. M. tuberculosis H37Rv challenge was referred to [18] with slightly modifications. Briefly, BALB/c mice were orally administrated three times at 2-week intervals either with saline control, pcDNA3.1 Cyclopamine or pcDNA3.1+/Ag85A DNA encapsulated by liposome. Mice were then rested for 6 weeks after the third DNA immunization and challenged

intravenously in a lateral tail vein with 106 CFU of M. tuberculosis H37Rv grown as a surface pellicle for 2 weeks on synthetic Sauton medium and stored as a stock solution at −70 °C in glycerol. 3 weeks after challenge, mice were sacrificed, lung homogenate dilutions were plated on 7H11 Middlebrook Trametinib clinical trial agar supplemented with albumin-oleic acid-dextrose-catalase-enrichment broth (Difco, Detroit, MI). Petri dishes were incubated for 4 weeks in sealed plastic bags at 37 °C, and colonies were counted

visually. For statistical analysis (Student’s t test), data obtained from two or three dilutions were used to calculate the mean log10 CFU values per lung. Data are expressed as mean log10 values per experimental group (each consisting of 5 mice). Statistical analysis (SPSS 11.0) of the microscopic significance was applied to evaluate the excitation intensity of fluorescence between experimental and control areas. Initially, we try to investigate efficacy of delivery system of liposomal-pcDNA3.1+/Ag85A DNA to intestinal tract. C57BL/6 mice were orally administrated 3 times at 2-week intervals nearly with either saline, pcDNA3.1 or pcDNA3.1+/Ag85A DNA encapsulated in liposome. Expression of Ag85A antigen in the epithelium of small intestine was examined after final immunization by immunohistochemistry method. As shown in Fig. 1, Ag85A protein was intensively expressed in Peyer’s patches (Fig. 1 A-c, black arrows) and epithelium (Fig. 1, black and white arrows) of the small intestine. In contrast, no positive staining cells in Peyer’s patches (Fig. 1A (a and b)) and epithelium (Fig. 1A (d and e)) were found in those of two control mice. The quantitatively calculated density of positive staining cells in Peyer’s patches (Fig. 1B (c)) and epithelium (Fig. 1B (f1 and f2)) were also significantly higher as compared to those in normal control mice and plasmid control mice. These results indicated that the pcDNA3.