Figure 4: Immunization with rSSP4 induces TGF-β mRNA expression and TGF-β, IL-10, and IFN-γ production. RNA from 24 h cultured spleen cells was extracted and used to determine TGF-β expression by RT-PCR, and culture supernatants were harvested and assessed for cytokines production by ELISA ( ). (a) TGF-β mRNA expression by wild type spleen cells (upper panel); densitometric analysis of TGF-β mRNA expression (lower panel). Culture supernatants were harvested after 72 h of culture and assessed for TGF-β, IL-10, and IFN-γ production by ELISA. (b) TGF-β production was measured in culture supernatants (SNs) of spleen cells from rSSP4 immunized mice under rSSP4 restimulation condition and in sera from immunized mice. Graphs show values in pg/mL . (c) and (d) IL-10 and IFN-γ production, respectively, by wild type mice in immunized (full bars) and nonimmunized mice (open bars) under different conditions: NS (non-stimulated), ConA, or rSSP4. Results shown are the average of at least six independent experiments done in triplicate. Differences between groups are considered significant at less than 0.05 and are represented by * , and *** in a Student -test.