Schistosomicidal effects of PZQ, ART and OXA on 14-day-schistosomula.

<p>Magnification of the <i>in vitro</i>-cultured schistosomula treated with PZQ, ART, and OXA at 10 μM. Drugs were added at day 14 and schistosomula were analyzed at day 21 (late treatment). 0.1% DMSO in medium (solvent of the drugs; used as negative control) had no effect on schistosomula development (representatives out of triplicate wells are shown; see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003593#pntd.0003593.g002" target="_blank">Fig. 2</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003593#pntd.0003593.s002" target="_blank">S2</a>–<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003593#pntd.0003593.s005" target="_blank">S5</a> Videos).</p

Development of <i>in vitro</i>-cultured schistosomula.

<p><b><i>S</i>. <i>mansoni</i> larvae were cultured <i>in vitro</i> in IMDM cell culture medium supplied with human PBMC, serum and whole blood.</b> Images were taken at day 2, 7, 14 and 21 after transformation. By day 7 the gut (filled with black hemozoin) had developed; by day 14 the typical divided gut became clearly visible, and by day 21 oral and ventral suckers appeared. PMBC (P), s (schistosomula), g (gut), os (oral sucker), vs (ventral sucker).</p

SmCD59a and SmKK7 on living, intact schistosomula are not accessible by anti-SmCD59a and anti-SmKK7 antisera.

<p>Binding of rat anti-SmCD59a antiserum (upper panel), rat anti-SmKK7 antiserum (median panel) and serum from infected rat (RIS; lower panel) to schistosomula at day 2, day 7 and day 14 after transformation. Antibody binding was detected with a fluorophore-labeled anti-rat IgG (IF). Corresponding lower images show the bright field images for control larval development (BF). Anti-SmCD59a and anti-SmKK7 antisera do not recognize the antigens on the surface of the live schistosomula, while the RIS shows strong binding, strongest on day 2. Scale bars = 100 μm.</p

Immunohistochemical analysis of SmCD59a and SmKK7 expression in schistosomula.

<p>Formalin-fixed and permeabilized schistosomula were analyzed by immunofluorescence microscopy at day 2, 7 and 14 following transformation. Schistosomula were incubated with rat anti-SmCD59a antiserum and rat anti-SmKK7 antiserum, binding of IgG antibodies were detected with fluorophore-labeled anti-rat IgG. The IF marked panels show the SmCD59a and SmKK7 fluorescence signal. Corresponding lower panels show the bright field image for control of the larval development (BF). Scale bars = 100 μm.</p

Differential unmasking of the antigen SmKK7 by treatment with PZQ and OXA.

<p>Drugs were added at day 2 and analyzed 2 days after administration of PZQ and 6 days after OXA application. For the analysis of antigen accessibility, schistosomula were incubated with anti-SmKK7 antiserum. Antibody binding was detected with a fluorophore-labeled anti-rat IgG (IF). Corresponding lower images show the bright field images (BF). Scale bars = 100 μm.</p

3D projection of schistosomula reveals differential localization of SmCD59a and SmKK7.

<p>Formalin-fixed and permeabilized schistosomula were analyzed at day 14 for SmCD59a localization and at day 7 for SmKK7 localization. Schistosomula were incubated with rat anti-SmCD59a antiserum, rat anti-SmKK7 antiserum, a control sample from uninfected rats (NRS) and a control sample from infected rats (RIS). Binding of IgG antibodies were detected with fluorophore-labeled anti-rat IgG. Immunofluorescence was visualized by projection overlay of a confocal z-stack. Corresponding panels show the bright field image for control of larval development.</p

Differential unmasking of the surface antigen SmCD59a by treatment with PZQ, ART and OXA.

<p>Drugs were added either at day 7 or day 14 and analyzed 2 days after administration of PZQ and ART and 6 days after OXA application. For the analysis of antigen accessibility, schistosomula were incubated with anti-SmCD59a antiserum. Antibody binding was detected with a fluorophore-labeled anti-rat IgG (IF). Corresponding lower images show the bright field images (BF). Scale bars = 100 μm.</p

Percentage of unmasked schistosomula after treatment with different OXA doses.

<p>14-day schistosomula were treated with 10, 1 or 0.1 μM OXA. 6 days after drug application schistosomula were incubated with anti-SmCD59a antiserum. Antibody binding was detected with a fluorophore-labeled anti-rat IgG and positive larvae were counted under an inverted fluorescence microscope. Data from three independent experiments are shown.</p

Expression of SmCD59a and SmKK7 in extracts of 2-, 7- and 14-day schistosomula.

<p>Western blots of extracts of 2-, 7- and 14-day schistosomula were incubated with rat anti-SmCD59a antiserum (upper panel A) and rat anti-SmKK7 antiserum (upper panel B), binding of IgG antibodies were detected with labeled anti-rat IgG. No cross-reactivity was observed with extract of human PBMC (huPBMC). Same blots subsequently stained with India Ink for protein detection (lower panel A+B).</p