TCDD reduces parasite burdens and slows the progression of cutaneous leishmaniasis in SCID mice.

<p>Female SCID mice were treated with peanut oil (vehicle) or TCDD (160 µg/Kg body weight) per os one day prior to infection with one million stationary phase L. major promastigotes in one rear footpad. <b>(A)</b> Lesion size is shown as mean ± SEM for five mice per group. *Indicates a statistically significant difference from vehicle-treated mice on that day (p < 0.05). The results are representative of four separate experiments. <b>(B)</b> Parasite burdens in individual infected feet were analyzed at four weeks post infection (six mice per treatment group; data represent mean ± SEM). Parasite burdens in individual spleens were analyzed at four weeks post infection (three mice per treatment group; data represent mean ± SEM). *Indicates a statistically significant difference from vehicle-treated mice on that day (p < 0.05). The results are representative of 2-3 separate experiments.</p

TCDD reduces parasite burdens and slows the progression of cutaneous leishmaniasis in BALB/c mice.

<p>Female BALB/c mice were treated with peanut oil (vehicle) or TCDD at various doses (per os) one day prior to infection with one million stationary phase L. major promastigotes in one rear footpad. Data are shown for 3-5 mice per treatment group on days 2-35 and are representative of three independent experiments. Data for two mice from one experiment are shown after day 35. <b>(A)</b> Lesion size is shown as mean ± SEM. Symbols with internal plus marks (+) indicate a statistically significant difference from vehicle-treated mice on that day (p < 0.05). <b>(B)</b> Parasite burdens in infected feet are shown (mean ± SEM) for mice euthanized on the days indicated: three mice per group up to day 35; after day 35, two mice were pooled (n = 1) . *Indicates a statistically significant difference from vehicle-treated mice on that day (p < 0.02).</p

Effects of TCDD in SCID mice after low dose infection.

<p>Female SCID mice were treated with peanut oil (vehicle) or TCDD at various doses (per os) one day prior to infection with ten thousand stationary phase <i>L. major</i> promastigotes in one rear footpad. (<b>A</b>) Lesion size is shown as mean ± SEM for 3-5 mice per time point. Symbols with internal plus marks (+) indicate a statistically significant difference from vehicle mice on that day (<i>p</i> < 0.05). All animals within a treatment group were euthanized following the last indicated measurement. (<b>B</b>) Mice were euthanized on the days indicated, and the infected feet of 2-3 mice per group were analyzed as a pool (n=1) for parasite burdens. (<b>C</b>) Percent body weight change is shown as mean ± SEM for 5 mice per time point. Data shown on day 35 reflects body weight change of animals measured either on day 35 or day 37.</p

Localization and appearance of the BluePort tissue compartment.

<p>Macroscopic appearance of the blue nodule (BluePort) formed one month after injection of CBa-beads in the subcutaneous tissue of mice (A and B). Macroscopic (C) and microscopic appearance (D) of BluePort-associated skin 24 hours after exposure of naïve mice to the bite of <i>L. longipalpis</i> sand flies. Black arrows indicate skin erythema in (C), and vasodilatation of dermal blood vessels in (D). White arrow indicates vasodilatation of blood vessels inside the BluePort parenchyma in (D). Hematoxylin-eosin stain, magnification 100x in (D).</p

Chronic inflammatory response to CBa-beads.

<p>Histopathological changes observed 30 days (A and B), 60 days (C and D), 90 days (E and F) and 120 days (G and H) days after injection of CBa-beads. Arrows indicate endothelial cells in (A), macrophages in (B), neutrophils inside a blood vessel in (C), capsule in (D), multinucleated foreign-body giant cells in (E), mast cells in (F), eosinophils in (G) and lymphocytes in (H). Hematoxylin-eosin stain, 1000x magnification.</p

Inflammatory response in mice exposed twice to sand fly bites on BluePort-associated skin.

<p>Evolution of the inflammatory response in samples collected 24 hours (A, E and I), 48 hours (B, F and J), 72 hours (C, G and K) and 96 hours (D, H and L) after exposure for the second time to the bite of sand flies. Black arrows indicate neutrophils and white arrows indicate eosinophils. Hematoxylin-eosin stain. Magnification: 100x in (A–D), 1000x in dermis (E–H) and hypodermis (I–L).</p

Lectin-blot and immuno-blot analysis of <i>L. longipalpis</i> salivary glycoproteins.

<p>Effect of enzymatic deglycosylation with PNGase F on two sources of sand fly salivary glycoproteins, SGL and HSL. Samples were stained for protein composition profile (Imperial), probed for the presence of N-linked glycans (GNL, AAL) and O-linked glycans (VVL, PNA), or probed for reactivity with IgG antibodies of animals exposed to the bite of sand flies on BluePort-associated skin. Arrows indicate the main antigenic salivary glycoproteins.</p

Inflammatory response of naïve and immune mice to sand fly bites on normal skin.

<p>Inflammatory response detected 48 hours after exposure to the bite of <i>L. longipalpis</i> sand flies on naïve mice (A and B) and immune mice (C and D). Black arrows indicate neutrophils and white arrows eosinophils. Hematoxylin-eosin stain. Magnification, 200x in (A and C), 1000X in (B and D).</p

Interfering with arginase-induced L-arginine metabolism restores CD4⁺ T cell responses in nonhealer BALB/c mice.

<p>(A) Two groups of BALB/c mice (n = 8) were infected with <i>L. major</i> parasites and one group was injected daily i.p. with a competitive inhibitor of arginase (nor-NOHA, 1 mg/mouse) starting from the day of infection. Two weeks later, the number of viable parasites was measured in individual footpads (left panel) and the % of CD4⁺ BrdU⁺ T cells and IFN-γ-expressing CD4⁺T cells was determined by flow cytometry in popliteal lymph nodes and lesions (pool of at least 2 footpads). Error bars represent standard deviations. Data show the results of one representative experiment out of two independent experiments. (B) Two groups of BALB/c mice (n = 8) were infected with <i>L. major</i> parasites in one hind footpad and 2 weeks post infection, one group was injected i.p. with L-arginine (10 mg/mouse) 3 times a week, for 3 weeks. The lesion development was monitored at weekly intervals (left panel) and 33 days post infection, number of viable parasites and the arginase activity was measured in individual footpads (right panel). (C) % of CD4⁺ BrdU⁺ T cells and IFN-γ-expressing CD4⁺T cells in popliteal lymph nodes and lesions (pool of at least 2 footpads), error bars represent standard deviations. Data show the results of one representative experiment out of four independent experiments.</p

Impaired capacity of antigen-specific CD4⁺ T cells to express cytokines.

<p>Groups of BALB/c and CBA mice (n = 8) were infected with <i>L. major</i> parasites for two (A and B) or four (C) weeks. Individual popliteal lymph nodes and footpads (pool from at least two footpads) were harvested and the % of cytokine-expressing CD4⁺ T cells was determined by flow cytometry. (3A) dot plot profiles of cytokine-expressing CD4⁺ T cells isolated from the popliteal lymph nodes and the footpads two weeks post infection; (3B) % of cytokine-expressing CD4⁺ T cells two weeks post infection. (3C) % of cytokine-expressing CD4⁺ T cells four weeks post infection. Error bars represent standard deviations. Isotype control for IFN-γ: 0.23±0.05%, IL-4: 0.31±0.08% and IL-10: 0.26±0.06%. All cytokines were below the detection limit when the cells were stimulated in the absence of PMA/ionomycin stimulation. The detection limits were defined as % isotype control +3 standard deviations: IFN-γ = 0.38%, IL-4 = 0.55% and IL-10 = 0.44%. Data show the results of one representative experiment out of three independent experiments.</p