Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30–120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed new therapeutic strategies and unearth stage-specific diagnostic biomarkers. Lymphatic filariasis is caused by parasitic nematodes that invade and occupy the host lymphatic system. The extent of lymphatic filariasis is staggering, with over 120 million people infected in 73 endemic countries and an estimated 40 million people suffering from a range of disfiguring and debilitating clinical manifestations of this disease. The mechanisms by which these medically important parasites navigate the host immune response to establish infection are not yet fully understood. In this study, we identify exosome-like vesicles (ELVs) that are abundantly released from infective stage L3 Brugia malayi, an etiological agent of human lymphatic filariasis. We show that these vesicles have a narrow size distribution and morphology consistent with classical exosomes, and that they contain common exosomal protein markers, putative effector proteins, as well as small regulatory RNAs. We show that ELVs are enriched with microRNAs that are perfectly conserved between parasite and host, suggesting a potentially novel mechanism by which filarial worms can actively manipulate host gene expression. We demonstrate that parasite ELVs are internalized by macrophages and elicit a classically activated phenotype in these host cells. The discovery of exosome-like vesicle release by human nematode parasites newly enlightens the roadmap to understanding the pathology of LF and related helminthiases. These vesicles also present promising new targets for intervention and diagnostics.

Schistosomiasis is a parasitic flatworm disease that infects 200 million people worldwide. The drug praziquantel (PZQ) is the mainstay therapy but the target of this drug remains ambiguous. While PZQ paralyses and kills parasitic schistosomes, in free-living planarians PZQ caused an unusual axis duplication during regeneration to yield two-headed animals. Here, we show that PZQ activation of a neuronal Ca²⁺ channel modulates opposing dopaminergic and serotonergic pathways to regulate ‘head’ structure formation. Surprisingly, compounds with efficacy for either bioaminergic network in planarians also displayed antischistosomal activity, and reciprocally, agents first identified as antischistocidal compounds caused bipolar regeneration in the planarian bioassay. These divergent outcomes (death versus axis duplication) result from the same Ca²⁺ entry mechanism, and comprise unexpected Ca²⁺ phenologs with meaningful predictive value. Surprisingly, basic research into axis patterning mechanisms provides an unexpected route for discovering novel antischistosomal agents. Schistosomiasis (Bilharzia) is one of the most burdensome parasitic worm infections, encumbering third world economies with an annual loss of several million disability-adjusted life years. The key treatment for schistosome infections is the drug praziquantel but the mechanism of action of this drug remains controversial hampering targeted development of next generation antischistosomal agents. Here we provide fresh insight into the signaling pathways engaged by PZQ, by resolving commonalities in the action of PZQ with the process of regenerative signaling in free-living planarian flatworms. A similar calcium-dependent network is engaged in both model systems, but with divergent phenotypic outcomes. This relationship provides predictive insight such that basic research on signaling pathways involved in tissue regeneration reveals novel drug leads for schistosomiasis, and reciprocally schistosomal drug screens reveal targets involved in regenerative signaling. We believe this phenology will be helpful for uncovering new antischistosomal drug targets by exploiting broader vulnerabilities within the PZQ interactome.

G protein-coupled receptors (GPCRs) represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi) was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα_i/o. The method was then extended to deorphanize a novel Gα_s-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.

Objective—To evaluate the effect of IV administration of polymyxin B on clinical and serum biochemical variables in foals with experimental endotoxemia. Design—Prospective experimental study. Animals—14 healthy neonatal foals. Procedures—Foals were randomly assigned to a treatment or control group and were administered a single dose of lipopolysaccharide (0.5 μg/kg [0.23 μg/lb]) IV over 30 minutes. The treatment group received polymyxin B (6,000 U/kg [2,727 U/lb], IV) immediately after completion of lipopolysaccharide infusion; the control group was administered an equal volume of saline (0.9% NaCl) solution. Subsequent doses of polymyxin B or saline solution were administered IV at 8 and 16 hours. Blood was collected at various time points, and outcome variables, including heart rate, respiratory rate, rectal temperature, attitude score, WBC count, neutrophil count, lymphocyte count, monocyte count, platelet count, Hct, blood lactate concentration, blood glucose concentration, serum tumor necrosis factor-α concentration, and plasma thromboxane B₂ concentration, were measured. Urine was collected prior to and after experimentation to determine whether nephrotoxicosis was associated with treatment. Results—The treatment group had significantly lower blood lactate concentration and serum tumor necrosis factor-α and plasma thromboxane B₂ concentrations and had higher blood glucose concentrations and better attitude scores, compared with the control group, at various time points during the study. No other significant differences and no evidence of overt nephrotoxicosis were detected. Conclusions and Clinical Relevance—Administration of polymyxin B IV in healthy neonatal foals challenged with lipopolysaccharide attenuated some clinical and serum biochemical derangements associated with endotoxemia.

Praziquantel (PZQ) is a key therapy for treatment of parasitic flatworm infections of humans and livestock, but the mechanism of action of this drug is unresolved. Resolving PZQ-engaged targets and effectors is important for identifying new druggable pathways that may yield novel antiparasitic agents. Here we use functional, genetic and pharmacological approaches to reveal that serotonergic signals antagonize PZQ action in vivo. Exogenous 5-hydroxytryptamine (5-HT) rescued PZQ-evoked polarity and mobility defects in free-living planarian flatworms. In contrast, knockdown of a prevalently expressed planarian 5-HT receptor potentiated or phenocopied PZQ action in different functional assays. Subsequent screening of serotonergic ligands revealed that several ergot alkaloids possessed broad efficacy at modulating regenerative outcomes and the mobility of both free living and parasitic flatworms. Ergot alkaloids that phenocopied PZQ in regenerative assays to cause bipolar regeneration exhibited structural modifications consistent with serotonergic blockade. These data suggest that serotonergic activation blocks PZQ action in vivo, while serotonergic antagonists phenocopy PZQ action. Importantly these studies identify the ergot alkaloid scaffold as a promising structural framework for designing potent agents targeting parasitic bioaminergic G protein coupled receptors. The parasitic infection schistosomiasis afflicts millions of people worldwide and is clinically treated using a single drug, praziquantel (PZQ). Despite the fact that PZQ has served as a stalwart anthelmintic for decades, the molecular basis of action of this clinical agent is poorly understood. This lack of mechanistic information impedes the rational design of alternative therapies and highlights the need for new approaches for studying the target(s) and effectors engaged by PZQ in vivo. Here, we exploit the predictive phenology between free-living planarian regenerative screens and parasitic neuromuscular physiology to reveal a broad efficacy of ergot alkaloids in phenocopying the action of PZQ. In planarian regenerative screens, data highlight structural features of the ergoline scaffold that yield specific regenerative effects to promote or inhibit head regeneration. Ergot alkaloids with efficacy in regenerative assays were also found to modulate the contractility of schistosomules. Overall, these data highlight a possible therapeutic potential of ergot alkaloids as antischistosomals and the action of PZQ as an ergomimetic.

Background: Sport injuries have been common among athletes across the globe for decades and have the potential to disrupt athletic careers, performance, and psyche. Many health professionals and organizations have undertaken injury mitigation strategies to prevent sport injuries through protective equipment, training protocols, and a host of other evidence-based practices. Many of these specialized training methods were disrupted due to protocols to mitigate the spread of COVID-19. This research examines the effects of the COVID-19 pandemic in relation to the prevalence of athletic injuries in the National Football League (NFL). Objective: During the COVID-19 pandemic, NFL teams and athletes across all levels of sport were reported to have reduced training in preparation for their seasons due to protocols to mitigate the spread of COVID-19. This study compares the prevalence of injury during the 2018, 2019, and 2020 NFL seasons, with the aim to determine the potential causes of the differences in injury prevalence. Methods: Official injury reports from each team were counted during the 17-week regular season of each year (2018, 2019, and 2020). The data were analyzed using an unpaired t test to compare the injury prevalence between each of the three seasons. Results: The 2018 season produced a total of 1561 injuries and a mean of 48.8 injuries per team. The 2019 season produced a total of 1897 injuries and a mean of 59.3 injuries per team, while the 2020 season produced a total of 2484 injuries and a mean of 77.6 injuries per team. An unpaired t test was performed using the data to compare the mean number of injuries per team during each of the seasons. Comparison of the 2020 season against the 2019 season showed a statistically significant difference (P<.001); comparison of the 2020 season to the 2018 season found a statistically significant difference (P<.001); and comparison between the 2019 and the 2018 seasons found a statistically significant difference (P=.03). Conclusions: Although the 2019 and 2018 seasons showed a statistically significant difference (P=.03), this difference is not as large when we compare the 2020 seasons versus the 2019 (P<.001) and 2018 (P<.001) seasons. The astronomical increase in injury prevalence during the 2020 season over the previous years raises the possibility that there was a reduced physiological adaptation to stress, due to the limited amount of training as a result of the closure of practice facilities in order to slow the spread of COVID-19.