Structure of compounds [21].

<p>(3<i>R</i>,6<i>R</i>)-linalool oxide acetate (<b>1</b>), (<i>E</i>)-spiroether (<b>2</b>), luteolin (<b>3</b>), luteolin-7-<i>O</i>-β-D-glucopyranoside (<b>4</b>) were derived from <i>A</i>. <i>nubigena</i> and tested for their anthelmintic activities using xWORM. Plant photo courtesy–PW.</p

Scanning electron micrographs of <i>S</i>. <i>mansoni</i> adult fluke teguments after treatment with 4 μg/mL concentration of luteolin (3) and praziquantel.

<p>A, B and C represent flukes treated with solvent (1% DMSO in culture media). A) Male tegument with well defined tubercles. B) Female tegument with clearly defined grooves and sensory papillae. C) Oral sucker with well formed pits containing spines. D, E and F represent flukes treated with praziquantel. D) Male tegument with damaged tubercles and spines. E) Female tegument with damaged papillae and swollen grooves. F) Oral suckers with eroded pits and detached spines. G, H and I represent flukes treated with luteolin (<b>3</b>). G) Male tegument with destroyed tubercles and holes. H) Female tegument with burst sensory papillae and holes. I) Oral suckers with partially eroded pits. Arrows in panels highlight treatment effects described in results text. All images at ×4000 magnification and 1 μm are scale bars shown.</p

<i>In vitro</i> anti-schistosome activities of four compounds against adult <i>S</i>. <i>mansoni</i>.

<p>A) Motility index dose response curve of worms at the 12 hr time point when treated with (3<i>R</i>,6<i>R</i>)-linalool oxide acetate (<b>1</b>), (E)-spiroether (<b>2</b>), luteolin (<b>3</b>), luteolin-7-<i>O</i>-β-D-glucopyranoside (<b>4</b>) (abbreviated as Com-1, Com-2, Com-3 and Com-4) at different concentrations (0.1–1000 μg/mL). Motility index was calculated as the standard deviation (SD) over 800 data points (i.e. 4 readings per min for 200 min) of the cell index (CI) difference from the rolling average over 20 data points (10 proceeding and preceding CI values—5 min total). B) 50% inhibitory concentration (IC₅₀) curves over time. Error bars represent 95% confidence intervals of nonlinear curve fit. The curves were marginally shifted on the x-axis to aid viewing. These figures represent the data from three independent studies.</p

Effects of luteolin (3) and praziquantel on the morphology of adult <i>S</i>. <i>mansoni</i> at 4–20 μg/mL concentration visualized using scanning electron microscopy.

<p>A, C and E show male flukes. B, D and F depict female flukes. (A, B) Flukes in DMSO/media control. (C, D) Flukes treated with praziquantel (tightly coiled). (E, F) Flukes treated with luteolin (<b>3</b>) (moderately coiled). Three separate wells (with 3–5 adult flukes per well) were treated with different doses of test samples as above and each of them was examined under SEM.</p

Effects of luteolin (3) and mebendazole on the cuticle of adult <i>T</i>. <i>muris</i>.

<p>A) Normal appearance of control worms cultured in 1% DMSO/media (x100) and at higher magnification focusing on the cuticle (B) and bacillary gland (C), respectively. D) Shrinkage of the anterior regions of worms after treatment with mebendazole and damage to the cuticle (E) and bacillary glands (F) were observed at higher magnification. G) Shrinkage of the anterior regions of worms after treatment with luteolin (<b>3</b>) and damage to the cuticle (H) and swelling of bacillary glands (I) observed at higher magnification. Red arrows in all panels highlight treatment damage described in results text.</p

Effects of luteolin (3) and mebendazole on <i>T</i>. <i>muris</i> burden in mice.

<p>The graph represents the percentage of worms recovered from the STAT6^<i>-1-</i> mice five days after a single oral dose of luteolin (<b>3</b>) (27.1% reduction with <i>p</i>-value of 0.0087) and the positive control drug, mebendazole (93.1% reduction with <i>p</i>-value of 0.0005). The <i>p</i>-values were determined by one-way ANOVA Holm-Sidak’s multiple comparisons test. Each experimental group consisted of nine mice.</p

Anti-<i>Trichuris</i> activity of compounds (1–4) on adult <i>T</i>. <i>muris</i>.

<p>A) Motility index dose response curve of worms at the 12 hr time point when treated with (3<i>R</i>,6<i>R</i>)-linalool oxide acetate (<b>1</b>), (<i>E</i>)-spiroether (<b>2</b>), luteolin (<b>3</b>) and luteolin-7-<i>O</i>-β-D-glucopyranoside (<b>4</b>) (abbreviated as Com-1, Com-2, Com-3 and Com-4) at different concentrations. Motility index was calculated as the standard deviation (SD) over 800 data points (i.e. 4 readings per min for 200 min) of the cell index (CI) difference from the rolling average over 20 data points (10 proceeding and preceding CI values—5 min total). B) Combined IC₅₀ values of these four compounds calculated for three different doses at 1 hr, 6 hr, and 12 hr time points. Error bars represent 95% confidence intervals of nonlinear curve fit. The curves were marginally shifted on the x-axis to aid viewing. These figures represent the data from three independent studies.</p

Effect of compounds on the survival of schistosomula.

<p>A) Images (20x) of wells containing schistosomula that were treated with linalool oxide acetate (<b>1</b>) and luteolin (<b>3</b>) (abbreviated as Com-1 and Com-3) at the maximum concentration tested of 250 μg/mL and stained with trypan blue. Deep blue staining signifies a dead parasite. B) The effect of (3<i>R</i>,6<i>R</i>)-linalool oxide acetate (<b>1</b>) and luteolin (<b>3</b>) (abbreviated as Com-1 and Com-3) on schistosomula mortality observed at two-fold dilutions (250–1.9 μg/mL). The data was generated from triplicate samples obtained from two independent studies.</p

image_4_Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.tif

<p>Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite–host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.</p

image_1_Hookworm Secreted Extracellular Vesicles Interact With Host Cells and Prevent Inducible Colitis in Mice.tif

<p>Gastrointestinal (GI) parasites, hookworms in particular, have evolved to cause minimal harm to their hosts, allowing them to establish chronic infections. This is mediated by creating an immunoregulatory environment. Indeed, hookworms are such potent suppressors of inflammation that they have been used in clinical trials to treat inflammatory bowel diseases (IBD) and celiac disease. Since the recent description of helminths (worms) secreting extracellular vesicles (EVs), exosome-like EVs from different helminths have been characterized and their salient roles in parasite–host interactions have been highlighted. Here, we analyze EVs from the rodent parasite Nippostrongylus brasiliensis, which has been used as a model for human hookworm infection. N. brasiliensis EVs (Nb-EVs) are actively internalized by mouse gut organoids, indicating a role in driving parasitism. We used proteomics and RNA-Seq to profile the molecular composition of Nb-EVs. We identified 81 proteins, including proteins frequently present in exosomes (like tetraspanin, enolase, 14-3-3 protein, and heat shock proteins), and 27 sperm-coating protein-like extracellular proteins. RNA-Seq analysis revealed 52 miRNA species, many of which putatively map to mouse genes involved in regulation of inflammation. To determine whether GI nematode EVs had immunomodulatory properties, we assessed their potential to suppress GI inflammation in a mouse model of inducible chemical colitis. EVs from N. brasiliensis but not those from the whipworm Trichuris muris or control vesicles from grapes protected against colitic inflammation in the gut of mice that received a single intraperitoneal injection of EVs. Key cytokines associated with colitic pathology (IL-6, IL-1β, IFNγ, and IL-17a) were significantly suppressed in colon tissues from EV-treated mice. By contrast, high levels of the anti-inflammatory cytokine IL-10 were detected in Nb-EV-treated mice. Proteins and miRNAs contained within helminth EVs hold great potential application in development of drugs to treat helminth infections as well as chronic non-infectious diseases resulting from a dysregulated immune system, such as IBD.</p