Unpolarized Release Enhances Mucosal Responses Figure 4. Distribution of p24 antigen and vaccinia virus in mice. Six-week-old female BALB/c mice were inoculated i.n. with 16107 PFU of rVTT-gagpol per mouse. For determination of infection and expression kinetics, nose samples, nasal washes, lung samples, lung bronchial washes, and plasma were collected at specific times. p24 and virus titer were determined by ELISA and plaque assay, respectively (A, B, C, D, E, and F). For immunoperoxidase staining of p24 antigen, nose and lung tissues were fixed and prepared into paraffin slides, which were stained with immunoperoxidase (G, H, I, and J). Rabbit anti-p24 served as the first antibody. In detail, p24 distribution in (A) nose samples and nasal washes, (C) lung samples and lung bronchial washes, and (E) plasma. Virus distribution in (B) nose samples and nasal washes, (D) lung samples and lung bronchial washes, and (F) plasma. Nose section (G) 24 hours and (H) 72 hours after i.n. inoculation with rVTT-gagpol. (I) Nose section after PBS i.n. inoculation. (J) Lung section 72 hours after i.n. inoculation. Magnification, 1006. (x) designates epithelium, ( ) designates lamina propria, ( ) and designates lumen. These images are typical of those observed. There were five mice in each group. The experiments were performed in triplicate. doi:10.1371/journal.pone.0024296.g004 shown), colchicine treatment induced basolateral viral and p24 release. In conclusion, colchicine treatment 72 hours before rVTT-gagpol infection was found to cause unpolarized release of virus and p24 in vitro. Next, the impact of colchicine on antigen distribution was explored in mice epithelia. Colchicine was i.n. inoculated 72 hours before i.n. administration of rVTT-gagpol. PBS was used as a substitute for colchicine in the control group. Mice were euthanized at 1, 10, 24, 48, 72, and 96 hours. Then plasma, nasal washes, lung bronchial washes, and homogenized nose and lung tissues were collected. Levels of virus and p24 in these samples were measured by plaque assay and ELISA, respectively. The results demonstrated that, relative to the rVTT-gagpol group, the colchicine-treated group showed consistently higher p24 expression and infection rates in both nose and nasal washes (Figs. 6A and B), about 70 times higher at 24 hours and about 5 times higher at 72 hours. In the lung and lung bronchial washes, both p24 and virus levels were much higher after 48 hours. After 72 hours, both began to decrease, reaching their lowest points at 96 hours (Figs. 6C and D). No virus or p24 was detected in the blood of colchicine-treated or control mice (data not shown). Regarding p24 distribution in the nose, immunoperoxidase staining of p24 antigen showed that, at 24 hours, the colchicine- treated group expressed p24 in the epithelium at a much higher rate than the group immunized with rVTT-gagpol alone did. The level of p24 in the lamina propria of both groups was low (Figs. 6E and F). At 72 hours, the colchicine-treated group expressed p24 at much higher levels than the rVTT-gagpol immunization group in both the epithelia and the lamina propria (Figs. 6G and H). Figure 5. Comparison of distribution of differently treated cell layers after rVTT-gagpol infection in vitro. Caco-2 cell layers were infected with rVTT-gagpol 14–16 days after polarization to form the polarized group. A similar group was treated with colchicine (0.08 mg/ml) apically 72 hours before rVTT-gagpol infection to form the polarized + colchicine group. A 3-day Caco-2 transwell culture was infected with rVTT- gagpol to form the unpolarized group. The data shown are the sums of the 48-hour and 72-hour values. (A) p24 distribution. (B) Virus distribution. (C) TER. Apical, cellular, and basolateral samples of the three groups were compared. T-testing was conducted to compare the polarized + colchicine group and unpolarized group to the polarized group. Statistical significance: (P,0.05) in an unpaired test is indicated by an asterisk. There were two wells for every group. The experiments were performed twice. doi:10.1371/journal.pone.0024296.g005 PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24296