Figure legends
Figure 1 Schedule of therapeutic (A), prophylactic (B), vaccination and grouping (C) for For t 2 DNA with skin patches. Time points of sensitization with rFor t 2/alum, videotaping, and sacrificing are indicated.
Figure 2 Scratching bouts of mice from (A) therapeutic and (B) prophylactic groups. The scratching counts were recorded for 20 minutes after induction by intradermal injection of rFor t 2 at the endpoints of the two experiments. **p <0.01 by one-way analysis of variance with the Bonferroni multiple range test.
Figure 3 Change in serum total IgE and For t 2-specific IgG2a antibodies of mice from (A) therapeutic or (B) prophylactic groups as indicated in weeks. * denotes p <0.05, **denotesp <0.01, ns denotes not statistically significant by one-way analysis of variance with the Bonferroni multiple range test.
Figure 4 Effects of For t 2 vaccine on histopathology of challenged skins from (A) therapeutic or (B) prophylactic groups by H&E staining. Figures showed the representative abdominal skin sections obtained 48 hours after intradermal challenge under 100 x and 400 x light microscope. Moreover, the infiltrating inflammatory cells were quantified under 400-fold view from each group. The statistical significance of differences between groups was assessed by the Bonferroni multiple range test. ** denoted p<0.01, ns denotes not statistically significant.
Figure 5 mRNA and protein expression levels of Th2 and Th1 cytokines in For t 2-stimulated splenocytes from (A) therapeutic and (B) prophylactic groups by real-time PCR and ELISA. Real-time PCR data are expressed as mean of increasing folds ± SEM. The statistical significance of differences between groups was assessed by the Bonferroni multiple range test. * denotes p<0.05, ** denotes p<0.01, ns denotes not statistically significant.
Figure 6 mRNA and protein expression levels of IL-10 and mRNA of the FOXP3 transcription factor in For t 2-stimulated splenocytes from (A) therapeutic or (B) prophylactic groups by real-time PCR and ELISA, respectively. Real-time PCR data are expressed as mean of increasing folds ± SEM. The statistical significance of differences between groups was assessed by the Bonferroni multiple range test. * denotes p<0.05, ns denotes not statistically significant.
Figure S1 Dose finding schedule of For t 2 DNA patch vaccine on rFor t 2-sensitized mice in the pilot study.
Figure S2 The representative skin images show before (A) and after (B) patch application. The For t 2 DNA vaccine was delivered onto the pre-shaved abdominal skin through a Fin chamber for 1 hour.
Figure S3 H&E and immunohistochemotry (IHC) staining with rabbit anti-For t 2 polyclonal antibody of skin and spleen sections under 400x light microscope. For t 2 protein expression of skin and spleen after vaccination was examined after skin patch for 24 and 48 hours in mouse abdominal skin.
Figure S4 Counts of scratching bouts from each group of mice before (day 0) and after (day 61) induction by intradermal injection of rFor t 2. The scratching behavior was videotaped and data represent the number of scratches for 20 minutes after allergen challenge. **p <0.01; ***p <001 by one-way analysis of variance with the Bonferroni multiple range test.
Figure S5 (A) Total IgE and (B) For t 2-specific IgG2a antibodies in the sera at week 9 determined by ELISA. Results are mean ±SD of 5 mice from each group. * denoted p <0.05, **denotedp <0.01, ns denotes not statistically significant by one-way analysis of variance with the Bonferroni multiple range test.
Figure S6 A. Effects of For t 2 vaccine on histopathology of abdominal skins by H&E staining. Figures showed the representative skin sections obtained 48 hours after intradermal challenge under 100 x and 400 x light microscope. B. The infiltrating inflammatory cells were quantified under 400-fold view from each mouse (n=5 per group). The statistical significance of differences between non-vaccinated group and vaccinated groups was assessed by the Bonferroni multiple range test. * denotes p<0.05, ** denotes p<0.01, ns denotes not statistically significant.