rTgPI-1 treatment ameliorates lung histopathology.

<p>(A) BAL eosinophil count was performed on cytocentrifuge slides stained with a modified Wright-Giemsa stain. (B) Sections for microscopy were stained with Haematoxylin and PAS. Original magnification 100X. (C) A histological goblet cell score was obtained in Periodic acid- Schiff (PAS)-stained lung sections by examining 20 consecutive airways at 400x magnification. (D) An index of pathologic changes in H&E slides was obtained by scoring the inflammatory infiltrate around the airways and vessels for greatest severity and overall extent. Results for each group are expressed as the mean ± SEM.*<i>p</i> <0.05 and **<i>p</i> <0.01, α <i>p</i><0.01 vs N, ns: non-significant; ANOVA with Bonferroni's test <i>a posteriori</i>.</p

Schematic of asthma model and treatment protocol.

<p>Mice were sensitized by two injections of OVA/alum and challenged by exposure to aerosols of allergen on 3 consecutive days. Two days later mice were intranasally (in) treated with rTgPI-1 (PI) or rTgPI-1 plus OVA (OPI). Controls included non-sensitized mice treated with rTgPI-1 alone (N), and sensitized mice exposed intranasally to PBS (O) or OVA (OO). One week later, mice were re-challenged and analysis was performed 48 h after the last exposure.</p

rTgPI-1 treatment attenuates airway hyperresponsiveness.

<p>Changes in pulmonary resistance in response to increasing doses of methacholine (0–30 mg/ml) were evaluated by invasive plethysmography. Results for each group are expressed as the mean ± SEM. ψ <i>p</i><0.01 OPI vs OO and O, Ѡ <i>p</i><0.001 OPI vs OO and O, φ <i>p</i><0.05 PI vs O, α <i>p</i><0.05 O and OO vs N; ANOVA with Bonferroni's test <i>a posteriori</i>.</p

Effect of rTgP-1 treatment on allergen-specific humoral response.

<p>Serum levels of OVA-specific IgE (dilution, 1:40), IgG1 (dilution, 1:64x10⁶) and IgG2a (dilution, 1:16x10³) antibodies were quantified in all experimental groups. Results are expressed as the mean ± SEM. *<i>p</i> <0.05, α <i>p</i><0.05 vs N, ns: non-significant; ANOVA with Bonferroni's test <i>a posteriori</i>.</p

Local production of cytokines.

<p>Cytokine production by TLNC cells cultured <i>ex vivo</i> with OVA were analysed in all group of mice by ELISA. Results for each group are expressed as the mean ± SEM *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, α p<0.001 vs N, ns: non-significant; ANOVA with Bonferroni’s test <i>a posteriori</i>.</p

Co-administration of rTgPI-1 plus allergen enhances generation of Tregs both local and systemically.

<p>Tregs were identified by flow cytometry. The percentage of FoxP3⁺CD4⁺ among total CD4⁺ cells from TLNC (A) or splenocytes (B) was calculated. Results for each group are expressed as the mean % CD4⁺FoxP3⁺/CD4⁺ ± SEM. *<i>p</i><0.05, **<i>p</i><0.01, ns: non-significant; ANOVA with Bonferroni's test <i>a posteriori</i>.</p

Purification and activity of rTgPI-1.

<p>(A) SDS-PAGE of rTgPI-1 stained with Coomassie Blue. (B) Western blot of rTgPI-1 revealed with anti-His-Tag (lane 1) antibody or mouse anti-TgPI-1 serum (lane 2). (C) Analysis of inhibitory effect of rTgPI-1 on trypsin by gelatin substrate-SDS PAGE. All lanes contain 50 ng of trypsin. Lane 1, 1 μg of rTgPI-1; lane 2, control without rTgPI-1. Gelatinolytic activity was visualized by staining with Coomassie Brilliant Blue. The image was digitally inverted.</p

rTgPI-1 treatment suppresses cell proliferation.

<p>Proliferative responses of TLNC (A and B) or splenocytes (C and D) were determined by ³H-thymidine incorporation upon stimulation with OVA. Results for each group are expressed as the mean ± SEM. *<i>p</i><0.05, **<i>p</i><0.01, α <i>p</i><0.001 vs N, ns: non-significant; ANOVA with Bonferroni's test <i>a posteriori</i>.</p