14 research outputs found
A Principal Molecular Target of K11777 in S. mansoni is the Cathepsin B1 Cysteine Protease
<div><p>(A) Extracts of worms exposed to long-course treatment with K11777 (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-g001" target="_blank">Figure 1</a>) had significantly less protease activity (relative fluorescence units/min/μg of extract) against the cathepsin B–selective substrate Z-Arg-Arg-AMC (Z-RR-AMC) or the cathepsins B- and L-specific substrate Z-Phe-Arg-AMC (Z-FR-AMC).</p>
<p>(B) By SDS-PAGE, a major 31 kDa protease species in extracts of control worms (lane 2) or worms exposed to K11777 (lane 4) reacted with the radiolabeled CP inhibitor <sup>125</sup>I-DCG-04. The reaction was approximately 10-fold less intense in the latter by scanning densitometry. For both extracts, the reaction with <sup>125</sup>I-DCG-04 could be inhibited by prior incubation with the cathepsin B–selective inhibitor CA-074 (lanes 1 and 3), thus confirming the 31 kDa reactive species as cathepsin B1 [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-b031" target="_blank">31</a>]. The second, faster-migrating protease species in lane 1 is consistent with a schistosome cathepsin L [<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-b031" target="_blank">31</a>].</p></div
Early and Late Treatment of Mice with K11777 Significantly Decreases Egg and Worm Burdens in Mice Infected with S. mansoni
<p>Results for egg (A) and worm (B) burdens following the experimental protocol depicted in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-g001" target="_blank">Figure 1</a> are shown. Points represent data from individual mice which were combined from either two early or two late treatment experiments. The horizontal bars represent median values: **<i>p</i> = 0.002, *** <i>p</i> = 0.0007. The data comparing female worm burdens between late treatment and untreated controls were not statistically different (<i>p</i> = 0.2).</p
Long-Course Treatment with K11777 Resolves Egg-Induced Hepato- and Splenomegalies in S. mansoni–Infected Mice
<p>Points represent data from individual mice that were infected and treated, infected and untreated (control), or uninfected (<a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-g001" target="_blank">Figure 1</a>). The horizontal bars represent median values: <b><sup>**</sup></b><i>p =</i> 0.007<i>.</i> The hatched lines represent the baseline for age-matched uninfected mice.</p
Experimental Design for Assessing the Therapeutic Effect of the Cysteine Protease Inhibitor K11777 on Mice Infected with S. mansoni
<p>All mice were infected with 150 S. mansoni cercariae injected subcutaneously. K11777 was administered intraperitoneally in 100 μl of water at the dosages and timings indicated. Control mice received water alone. All mice were perfused at 49 days p.i., and worm and egg burdens and organ pathology were assessed.</p
Early Treatment of S. mansoni–Infected Mice with K11777 Retards Hepatocellular Damage Arising from Parasite Eggs
<p>Plasma samples from mice in experiments outlined in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-g001" target="_blank">Figure 1</a> were quantitatively assayed for the presence of ALT activity as a marker for hepatocellular damage. Points represent data from individual mice which were combined from either two early or late treatment experiments. The horizontal bars represent median values; ***<i>p</i> = 0.002. For plasma from late-treatment mice, ALT activity was not significantly different (<i>p</i> = 0.9) from untreated controls, most likely due to the re-emergence of hepatocellular toxicity upon cessation of treatment at day 37 p.i.—i.e., 12 days prior to the end of the experiment and recovery of worms. The hatched line represents the baseline for age-matched uninfected mice.</p
Long-Course Treatment with K11777 of Mice Infected with S. mansoni Retards Worm Growth
<p>Representative worms treated with K11777 in the experiment outlined in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-g001" target="_blank">Figure 1</a> are shown.</p
Late Treatment of <i>S. mansoni–</i>Infected Mice with K11777 Decreases the Granulomatous Reaction to Parasite Eggs Trapped in the Liver
<p>After necropsy of mice from experiments outlined in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040014#pmed-0040014-g001" target="_blank">Figure 1</a>, livers were sectioned at 5 μm and stained with hematoxylin and eosin. Arrows in representative sections indicate schistosome eggs; the granulomatous reaction to the eggs is much reduced after the late-treatment regimen (A), compared to the intense reaction visible in sections from untreated control mice (B). Note that after early K11777 treatment, eggs or egg-associated granulomas were not found (unpublished data). Scale bar = 100 μm.</p
Parasite traversal across human BMEC.
<p>Transwell inserts containing human BMEC (initial TEER = 26.3 Ω) were incubated with 3×10<sup>5</sup> pZJMTbRho RNAi trypanosomes (+/−tetracycline) and the number of parasites that crossed the BMEC monolayers into the bottom wells determined. All values represent the mean±SEM of triplicate determinations.</p
RNAi reduces brucipain protein and protease activity.
<p>(A) Equal amounts of protein were resolved by 15% SDS-PAGE, stained with anti-rhodesain antibody, and visualized by Western blot. Brucipain protein level is decreased after RNAi induction in pZJMTbRho parasites recovered from infected mice but the level of cathepsin B is not decreased after brucipain RNAi induction in pZJMTbRho parasites. (B) Brucipain activity is also decreased by 60% with brucipain RNAi induction. The level of brucipain activity in pZJMRho transfected parasites purified from mice was determined with the active site tag <sup>125</sup>I-DCG-04, visualized by autoradiography, and quantified by PhosphorImager analysis. (C) In the absence of RNAi bands of brucipain and TbCatB activity can be identified in purified parasites from mice infected with 90-13 labeled with the active site tag <sup>125</sup>I-DCG-04 and visualized by autoradiography.</p
RNAi reduces expression of brucipain mRNA in parasites isolated from mice.
<p>(A) Evidence that RNAi reduces expression of brucipain mRNA in parasites isolated from mice. Mice infected with the brucipain RNAi-containing plasmids were either induced with doxycycline or left un-induced. (B) RNAi induced against brucipain did not decrease mRNA level for cathepsin B.</p