MSD analysis of cytokine (pg/ml) release from hESC-EC.

<p>Data mean are ± SEM for n = 3. hESC-EC were treated for 24 hours with vehicle, LPS (1 µg/ml), or C12-iE-DAP (10 µg/ml). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05). ND = non-detectable.</p

Responses of hESC-EC and HUVEC to 24 hour infection with <i>Heamophilus influenzae.</i>

<p>(A) Effect of LPS (1 µg/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release from hESC-EC and HUVEC after 24 hours. (B) Effect of <i>Haemophilus influenzae</i> (HIN) (10⁵–10⁸ CFU/ml) on CXCL8 release from hESC-EC (solid line) or HUVEC (dashed line) after 24 hours. Data are mean ± SEM; n = 3 representative of 6 hESC-EC isolations. Statistical significance for responses to drugs or bacteria was determined by one-way ANOVA followed by Dunnett's multiple comparison test (p<0.05).</p

MSD analysis of cytokine (pg/ml) release from HUVEC.

<p>Data are mean ± SEM for n = 3. HUVEC were treated for 24 hours with vehicle, LPS (1 µg/ml), or C12-iE-DAP (10 µg/ml). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05).</p

Effect of <i>in vivo</i> ‘conditioning’ on TLR4 and NOD1 expression.

<p>TLR4 and NOD1 expression in (A) hESC-EC and (B) HUVEC before (pre-implant; open bars) and 21 days after (post-implant; filled bars) implantation <i>in vivo</i> (‘conditioning’). Data are mean ± SEM and are normalized at unity (1) to gene levels in pre-implant cells. HUVEC; NOD1 pre-implant n = 8, post implant n = 4: HUVEC; TLR4 pre-implant n = 10, post implant n = 3. hESC-ECs; NOD1 pre-implant n = 6, post implant n = 5: hESC-ECs; TLR4 pre-implant n = 10, post implant n = 6. Data was obtained from 2 independent experiments (using up to 12 rats per group). Statistical significance was determined by one-sample t-test where results were compared to a theoretical control of 1 (*p<0.05). ND = none detectable.</p

Effect of pharmacological inhibition of RIP2 and NOD1 siRNA mediated knockdown on responses of hESC-EC to <i>Haemophilus influenzae</i> (HIN) and C12-iE-DAP.

<p>(A) Relative expression (vs. GAPDH) of NOD1 following 48 hour incubation with NOD1 siRNA normalized to non-targeting siRNA; n = 6. (B) CXCL8 release from hESC-EC following 48 hour pre-incubation with non-targeting siRNA (open bars) or NOD1-siRNA (filled bars) and 24 hour treatment +/− C12-iE-DAP (10 µg/ml) or <i>Haemophilus influenzae (HIN)</i> (10⁷–10⁸ CFU/ml); n = 7–8. (C) Effect of GSK'214 (300 nM; RIP2 inhibitor) or GSK'217 (300 nM; NOD1 inhibitor), given 30 minutes before a 24 hour treatment with HIN (10⁷ CFU/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release; n = 4. It should be noted that GSK drugs increased CXCL8 release under basal conditions; for each experiment this was subtracted from treatment groups. For panel A, statistical significance was determined by one-sample t-test. For panel B statistical significance within siRNA groups was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05), and between groups by two-way ANOVA followed by Bonferroni's post-test (+p<0.05). For panel C statistical significance for the effects of inhibitor of C12-iE-DAP or HIN induced CXCL8 was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05).</p

Pulmonary Innate Lymphoid Cell Responses during Rhinovirus-induced Asthma Exacerbations In Vivo

Rationale Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well-characterized. Objectives To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. Methods Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16, and underwent bronchoscopy at baseline, day 3 and day 8 post-inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage (BAL) using flow cytometry. The ratio of BAL ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. Measurements and Main Results At baseline, ILC2s were significantly higher in patients with asthma than healthy subjects. At day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in asthma than in healthy subjects (all comparisons P<0.05). In healthy subjects, ILC1s increased from baseline at day 3 (P=0.001), while in patients with asthma, ILC1s increased from baseline at day 8 (P=0.042). Patients with asthma had significantly higher ILC2:ILC1 ratios at baseline (P=0.024) and day 8 (P=0.005). Increased ILC2:ILC1 ratio in asthma correlated with clinical exacerbation severity and type 2 cytokines in nasal mucosal lining fluid. Conclusions An ILC2-predominant inflammatory profile in asthma was associated with increased severity and duration of rhinovirus infection compared with healthy subjects, supporting the potential role of ILC2s in the pathogenesis of virus-induced asthma exacerbations

Effect of systemically dosed 14C11 antibody on HRV16 infection in vivo.

<p>Mice were dosed intravenously with 14C11 24 hours prior to intranasal infection with HRV16 (n = 9 for tg− group; n = 6 for tg+ groups). (A) Total BAL cells, amacrophageslymphocytes and neutrophils were assessed by cytospin 2 days after infection. (B) The chemokines CXCL1, CXCL11 and CXCL10 in BAL were determined by MSD or quantitative ELISA 2 days after infection. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. **p<0.01 and ***p<0.001 vs HRV16 infected transgenic negative mice; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs HRV16 infected transgenic positive mice; Data are representative of 3 independent experiments.</p

14C11 antibody inhibits major group HRV16 infection <i>in vivo</i>.

<p>Groups of 7 mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16. (A) Total BAL cells, macrophages, lymphocytes and neutrophils were assessed with differentially cytospin counts day 2 after infection. (B) Levels of proinflammatory cytokines and chemokines IL-1β, IL-6, CXCL1, IFNλ2/3, CXCL11 and CXCL10 in cell-free BAL were determined by MSD or quantitative ELISA 2 days after infection. (C) Proinflammatory cytokines and chemokines IL-1β, IL-6 and CXCL1 in lung homogenate were assessed with MSD 2 days after infection. (D) Groups of 3–5 mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16 and vRNA in lung tissue was assessed by qPCR at the timepoints indicated. (E) Inflammatory cells in the lungs prior to infection (uninfected) and at 12 hours post-infection (12 p.i.) of huICAM negative (tg−) and huICAM-expressing (tg+) mice without antibody treatment, or for tg+ mice following isotype control antibody (tg+ iso) or 14C11 antibody treatment (tg+ 14C11) assessed by H&E staining Arrows indicate peribronchial cellular inflammation. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. ***p<0.001 and **p<0.01 vs HRV16 infected transgenic negative mice; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs HRV16 infected transgenic positive mice. Data are representative of 3 independent experiments each.</p

14C11 inhibits major group HRV replication <i>in vitro</i>.

<p>Ohio HeLa cells were pre-incubated with serial dilutions of 14C11 or isotype control and infected with (A) HRV16, (B) HRV14 and (C) minor group HRV25. The antiviral effect of 14C11 was determined by CPE reduction assay and expressed as % of control. (D) IC₅₀s for 14C11 were determined for major HRVs by CPE assay as indicated in. Experiment (A), (B) and (C) were performed in sextuplicates and (D) is a pool of two independent experiments. Data are expressed as mean (± SEM).</p

14C11 does not inhibit inflammation induced via mechanisms independent of human ICAM-1.

<p>Mice were dosed intravenously with 14C11 24 hours prior to intranasal dosing with HRV1B, UV-inactivated HRV1B (UV) or HRV16 (n = 4 for tg− UV, tg− 1B, tg+ 16 iso and tg+ 16 14C11 groups; n = 6 for tg+ UV, tg+ 1B, tg+ 1B iso and tg+ 1B 14C11 groups). (A)Total BAL cells, neutrophils (on day 1) and lymphocytes (on day 4) were assessed with differentially stained cytospins. Data expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. ***p<0.001 vs UV-RV1B in transgenic negative mice; ^###p<0.001 vs UV-RV1B in transgenic positive mice; ^§p<0.05 and ^§§§p<0.001 vs isotype treated HRV16 infected transgenic positive mice. Data are representative of 2 independent experiments. (B) Groups of 7 mice were dosed intravenously with 14C11 24 hours prior to intranasal dosing with 1 µg LPS/mouse. Total BAL cells, lymphocytes and neutrophils were assessed with differentially stained cytospins day 1 after infection. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. *p<0.05, **p<0.01 and ***p<0.001 vs transgenic positive mice without treatment. Data are representative of 2 independent experiments.</p