Presence of human serum <i>in vitro</i> enhances NTS viability and induces worm development past the LuS.

NTS were cultured in HM and DMEM supplemented with 200 U/ml Penicillin and 200 μg/ml Streptomycin with or without additional supplementation of 20% HSe. (A) Viability scoring was performed at the indicated time points. The percentage of developmental stages in culture with either (B) HM, (C) HM + 20% HSe, (D) DMEM or (E) DMEM + 20% HSe was calculated for the indicated time points by bright field microscopy. (F) Representative photomicrographs were taken on day 35 post-transformation. Scale bar applies to all shown pictures. Arrowheads indicate dead NTS. Arrows indicate early and late LiS. Each data point is shown as a mean ± SD of pooled data from three independent experiments with three biological replicates each. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 comparing HM with HM + 20% HSe, × p ≤ 0.05, ×× ≤ 0.01, ××× ≤ 0.001 comparing DMEM with DMEM + 20%. HSe, human serum; SkS, skin stage; LuS, lung stage; LiS, liver stage; s.p., score points; p.t., post-transformation.</p

Generation of late LiS worm in medium supplemented with human serum increases in a concentration-dependent manner.

NTS were cultured in HM supplemented with 200 U/ml Penicillin and 200 μg/ml Streptomycin as well as 1%, 5%, 10%, 20% and 50% of HSe. The percentages of the developmental stages as well as dead parasites in culture with (A) HM alone or supplemented with (B) 1%, (C) 20% and (D) 50% HSe were calculated at indicated time points and (E) their viability was scored during bright field microscopy. (F) Representative photomicrographs were taken on day 35 post transformation. Scale bar applies to all pictures shown. Arrowheads indicate dead NTS. Arrows indicate LiS (early LiS in 5% and 10% HSe) and late liver stage (in 20% and 50% HSe). Results are pooled from three independent experiments with at least three biological replicates each. ×× p ≤ 0.01 comparing HM vs. 1% HSe; ┴┴ p ≤ 0.01 comparing HM vs. 5% HSe; ∨∨ p ≤ 0.01 comparing HM vs. 10% HSe; ≠≠ p ≤ 0.01 comparing HM vs. 20% HSe; ** comparing p ≤ 0.01 HM vs. 50% HSe. HSe, human serum; s.p., score points; p.t., post-transformation; SkS, skin stage; LuS, lung stage; LiS, liver stage.</p

Culture media selectively ensure long-term viability of <i>S</i>. <i>mansoni</i> NTS without serum or cell supplementation.

NTS were cultured in HM, M199, DMEM or RPMI supplemented with 200 U/ml Penicillin and 200 μg/ml Streptomycin for 4 weeks. (A) The viability of the parasites was scored at the indicated time points and (B) the morphology was observed under light microscopy. Photomicrographs were taken 2 and 4 weeks p.t. Scale bar applies to all shown images. Results are representative of at least three independent experiments. Each data point is shown as mean ± SD of at least three biological replicates. p.t., post-transformation; s.p., score points; HM, HybridoMed Diff 1000.</p

Efficient drug screening of advanced larval stages of <i>S</i>. <i>mansoni</i> generated in human serum.

NTS were cultured in HM supplemented with 200 U/ml Penicillin and 200 μg/ml Streptomycin and 20% HSe for 1 week to generate LuS (B, D, F and H) and 6 weeks to obtain LiS (A, C, E, G) schistosomula. (A, B) Praziquantel (PZQ), (C, D) Oxamniquine (OXM), (E, F) Mefloquine (MFQ) and (G, H) Artemether (ART) were dissolved in DMSO and added at indicated concentrations for the entire duration of the experiment. 1% DMSO in culture medium served as control. Viability was scored before treatment (0 h) and 3 hours, 1, 2, 3 and 7 days a.t. Each data point is shown as a mean ± SD of three independent experiments with at least three biological replicates each. ××× p ≤ 0.001, ×× p ≤ 0.01, × p ≤ 0.05 comparing control with 1 μg/ml drug; ┴┴┴ p ≤ 0.001 ┴┴ p ≤ 0.01 ┴ p ≤ 0.05 control with 10 μg/ml drug; ***p ≤ 0.001, **p ≤ 0.01 *p ≤ 0.05 control with 100 μg/ml drug. LuS, lung stage; LiS, liver stage; s.p., score points; a.t., after treatment.</p

HSe supplemented HM increased the development to late liver stage compared to Basch medium 169.

NTS of Schistosoma mansoni (NMRI strain) were cultured in HM and Basch-Medium 169 supplemented with 200 U/ml Penicillin and 200 μg/ml Streptomycin as well as additional supplementation of either 20% FCS or 20% HSe. (A) Viability scoring was performed at the indicated time points. The percentage of developmental stages in culture with (B) Basch medium 169 supplemented with (C) 20% FCS or (D) 20% HSe as well as (E) HM supplemented with (F) 20% FCS or (G) 20% HSe were calculated per well for the indicated time points by bright field microscopy. (H) Representative photomicrographs were taken on day 28 p.t. Scale bar applies to all images shown. Arrowheads indicate dead NTS. Arrows indicate early and late LiS. Each data point is shown as a mean ± SD of an experiment with three biological replicates each. FCS, fetal calf serum; HSe, human serum; SkS, skin stage; LuS, lung stage; LiS, liver stage; s.p., score points; p.t., post-transformation.</p

Drug sensitivity is dependent on the developmental stage of <i>S</i>. <i>mansoni</i> larvae generated <i>in vitro</i>.

NTS were cultured and matured in HM supplemented with 200 U/ml Penicillin and 200 μg/ml Streptomycin only or additionally supplemented with 20% HSe. 100, 10 and 1 μg/ml of (A) praziquantel, (B) oxamniquine, (C) mefloquine and (D) artemether were then added to the culture 1 day p.t. to test the SkS, 7 days p.t. to test the LuS and 6 weeks p.t. to test the LiS. Viability was scored 72h a.t. Each data point is shown as a mean ± SD of three independent experiments with at least three biological replicates each. ××× p ≤ 0.001, ×× p ≤ 0.01, × p ≤ 0.05 comparing control with 1 μg/ml drug; ┴┴┴ p ≤ 0.001, ┴┴ p ≤ 0.01, ┴ p ≤ 0.05 comparing control with 10 μg/ml drug; ***p ≤ 0.001, **p ≤ 0.01 *p ≤ 0.05 control with 100 μg/ml drug. SkS, skin stage; LuS, lung stage; LiS, liver stage; PZQ, praziquantel; OXM, oxamniquine; MFQ, mefloquine; ART, artemether; s.p., score points.</p

Suppression of AAI in <i>S. mansoni</i>-infected mice requires patency.

<p>Groups of BALB/c female mice were infected with <i>S. mansoni</i>. Left PiP experiments: After the 5th week of infection, OVA sensitizations were administered either i.p. with alum or s.c. in the absence of adjuvant (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002379#pntd-0002379-g001" target="_blank">Figure 1A</a>). Middle PP experiments: sensitizations were given during the first weeks of infection (before patency) but challenge was performed at the same time-point of infection as in PiP investigations (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002379#pntd-0002379-g001" target="_blank">Figure 1B</a>). Right EP studies: both sensitizations and challenge were performed before the onset of patency (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002379#pntd-0002379-g001" target="_blank">Figure 1C</a>). Upon AAI analysis, individual mice were assessed for leucocyte numbers (A–C) and eosinophil levels (D–F). Lung sections were also scored for inflammation (G–I). In i.p.+Alum experiments displayed in A, D and G, bars show mean + SD of individual mice from 5 independent infection/allergy experiments (PBS, n = 21; OVA, n = 34; Inf/OVA, n = 29 and Inf., n = 28). In s.c.-Alum experiments OVA, n = 8 and Inf/OVA, n = 7. In PP experiments B, E and H, bars show mean + SD of individual mice from 2 independent infection/allergy experiments (PBS, n = 2; OVA, n = 14; Inf/OVA, n = 13 and Inf., n = 10). In EP experiments C, F and I, bars show mean + SD of individual mice from 1 or 3 similar independent infection/allergy experiments (PBS, n = 2; OVA, n = 10; Inf/OVA, n = 10 and Inf., n = 4). Asterisks show statistical differences (ANOVA or Student's t test) between the groups indicated by the brackets (*p<0.05, **p<0.01, ***p<0.001).</p

Depletion of Treg during sensitization reverts schistosome-mediated dampening of AAI.

<p>As depicted in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002379#pntd-0002379-g001" target="_blank">Figure 1F</a>, groups of C57BL/6 DEREG mice were infected with <i>S. mansoni</i>. On the 9^th and 10^th week of infection mice were sensitized i.p. with OVA in the presence of alum. Prior to sensitization, Treg were depleted by the administration of DT. During the 12th week of infection, mice were challenged and assessed for the number of leucocytes (A) and eosinophils (B) in the BAL. Levels of inflammation were also scored using lung sections stained with PAS (C–G). OVA-specific IgE levels were measured in the sera of individual mice via ELISA (H). OVA-specific responses were measured following stimulation of spleen cells (2×10⁵) with 10 µg/ml OVA (I). In addition, schistosome-specific responses were evaluated upon re-stimulation of spleen cells with SEA (25 µg/ml) (J). After 72 hours, IL-5 levels were determined in the culture supernatant by ELISA (I and J). Egg burden was measured in livers of individually infected mice (K). Bars show data from one of two similar experiments (OVA, n = 7; OVA^DT, n = 7; Inf/OVA^DT, n = 5; Inf/OVA n  = 8; PBS^DT n = 3). Asterisks show statistical differences (ANOVA or Student's t test) between the groups indicated by the brackets (*p<0.05, p<0.01, ***p<0.001).</p

Experimental models for examining <i>S. mansoni</i>-mediated suppression of AAI.

<p>A <i>Patent Infected Protection (PiP)</i>. Groups of BALB/c female mice were infected with <i>S. mansoni</i>. Inf/OVA and OVA groups of mice were then sensitised with OVA either i.p. with Alum or s.c. without adjuvant at the indicated timepoints. Challenge occurred over three consecutive days in the 9th week of infection and analysis 5 days thereafter. B <i>Prepatent (PP</i>). Sensitizations were started in Inf/OVA and OVA groups during the first weeks of infection, before eggs are released from fecund females. Challenge and analysis were performed as in A. C <i>Early Phase (EP)</i>. AAI was performed entirely over the first 5 weeks of infection. D <i>Praziquantel therapy</i>. During the 6th week of infection, mice were orally treated with PZQ for 5 consecutive days. AAI was then begun after either 2 weeks (upper track) or 6 weeks (lower track). E <i>Depletion of Treg</i>. Groups of C57BL/6 DEREG mice were infected with <i>S. mansoni</i>. In some groups, Treg were depleted using DT injections prior to OVA sensitization at the indicated timepoints. Challenge and analysis occurred in the 12th week of infection.</p

Increased Foxp3⁺ Treg frequency in the draining lymph nodes of Inf/OVA mice is not reflected in lung sections.

<p>A) Total cell counts and B) the number of CD4⁺Foxp3⁺ T cells in the draining lymph nodes of individual mice on the day of analysis in PiP studies. Symbols depict the levels in each mouse and data shows values from 5 independent infection studies (PBS, n = 21; OVA, n = 34; Inf/OVA, n = 29 and Inf., n = 28). CD3⁺Foxp3⁺ T cells in the lung sections of C) OVA mice, D) Inf/OVA mice and E) control groups (PBS) were determined by immunohistochemical staining. Arrows depict positive cells. F) Quantification of CD3⁺Foxp3⁺ T cells in individual lung sections was determined within a 1 mm² area. Asterisks show statistical differences (ANOVA) between the groups indicated by the brackets (*p<0.05, **p<0.01, ***p<0.001).</p