Transgene expression in alveolar macrophages is restricted to the segment infected with adenoviral vector.

<p>Alveolar macrophages were collected on day 3, 7 and 10 from sheep infected on day 0 with Ad-GFP/calcium phosphate from both segments infected with adenovirus and from naïve segments and assessed for GFP by UV microscopy after 24 hours in culture. Data is shown as scatter plots with the horizontal bars indicating the median. Macrophages from Ad-GFP infected segments on day 14 were negative for GFP. No GFP+ve macrophages were identified in BALF from any naïve segments at any time point (data not shown). NB Zero values are not shown due to the use of a log scale.</p

Local lung response to the instillation of adenoviral vectors and/or LPS.

<p>Boxplots depicting the (a) log10 Total cell counts (cells/ml); (b) Alveolar macrophage phagocytic activity (AMPh) (geometric mean fluorescence units); (c) neutrophil phagocytic activity activity (geometric mean fluorescence units); (d) proportion of AM in BALF; (e) proportion of PMN in BALF; (f) proportion of lymphocytes in BALF; (g) log10 Elafin (pg/ml); (h) log10 MPO activity (absorbance units (au)/PMN * 10⁶); and (i) log10 TNF-α (pg/ml) data arising from analysis of BALF sampled from differentially treated lung segments of sheep exposed to either Ad-o-elafin (Virus E) or Ad-GFP (Virus G). The boxplot tails reflect the range of data, the bottom and tops of the boxes the 25th and 75th percentiles respectively, and the thick horizontal line the median values. The first pair of boxplots adjacent to the Y-axis reflect baseline data collected before lungs were exposed to treatment (Baseline). The next adjacent pair of boxplots depict data collected from non-treated control lung segments after other segments in the same lung had received different treatment regimes (Remote lung response). The third pair of boxplots reflect data collected from lung segments exposed to LPS six hours previously (LPS response). The last pair of boxplots reflect data collected from segments that had, in addition to LPS treatment six hours previously, been treated with adenoviral vectors ten days before, (Virus + LPS response). The results of statistical comparisons [i]-[iii] between these nested data sets are depicted by the interconnected horizontal lines above and below each separate graph (NS  =  no significant difference, *, ** & *** = P<0.05, 0.01 and 0.001 respectively). The bold font is simply used to emphasise the boxplot pairs across which the above statistical comparisons are directed. Whilst not explicitly annotated in this figure the virus-primed LPS-induced decrease in BALF MPO activity (6h) resulted in levels that were significantly greater for segments exposed to Ad-o-elafin (P = 0.016) when compared to segments treated with Ad-GFP, and levels of AM (6b) and PMN (6c) phagocytosis after LPS were significantly increased in segments exposed to Ad-o-elafin when compared to Ad-GFP (P<0.023).</p

Systemic haematological responses to intrapulmonary adenoviral vector and LPS administration.

<p>Heparinised blood was collected by venepuncture at various time points throughout the experimental period (Pre-treatment (immediately prior to collection of baseline BALF samples), d10 and d10+6h). This blood was analysed for total white blood cell numbers, neutrophil numbers and lymphocyte numbers as indicated in a, b and c respectively. The boxplot tails reflect the range of data, the bottom and tops of the boxes the 25th and 75th percentiles respectively, and the thick horizontal line the median values. A linear mixed-effect model, where sheep-specific responses were assumed a random effect, was used to analyse the influence of Ad-o-elafin and Ad-GFP and subsequent LPS instillation on blood parameters. *, ** and *** represent P<0.05 and P<0.01 and P<0.001 respectively.</p

Incorporation of adenovirus into calcium phosphate precipitate increases the infection efficiency of alveolar macrophages and pulmonary respiratory epithelium in vivo.

<p>Cryo-sections were prepared from lung tissue 48 hours after instillation of vehicle, vehicle and calcium phosphate, 1×10⁸ pfu Ad-GFP in PBS or 1×10⁸ pfu Ad-GFP after pre-incubation with calcium phosphate. 24 random fields were counted at high power using UV light and the number of GFP+ve cells recorded. (a) – The number of GFP+ve cells is represented here as mean with error bars indicating standard deviation. *, ** and *** indicate P<0.05, P<0.005 and P<0.0005 respectively either comparing values to relevant control (i.e. vehicle alone or vehicle with calcium phosphate) or comparing virus treatment with and without calcium phosphate (indicated by a bar). Significance was calculated by non-paired T Test. (b) – Representative fields from the Ad-GFP/calcium phosphate segment showing infection of Type II epithelial cells (filled arrows) and an alveolar macrophage in an airway (open arrow). Counterstaining of nuclei is with DAPI.</p

Incorporation of adenovirus into calcium phosphate precipitate increases the infection efficiency of alveolar macrophages in vitro.

<p>Ovine alveolar macrophages were cultured at a density of 250,000 per well in 48 well plates and were infected with Ad-GFP and Ad-o-elafin-FLAG at MOI 100 or 200 either pre-complexed with calcium phosphate or as virus alone. (a) – Ovine alveolar macrophages 24 hours after infection with Ad-GFP MOI 100 alone. (b) – Ovine alveolar macrophages 24 hours after infection withAd-GFP MOI 100/calcium phosphate co-precipitate. (c) – Western blot analysis of ovine alveolar macrophage supernatant using Trab-2O antibody 4 days after infection with Ad-o-elafin-FLAG at MOI 100 and 200 either with or without coprecipitation with calcium phosphate. Uninfected alveolar macrophage supernatant is included as a control.</p

Experimental regimens used for both short-term and long-term guinea pig studies.

<p>Experimental regimens used for both short-term and long-term guinea pig studies.</p

Experimental protocol for the examination of the effect of local up-regulation of ovine elafin on the response to bacterial LPS.

<p>A. Baseline sampling of BALF from a lung segment (3–4 weeks before the start of the experiment (day 0) B. Anaesthetised sheep were instilled on day 0 with either Ad-o-elafin-FLAG or Ad-GFP (as a control vector) co-precipitated with calcium phosphate in discrete lung segments. C. Ten days later, bacterial LPS was instilled into the same virus treated segments and also previously naïve segments (Pre-treatment blood samples were obtained immediately prior to anaesthetic induction on Day 10). D. 6 hours after LPS instillation, BALF was collected from these LPS treated segments and also a new ‘remote lung’ segment. The sample categories “Baseline”, “Remote lung response”, “LPS response” and “Virus + LPS response” correspond to the x-axes labels on the boxplots drawn in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107590#pone-0107590-g006" target="_blank">figure 6</a>. In this design four samples of bronchoalveolar lavage fluid were derived, one sample obtained before any treatment was administered to the lung (Baseline), one sample derived from an area of the lung not subject to any direct treatment (Remote lung response) but obtained after treatments had been administered to other parts of the lung, and further samples from two areas of the lung, the first subject to direct treatment with LPS only (LPS response) and the second subject to treatment with LPS and adenoviral vector (Virus + LPS response).</p

Comparison of Mean Days to Death of various treatment groups of guinea pigs.

<p>Mean Days to Death (MDD) was determined by dividing the summed total number of surviving days of each treatment group with the number of animals/group. Results are expressed as means±SEM of eight animals/group. Note: MDD for BCG/Ad groups may have been underestimated as 40–60% of animals in these treatment groups were still surviving at the time study termination (74 weeks). **p≤0.01, *p≤0.05 compared to saline control or between BCG and BCG/Ad i.n.</p

Kaplan-Meier curves of percent survival of <i>M.tb</i>-infected guinea pigs over the course of 74 weeks post-challenge.

<p>Groups of guinea pigs (eight animals per group) were treated as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005856#pone-0005856-g001" target="_blank">Fig. 1B</a> and their survival rate was determined on a weekly basis up to 74 weeks when the entire study was terminated. p≤0.01 BCG, BCG/Ad i.n and BCG/Ad i.m compared to saline control; p≤0.05 BCG vs. BCG/Ad i.n; p≤0.1 BCG vs. BCG/Ad i.m.</p

Ad/Tr-cells secrete Tr that is functionally active against HNE and both Tr/E are detected in Ad/Tr-cells supernatants in response to polyI∶C.

<p>HEC-1A cells were either treated with medium alone or with MOI 10–50 of Ad/dl or Ad/Tr. Supernatants were collected 36 h after Ad removal and tested for Tr/E expression by ELISA (<i>A</i>) or for antiprotease activity (<i>C</i>), where results are expressed as percent reduction over a positive control (media alone plus HNE and a substrate). The data are representative of two independent experiments performed in triplicate and are shown as the mean ± SD. Statistical analysis was performed using Student's <i>t</i> test with * representing significant difference between the groups, p<0.05. (<i>B</i>) Immunoblotting analysis of supernatants from HEC-1A cells developed using TRAB20 antibodies. Cells were either left untreated (lanes 1,2) or treated with MOI 50 of Ad/dl (lanes 3,4) or Ad/Tr (lanes 5,6) and then incubated for 24 h in presence of medium alone (−) or 25 µg/ml polyI∶C (+). Two recombinant reference proteins, namely in-house HAT-E (rE) (lane 7) and commercial 6×His-Tr (rTr) (lane 8), were used as comparative markers for E and Tr. Bands corresponding to the forms of Ad-induced Tr and E are indicated on the blot by arrows.</p