VBP15 reduces acute allergic lung inflammation.

<p>OVA-challenged mice were either left untreated or treated with oral doses of prednisone, VBP15 (20 mg/kg), or cherry syrup alone daily for 6 days. A group of non-challenged mice (naïve) was included in order to assess basal inflammatory parameters. Perfused whole lungs were processed for histological analysis and stained with H&E (A) or PAS (B). Images (10× magnification) represent areas of tissue surrounding bronchioles. Arrows on H&E sections indicate inflammatory foci. Percentage of PAS positive airways were counted via bright field microscopy (C). Bar graph represents mean (±SE)% PAS positive airways. *<i>p</i><.05; **<i>p</i><0.01 compared to the vehicle control group with n = 5 mice per group. IL-13 (D) and RANTES (E) were measured in BAL fluid by flow cytometric bead array. Bar graphs represent mean (±SE) cytokine concentration values. *<i>p</i><.05; **<i>p</i><0.01 compared to syrup group with n = 5 mice per group.</p

VBP15 reduces leukocyte degranulation.

<p>Anti-DNP-sensitized RBL-2H3 cells were treated with prednisolone (50 µM), VBP-15 (50 µM), or vehicle control (DMSO) for 7 minutes followed by addition of DNP to induce degranulation. The reaction was allowed to proceed for an additional 20 minutes before supernatant was removed and tested for β-hexosaminidase content. A well of untreated cells was lysed to gauge total β-hexosamindase content. Release percentage was determined using a formula described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063871#s2" target="_blank">Materials and Methods</a>. Bar graph represents mean (±SE) release percentage. **p<0.01 compared to vehicle control. Data represents 3 biological replicates with assay performed in triplicate. N.S. = Not statistically significant.</p

VBP15 does not induce tibia length shortening.

<p>Wildtype outbred CD1 mice were treated daily for 5 weeks with VBP15 (30 and 45 mg/kg), prednisolone (10 mg/kg) or vehicle control starting at 12 days of age. At the end of the treatment, tibias were harvested and measured. Bar graph represents mean (±SE) tibia length values. *p<0.05 compared to vehicle control with n = 10 mice/group.</p

VBP15 inhibits NFκB activity.

<p>A549 cells stably-transfected with a luciferase NFκB construct were exposed to increasing concentrations of VBP15 or prednisolone (3, 30, 300, 3000 nM) followed by TNFα stimulation before measuring luciferase activity. Bar graph represents mean (±SE) luciferase units. *<i>p</i><.012 (due to the Bonferroni adjustment for multiple comparisons) compared to treatment with vehicle control. Data represents 4 biological replicates with assay performed in triplicate.</p

Structure of VBP15 and schematic of the OVA-induced model of acute allergic lung inflammation.

<p>(A) Chemical structures of prednisone (left panel), prednisolone (mid panel), and VBP15 (right panel). VBP compounds include a delta-9,11 double bond and tail group modifications. (B) Diagram of OVA-induced mouse model of allergic lung inflammation.</p

VBP15 reduces basolateral cytokine secretion from human bronchial epithelial cells obtained from asthmatic patients.

<p>HBE cells from 3 separate human donors were pulse-treated with VBP15 (10 µM) or vehicle control (DMSO). Basolateral surface supernatant was tested for the presence of TGFβ1 (left panel) and IL-13 (right panel) by flow cytometric bead array. Bar graphs represent mean (±SE) concentration values. **, <i>p</i><0.01 compared to vehicle control with n = 3 donors. N.D. = Not Detectible (lower limit of detection = 4.5 pg/ml).</p