Figure 1.
Berenil treatment decreases the percentage of CD25+ and FoxP3+ cells in the spleens of infected mice.
Splenocytes from Trypanosoma congolense-infected BALB/c and C57BL/6 mice either treated or untreated with Berenil were routinely stained (see materials and methods) directly ex vivo with fluorochrome-conjugated mAb against CD4, CD25 and Foxp3 and analyzed by flow cytometry. Shown are representative dot plots showing expression of CD25 on total (A and D) and CD4+ (B and E) lymphocytes. Representative dot plot of CD25+ and Foxp3+ cells gated on CD4+ lymphocytes (C and F). The bar graphs represent the cumulative percentages of CD25+ and Foxp3+ cells from 3–5 mice per group. The results presented are representative of 4 different experiments with similar results. Bars show mean +/−SEM; *, p<0.05; **, p<0.01.
Figure 2.
Treatment with Berenil reduces serum pro-inflammatory cytokine levels in T. congolense-infected mice.
BALB/c (A–D) and C57BL/6 (E–H) mice were infected with T. congolense and treated with Berenil on day 5 post-infection. Eight days post-infection (3 days after Berenil treatment), mice were sacrificed and blood samples were taken via cardiac puncture to obtain serum. The levels of IL-6 (A and E), TNF (B and F), IL-12 (C and G) and IFN-γ (D and H) in the serum were determined by sandwich ELISA. Uninfected BALB/c mice were also treated with Berenil, sacrificed 3 days later and serum levels of IL-6 (I) and TNF (J) were determined by ELISA. The results presented are representative of 3 (A–H) and 2 (I and J) independent experiments (n = 4 mice per group) with similar results. Bars show mean +/−SEM; *, p<0.05; **, p<0.01; ***, p<0.001. N.D. = not detected (i.e. below 15 pg/ml).
Figure 3.
Berenil treatment suppresses IL-6, IL-12 and TNF production by CD11b+ spleen cells from T. congolense infected mice.
Highly enriched (by positive selection) CD11b+ splenocytes from Berenil treated or non-treated infected (A–F) and uninfected (G and H) mice were cultured for 18 hours with or without LPS (5µg/ml) and the culture supernatant fluids were assayed for IL-6 (A, D and G), TNF (B, E and H) and IL-12p40 (C and F) by ELISA. Top (A, B and C) and middle (D, E and F) panels are data obtained with splenocytes from infected BALB/c and C57BL6 mice, respectively. The bottom panel (G and H) shows data from uninfected (naïve) C57BL/6 mice. The results presented are representative of 3 independent experiments (n = 4 mice per group) with similar results. Bars show mean +/−SEM; *, p<0.05; **, p<0.01; ***, p<0.001. N.D. = not detected (i.e. below 15 pg/ml).
Figure 4.
Berenil treatment reduces spontaneous pro-inflammatory cytokine secretion by kupffer cells from T. congolense-infected mice.
BALB/c mice were infected with T. congolense and treated with Berenil on day 5 post-infection. Eight days post-infection (3 days after Berenil treatment), mice were sacrificed and hepatic mononuclear cells were isolated by Percoll gradient centrifugation. Kupffer cells were further enriched by positive selection using CD11b-coated beads, cultured for 24 hr and the concentration of IL-6 (A), TNF (B) and IL-12 (C) in culture supernatant fluids were measured by ELISA. The results presented are representative of 2 independent experiments (n = 4 mice per group) with similar results. Bars show mean +/−SEM; *, p<0.05; **, p<0.01.
Figure 5.
Berenil ameliorates LPS-induced toxicity and production of pro-inflammatory cytokines in vivo.
Naïve BALB/c mice were injected with Berenil and after 24 hr challenged with LPS (10 µg/ml) intraperitoneally. After 24 hr, mice were assessed for clinical (disease) score (A), sacrificed and serum levels of IL-6 (B), TNF (C), and MCP-1 (D) were determined by ELISA. The results presented are representative of 2 independent experiments (n = 4 mice per group) with similar results. Bars show mean +/−SEM; *, p<0.05.