Platelet granzyme B apoptosis surveyed by TUNEL in spleen, lung, and kidney.

<p>Representative frozen sections of end organs (i.e. spleen (top), lung (middle), and kidney (bottom)) from wild type (left) and granzyme B null mice (right) were stained for apoptosis with a TUNEL-based assay (TACS® 2 TdT In Situ Apoptosis Detection Kits, Trevigen, Gaithersburg, MD). Increased dark brown staining, evident of apoptosis, is seen in wild type spleens, lungs and kidneys. While the granzyme B null kidneys show apoptosis, there is no staining in the granzyme B null spleens and lungs. No apoptosis was noted in either set of heart and liver sections and is therefore not shown here. Photomicrographs were taken at 10X and 20X magnification. Apoptotic indexes, defined as the number of apoptotic cells per mm², are shown for quantification of tissue apoptosis.</p

Sepsis survival and severity in wild type and granzyme B null mice in a rapidly fatal (severe) CLP model.

<p><b>A.</b> Granzyme B null (−/−) mice had lower sepsis scores than wild type mice at every time point. For example, at 22 hours, the mean±SEM wild type score was 9.0±0.8 while the granzyme B null score was 6.8±0.7 (*p = 0.04) <b>B.</b> Kaplan-Meier survival curve for wild type and granzyme B null (−/−) mice in hours after CLP. Granzyme B null (−/−) mice survived longer following severe CLP than wild type mice (p = 0.0019 by Cox Proportional Hazard Regression). <b>C.</b> Endotoxin concentrations (EU/mL) were measured in granzyme B null and wild type mouse plasma. Differences between the two mouse strains were not statistically significant. <b>D.</b> Representative photomicrographs of lung and spleen in sepsis are shown. Platelet infiltration, assayed by CD41 (brown) staining, was visibly widespread and similar between wild type and granzyme B null mice in both organs. Photomicrographs were taken at 10X magnification.</p

Platelet induced splenocyte apoptosis is perforin independent and contact dependent. A.

<p>Platelets harvested from septic mice induce apoptosis in control CD4⁺ splenocytes in the absence of perforin. Percent apoptosis was significantly higher in splenocytes co-incubated with platelets harvested from septic wild type (i.e. C57BL6) mice (n = 5) than with platelets from healthy wild type mice (n = 5) and splenocytes without platelets. Repeat experiments with platelets from septic perforin null mice showed no reduction in induced splenocyte apoptosis. <b>B.</b> Direct platelet contact is necessary for granzyme B-mediated apoptosis. Incubation across a dividing semi-permeable membrane reduced splenocyte apoptosis to a rate indistinguishable from non-platelet treated controls.</p

Sepsis survival and severity in vehicle and eptifibatide treated mice in a cecal slurry sepsis model.

<p><b>A.</b> Eptifibatide treated mice had lower sepsis scores than vehicle treated mice at time points following drug administration. For example, at the 20 hour injection, the mean±SEM vehicle treated score was 8.4±0.3 while the eptifibatide treated score was 7.4±0.3 (p = 0.01) <b>B.</b> Kaplan-Meier survival curve for vehicle and eptifibatide treated mice in hours after cecal slurry injection. Eptifibatide treated mice survived longer following cecal slurry injection than vehicle treated mice (p = 0.019 by Cox Proportional Hazard Regression).</p

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

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 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

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