Effect of native versus pegylated IFN on IP10 gene induction in HLMVECs.

<p>Effect of native IFNα2a, IFNα2b versus pegylated (PEG) forms (40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) on IP10 gene induction in human lung microvascular endothelial cells (HLMVECs). Cells were treated with interferon (IFN) for 6 hours. Data are the mean ± standard error of the mean for n = 3.</p

Effect of Type I–III IFNs on ET-1 release from HPASMCs.

<p>In the presence of TNFα, type I and type II, but not type III, interferons (IFNs) induce the release of ET-1 from human pulmonary artery smooth muscle cells (HPASMCs). Cells were treated with IFNs for 24 hours. Data are the mean ± standard error of the mean for n = 8. Within-group analysis was performed using one-way ANOVA followed by a Dunnett's post-test, where * indicates p<0.05 compared to control. Between-group analysis, for the effect of TNFα, was performed using two-way ANOVA followed by Bonferroni's post-test where # indicates p<0.05.</p

Effect of native versus pegylated IFN on IP10 release from HLMVECs.

<p>Effect of native IFNα2a or IFNα2b versus pegylated (PEG) forms on IP10 release from human lung microvascular endothelial cells (HLMVECs; A, B), human pulmonary artery smooth muscle cells (HPASMCs; C, D) and type II pneumocytes (A549 cells; E, F). Cells were treated with interferons (IFNs) alone (A, C and E) or in the presence of TNFα (10 ng/mL; B, D and F) for 24 hours. Data shown are mean ± standard error of the mean for n = 6 and were analysed using two-way ANOVA where * indicates p<0.05 for PEG-IFNα2a versus the other three IFNs.</p

Effect of PEG-IFN α2a isoforms on IP10 release from HLMVECs.

<p>Effect of different isoforms of PEG-IFN α2a on IP10 release from human lung microvascular endothelial cells (HLMVECs) co-treated with TNFα (10 ng/mL). Cells were treated for 24 hours with PEGIFNα2a or isoforms of PEGIFNα2a (K31, K134, K121) each at 10 ng/mL. Data are mean ± standard error of the mean for n = 7. Data have been normalized using IP10 release in the presence of 40KDa-PEGIFNα2a as 100%, and were analysed using a paired one sample t-test for each isoform or combination compared to PEGIFNα2a, where * indicates p<0.05. Intergroup analysis was performed using one-way ANOVA followed by Bonferroni's Multiple Comparison Test, where # indicates p<0.05.</p

Effect of native versus pegylated IFN on ET-1 release from HPASMCs.

<p> = Effect of native 30 ng/mL IFNα2a or IFNα2b versus pegylated (PEG) forms on ET-1 release from human pulmonary artery smooth muscle cells (HPASMCs). Cells were treated with interferons (IFNs) for 24 hours. Data are the mean ± standard error of the mean for n = 8. Within-group analysis was performed using one-way ANOVA followed by a Dunnett's post-test, where * indicates p<0.05 compared to control. Between-group analysis, for the effect of TNFα was performed using two-way ANOVA followed by Bonferroni's post-test where # indicates p<0.05.</p

Medical Swab Analysis Using Desorption Electrospray Ionization Mass Spectrometry: A Noninvasive Approach for Mucosal Diagnostics

Medical swabs are routinely used worldwide to sample human mucosa for microbiological screening with culture methods. These are usually time-consuming and have a narrow focus on screening for particular microorganism species. As an alternative, direct mass spectrometric profiling of the mucosal metabolome provides a broader window into the mucosal ecosystem. We present for the first time a minimal effort/minimal-disruption technique for augmenting the information obtained from clinical swab analysis with mucosal metabolome profiling using desorption electrospray ionization mass spectrometry (DESI-MS) analysis. Ionization of mucosal biomass occurs directly from a standard rayon swab mounted on a rotating device and analyzed by DESI MS using an optimized protocol considering swab–inlet geometry, tip–sample angles and distances, rotation speeds, and reproducibility. Multivariate modeling of mass spectral fingerprints obtained in this way readily discriminate between different mucosal surfaces and display the ability to characterize biochemical alterations induced by pregnancy and bacterial vaginosis (BV). The method was also applied directly to bacterial biomass to confirm the ability to detect intact bacterial species from a swab. These results highlight the potential of direct swab analysis by DESI-MS for a wide range of clinical applications including rapid mucosal diagnostics for microbiology, immune responses, and biochemistry

Consort Flow Chart.

<p>Consort Flow Chart.</p

Idiosyncratic large nasal responses to lipopolysaccharide (LPS) on 2 occasions.

<p>Raw data for levels of chemokines and cytokines (pg/ml) in nasal mucosal lining fluid (MLF). Data is presented for individual subjects: A. Subject 2009 after nasal challenge with 10μg LPS. B. Subject 3101 after nasal challenge with 30μg LPS. Note that baseline levels of cytokines and chemokines at -0.5h are markedly raised, and come down to only a small extent after nasal lavage at 0.5h. It is suggested that these individuals may have had a non-symptomatic alteration in their nasal microbial flora prior to LPS challenge on these opccasions: possibly the individuals had a subclinical viral infection or bacterial colonisation.</p

Median levels of chemokines and cytokines (pg/ml) in nasal mucosal lining fluid (MLF) are shown after nasal spray challenge with 4 different doses of LPS and a placebo (see key below).

<p>Lower limits of detection (LLOD) for cytokines and chemokines were as follows: IL-1β (5.0pg/ml), IL-6 (5.0pg/ml), CXCL8/IL-8 (10.0pg/ml), CCL3/MIP-1α (5.0pg/ml), CCL2/MCP-1 (25.0pg/ml), IL-10 (5.0pg/ml). Time -0.5h refers to nasosorption performed 30 min prior to topical nasal challenge, and this varies considerably between individuals due to variations in individual’s microbial flora and mucosal immune responses. Nasal lavage was performed after nasosorption at -0.5h, but prior to nasal challenge, in order to partially wash the nose free of baseline inflammatory microbes and mediators, but causes detectable levels of inflammatory mediators at 0.5h to decrease markedly from -0.5h. There is a tendency for nasosorption levels of mediators to gradually increase from 0.5h to 10h in the placebo arm, but levels of inflammatory cytokines and chemokines after LPS are generally higher.</p

Levels of interleukins (IL) in nasosorption eluates presented as area under the curve (AUC) for times 0.5-10h in relation to topical nasal challenge (n = 14 or 15).

<p>AUC is derived from from 0.5 since the baseline sample at -0.5h is pre-nasal lavage and is highly variable due to difference in individuals in terms of nasal microbes and inflammatory mediators. A) IL-1β as medians with quartiles; B) IL-6 as medians with quartiles; C) IL-1β AUC data for individual subjects; D) IL- AUC data for individual subjects. Note the idiosyncratic large responses of subject 2009 after nasal challenge with 10μg LPS, and subject 3101 after nasal challenge with 30μg LPS, these responses being too large to be presented on the y axis.</p