Fig 1.
Clear and blue-stained capsules (EB capsules) on the surface and cut surfaces of brains from infected pigs.
Photographs of the surface and coronal sections of representative brains from an untreated pig (A and B) and a pig treated with praziquantel 48 h previously (C and D), showing cysts with clear (white arrowheads) and blue capsules (blue arrowheads) visible within the parenchyma and on the surface.
Fig 2.
EB capsules show increased inflammation and cyst wall damage compared to clear capsules.
Cysts were harvested from the brains of infected untreated pigs (n = 3; see Methods) or pigs treated with PZQ 48h (n = 4) or 120h (n = 4) earlier. For each cyst, scores reflecting the degree and extent of the inflammatory cell infiltrates (A) and cyst wall damage (B) at 48h (PZQ 48h) and 120h (PZQ 120h) after PZQ treatment (see Methods and S1 Fig.) are shown (open squares refer to clear capsules and closed squares, to EB capsules, bar = mean). Statistical significance in comparison between groups is represented by asterisks (*: p<0.05; **: p<0.01; ***: p<0.005).
Fig 3.
Upregulation of proinflammatory genes in EB capsules compared to clear capsules.
Expression levels of RNA in six types of brain tissue samples, uninfected brain tissue distant from cysts (no cysts), clear pericystic brain tissue in untreated pigs (UT Clear) and at 48h post treatment (PZQ 48 Clear) and EB capsules in untreated pigs (UT Blue) and at 48h (PZQ 48 h Blue) and 120h post treatment (PZQ 120 h Blue) were quantified using real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays. Gene expression levels are depicted as fold increase of studied RNA over reference (housekeeping) RNA (18S rRNA), TNF-α (A; number [n] of samples for the indicated types of capsules are shown below each bar) and IL-6 (B). The bars represent means and CI95 for the group. Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.
Fig 4.
Upregulation of both Th1 and Th2 markers, IFN-γ and IL-13, in blue capsules following PZQ treatment.
Expression levels of RNA in brain tissues and EB-stained and clear capsules for IFN-γ (A; number [n] of samples for the indicated types of capsules are shown below each bar), and IL-13 (B). In each graph, the bars represent means and CI95 for the group. Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.
Fig 5.
Increased expression of CD-25 and IL-10 in EB capsules following PZQ treatment.
Expression levels of RNA in brain tissue samples from EB-stained and clear capsules were quantified (See Methods). Shown are expression levels, depicted as fold increase over the RNA levels for the reference (housekeeping) gene (18S rRNA), encoding four representative regulatory T cells (Treg) phenotyping and functional markers: CD25 (A; number [n] of samples for the indicated types of capsules are shown below each bar), FoxP3 (B), IL-10 (C) and CTLA4 (D). Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.
Fig 6.
Increased expression of fibrosis/granuloma promoting genes in EB and clear capsules.
Methods and tissues examined are identical to those analyzed in Figs. 3–5. Shown are four genes representative of fibrosis and granuloma promoting molecules: MMP1 (A; number [n] of samples for the indicated types of capsules are shown below each bar; n = 7, 6, 3, 6, 6, respectively), MMP9 (B), TIMP1 (C) and TIMP2 (D). Statistically significant differences in levels of gene expression are indicated by asterisks (p<0.05, Mann-Whitney U test). Asterisks represent level of significance: *: p<0.05; **: p<0.01; ***: p<0.005.