Table 1.
In vitro activity of miltefosine against S. mansoni adult worms.
Fig 1.
In vitro lethal effect of miltefosine on S. mansoni adult worms.
LC50s of the drug (concentrations that kill 50% of worms) 24, 48, 72 and 96 hours after in vitro exposure to miltefosine were calculated from concentration-response curves at the respective time points and LC50 values were plotted against length of incubation.
Fig 2.
Immunofluorescent staining of miltefosine- and PZQ-treated S. mansoni adult worms.
The surface of worms treated with praziquantel (PZQ) (A-E), untreated worms (F) and worms treated with miltefosine (G-J) and exposed to: rabbit anti-S. mansoni infection (anti-SmI) IgG (i), rabbit anti-S. mansoni adult worm homogenate (anti-SmWH) antisera (ii). A, 2 μg/ml PZQ; B, 4 μg/ml PZQ; C, 6 μg/ml PZQ; D, 8 μg/ml PZQ; E, 10 μg/ml PZQ; G, 5 μg/ml miltefosine; H, 10 μg/ml miltefosine; I, 20 μg/ml miltefosine; J, 40 μg/ml miltefosine. Scale bar = 100 μm.
Fig 3.
Characterization of worm surface antigens recognized by rabbit anti-SmI antibodies after miltefosine- and PZQ-treatment.
(i) Immunofluorescent staining of PZQ- (6 μg/ml for 24 hours) (A and C) and miltefosine- (40 μg/ml for 48 hours) (B and D) treated S. mansoni adult worms detected with rabbit anti-SmI IgG antibodies (A and B) and IgG antibodies from a normal rabbit serum (C and D). Scale bar = 100 μm. (ii) Western immunoblots of a S. mansoni crude worm homogenate preparation (SmWH) were probed with rabbit anti-SmI antiserum (lane 1), rabbit anti-SmI antibodies eluted from PZQ- (6 μg/ml for 24 hours) (lane 2) and miltefosine- (40 μg/ml for 48 hours) (lane 3) treated worms. A blot of SmWH probed with a normal rabbit serum (lane 4) was used as a control. Lane M, protein molecular weight markers. Antigenic extract was loaded onto the gel with 10 μg protein/lane. Blots were detected using HRP-conjugated anti-rabbit IgG secondary antibodies. Detected antigens with the most intense reactivities are arrowed.
Fig 4.
Detection by rabbit anti-SmWH antibodies of worm surface antigens exposed by miltefosine or PZQ.
(i) Immunofluorescent staining of PZQ- (6 μg/ml for 24 hours) (A and C) and miltefosine- (40 μg/ml for 48 hours) (B and D) treated S. mansoni adult worms detected with rabbit anti-SmWH IgG antibodies (A and B) and IgG antibodies from a rabbit that received adjuvant alone (C and D). Scale bar = 100 μm. (ii) Western immunoblots of S. mansoni crude worm homogenate preparation (SmWH) were probed with rabbit anti-SmWH antiserum (lane 1), rabbit anti-SmWH antibodies eluted from PZQ- (6 μg/ml for 24 hours) (lane 2) and miltefosine- (40 μg/ml for 48 hours) (lane 3) treated worms. A blot of SmWH probed with a serum from a rabbit injected with Freund’s adjuvant alone (lane 4) was used as a control. Lane M, protein molecular weight markers. Antigenic extract was loaded onto the gel with 10 μg protein/lane. Blots were detected using HRP-conjugated anti-rabbit IgG secondary antibodies. Detected antigens with the most intense reactivities are arrowed.
Fig 5.
Purification and characterisation of the SmWH 23 kDa, 33 kDa and 37 kDa protein bands.
(i) Coomassie blue-stained reducing 12% SDS-PAGE gel of SmWH. Protein bands at ~23, 33 and 37 kDa (arrowed) were purified from by excision and elution from the gel. Lane M, protein molecular weight marker. (ii) Purified 37, 33 and 23 kDa protein bands in Coomassie blue-stained SDS-PAGE gel (1) and western immunoblots (2–5) probed with anti-SmI eluted antibodies from miltefosine- (2) and PZQ- (3) treated worms, a normal rabbit serum (4) and a rabbit anti-SmI antiserum (5). Samples were loaded onto the gel with 10 μg protein/lane.
Fig 6.
Detection of S. mansoni alkaline phosphatase (SmAP) after miltefosine treatment by immunofluorescence (i) and western blotting (ii).
(i) Immunofluorescence staining of drug-treated S. mansoni adult worms that had been incubated with a rabbit anti-S. mansoni alkaline phosphatase (anti-SmAP) antiserum. A, untreated worms; B, 6 μg/ml PZQ; C, 8 μg/ml PZQ; D, 10 μg/ml miltefosine; E, 20 μg/ml miltefosine; F, 40 μg/ml miltefosine. Scale bar = 100 μm. (ii) Western immunoblots of a detergent-soluble extract of S. mansoni adult worms (lanes 1 and 2) and SmWH (lanes 3 and 4) probed with the rabbit anti-SmAP-antiserum (lanes 1 and 3) and anti-SmAP antibody eluted from miltefosine-treated worms (lanes 2 and 4); M, protein molecular weight markers. Antigenic extract was loaded onto the gel with 10 μg protein/lane. Detected SmAP is boxed.
Fig 7.
Agglutination and indirect immunofluorescence reactivity of 3-hour schistosomula with rabbit anti-S. mansoni antibodies specific for drug-exposed antigens.
Representative microscopic images after incubation with the primary antibody under white light (A and B) and after processing for indirect immunofluorescence following incubation with anti-rabbit IgG conjugated to FITC (secondary antibody) (C). Schistosomula probed with: serum from naive rabbits (i); serum from rabbits immunized with adjuvant-alone (ii); anti-SmI antibodies eluted from PZQ-treated worms (iii); anti-SmI antibodies eluted from miltefosine-treated worms (iv); anti-SmWH antibodies eluted from PZQ-treated worms (v); anti-SmWH antibodies eluted from miltefosine-treated worms (vi). Scale bars = 100 μm.