Ultrastructure of the tegument of parasites treated with <i>Sm-tsp-2</i> dsRNA RNA observed using transmission electron microscopy.

<p>A. Tegument of adult female treated with <i>luciferase</i> dsRNA. B. Tegument of schistosomulum incubated for 7 days with <i>luciferase</i> dsRNA. C and E. Tegument of adult female incubated with <i>Sm-tsp-2</i> dsRNA. The tegument is more highly vacuolated (C) and thinner (E) compared with controls. D and F. Tegument of schistosomula incubated for 7 days with <i>Sm-tsp-2</i> dsRNA. Digitate extensions (arrows) are more abundant on the surface of the tegument. Abbreviations: Mus-muscles; teg-surface layer of tegument. The tegument of schistosomula were thinner, p<0.001 (G) and more dense, p = 0.014 (H) in <i>Sm-tsp-2</i> dsRNA treated schistosomula.</p

Expression of <i>Sm</i>-<i>tsp-1</i> and <i>Sm-tsp-2</i> at different stages of the <i>S. mansoni</i> life cycle.

<p>RNA levels of <i>Sm-tsp-1</i> (vertical bars) and <i>Sm-tsp-2</i> (horizontal bars) relative to <i>Sm</i>-α-<i>tubulin</i> were analyzed by qRT-PCR. Data are representative of mean±S.E. from three separate experiments.</p

Suppression of <i>Sm-tsp</i> mRNAs in adult parasites by RNAi.

<p><i>Sm-tsp-1</i> (A) and <i>Sm-tsp-2</i> (B) transcript levels relative to <i>Sm</i>-<i>paramyosin</i> (mean±S.E.) in adult parasites soaked for 7 days with 1 µg/ml of <i>Sm-tsp</i> or <i>luciferase</i> control dsRNAs.</p

Protein expression levels of parasites treated with <i>Sm-tsp-2</i> dsRNA.

<p>Protein extracts from adult parasites (A) and schistosomula (B) treated with <i>Sm-tsp-2</i> or <i>luciferase</i> dsRNAs for 7 days were loaded onto a 12% SDS-PAGE gel at 2, 1, 0.5 and 0.25 µg. Proteins were transferred onto nitrocellulose and immunoblotted with anti-<i>Sm</i>-TSP2 or anti-<i>Sm</i>-Pmy monoclonal antibodies. The intensity of paramyosin expression was evaluated to determine equal protein loading.</p

Expression of <i>Sm</i>-TSP-1 or <i>Sm</i>-TSP-2 on the surface of live schistosomula.

<p>Immunofluorescent labelling of live 3 h schistosomula with antisera raised against <i>Sm</i>-TSP-1 (A) and <i>Sm</i>-TSP-2 (B), and control pre-immune serum (C) followed by anti-mouse Ig-FITC. Schistosomula are shown in bright-field and FITC stained. Scale = 50 µm.</p

Suppression of <i>Sm-tsp</i> mRNAs in schistosomula by RNAi.

<p><i>Sm-tsp-1</i> (A) and <i>Sm-tsp-2</i> (B) transcript levels relative to <i>Sm-paramyosin</i> (mean±S.E.) in schistosomula soaked for 7, 14 and 21 days with 1 µg/ml of <i>Sm-tsp</i> or <i>luciferase</i> control dsRNAs.</p

Production of Th2 cytokines in the duodenal mucosa of hookworm infected individuals.

<p>Duodenal biopsies from Trial 1, taken from either the duodenum at week 20 post-infection or from directly adjacent to an adult hookworm attachment site (HW site – determined by endoscopy) at week 21 in the hookworm group only, were cultured for 24 h in tissue culture medium at 37°C with 95% O₂/5% CO₂. Cell supernatants were removed and levels of IL-4 (A), IL-5 (B) and IL-13 (C) were determined using a Cytometric Bead Array. Data were analysed by Mann-Whitney U test.</p

<i>Ex vivo</i> cytokine gene expression in the duodenal mucosa of hookworm infected individuals.

<p>Duodenal biopsies from Trial 2 were taken before (week 0) and 20 weeks after hookworm infection, and RNA was prepared <i>ex vivo</i>. Levels of <i>IL-4</i> (A) <i>IL-5</i> (B), <i>IL-13</i> (C), <i>IL-9</i> (D), <i>GATA-3</i> (E), <i>Foxp3</i> (F), <i>TGF-β</i> (G), <i>ALDH1A2</i> (H), <i>IFN-γ</i> (I), <i>IL-15</i> (J), <i>IL-17A</i> (K), <i>IL-23</i> (L) and <i>RORγt</i> (M) transcripts were determined by quantitative real time RT-PCR.</p

Trial design.

<p>For Trial 1, 20 patients were chosen according to selection criteria <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002520#ppat.1002520-Daveson1" target="_blank">[21]</a> and divided into two groups of 10, placebo control and hookworm infected (HW). All 20 patients completed the trial. For Trial 2, seven of the 10 patients from Group 1 in Trial 1 (placebo) chose to participate in the next trial and received hookworm infection and gluten challenge in an identical manner to that described for Group 2 in Trial 1. The only difference was that gut biopsies were taken at week 0 (prior to hookworm infection; denoted in bold font) in addition to biopsies taken at weeks 20 and 21. For the purposes of this study, sample collection ceased at week 20 (prior to gluten administration) and tissues from week 21 were not utilized, except for samples taken from the worm attachment site which were collected at week 21; week 21 tissues were critical for assessment of the effect of hookworm on the anti-gluten response in celiac disease and are therefore highlighted in italicised grey font and have been reported in relevant publications <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002520#ppat.1002520-Daveson1" target="_blank">[21]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002520#ppat.1002520-McSorley1" target="_blank">[22]</a>. ¹Forty millilitres of blood per person were collected at weeks 0, 4, 12 (Trial 1 only), 20 and 21. ²Gut biopsies were taken at weeks 0 (Trial 2 only), 20 (pre-gluten ingestion) and 21 (post-gluten ingestion) using a gastroscope as described elsewhere <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002520#ppat.1002520-Daveson1" target="_blank">[21]</a>.</p

Duodenal biopsies were taken from trial 2 participants pre-infection (Pre-Inf, week 0), pre-challenge (Pre-Ch, week 20) and post-challenge (Post-Ch, week 21) and cultured in either medium alone or with 50 µg/ml QE65 peptide for 24 hours at 37°C in 95% O₂/5% CO₂.

<p>Supernatants were then taken and levels of cytokines measured by CBA. Results shown in A–C are from the pre-infection timepoint only, showing both medium and QE65 levels. All other results (D–I) have had medium controls subtracted. Cytokines shown are IL-2 (A, D), IFN-γ (B, E), IL-17A (C, F), IL-10 (G), IL-5 (H) and IL-13 (I). A–C was analysed by paired t tests, D–I by 1-way ANOVA. Where data was not normally distributed, log-transformation was carried out and parametric tests used. Differences are not significant unless indicated. * = p<0.05, ** = p<0.01.</p