Quantifying metabolic activity of Ascaris suum L3 using resazurin reduction.

BACKGROUND: Helminth infections are an important public health problem in humans and have an even greater impact on domestic animal and livestock welfare. Current readouts for anthelmintic drug screening assays are stage development, migration, or motility that can be subjective, laborious, and low in throughput. The aim of this study was to apply and optimize a fluorometric technique using resazurin for evaluating changes in the metabolic activity of Ascaris suum third-stage larvae (L3), a parasite of high economic relevance in swine. METHODS: Ascaris suum L3 were mechanically hatched from 6- to 8-week embryonated and sucrose-gradient-enriched eggs. Resazurin dye and A. suum L3 were titrated in 96-well microtiter plates, and resazurin reduction activity was assessed by fluorometry after 24 h of incubation. Fluorescence microscopy was used to localize the resazurin reduction site within the larvae. Finally, we exposed A. suum L3 to various stress conditions including heat, methanol, and anthelmintics, and investigated their impact on larval metabolism through resazurin reduction activity. RESULTS: We show that the non-fluorescent dye resazurin is reduced inside vital A. suum L3 to fluorescent resorufin and released into the culture media. Optimal assay parameters are 100-1000 L3 per well, a resazurin concentration of 7.5 µg/ml, and incubation at 37 °C/5% CO2 for 24 h. An intact L2 sheath around the L3 of A. suum completely prevents the uptake of resazurin, while in unsheathed L3, the most intense fluorescence signal is observed along the larval midgut. L3 exposed to methanol or heat show a gradually decreased resazurin reduction activity. In addition, 24 h exposure to ivermectin at 0.625 µM, mebendazole at 5 µM, and thiabendazole from 10 to 100 µM significantly decreased larval metabolic activity by 55%, 73%, and 70% to 89%, respectively. CONCLUSIONS: Together, our results show that both metabolic stressors and anthelmintic drugs significantly and reproducibly reduce the resazurin reduction activity of A. suum L3, making the proposed assay a sensitive and easy-to-use method to evaluate metabolic activity of A. suum L3 in vitro.

Peripheral blood mononuclear cell transcriptomes reveal an over-representation of down-regulated genes associated with immunity in HIV-exposed uninfected infants.

HIV-exposed uninfected (HEU) infants are disproportionately at a higher risk of morbidity and mortality, as compared to HIV-unexposed uninfected (HUU) infants. Here, we used transcriptional profiling of peripheral blood mononuclear cells to determine immunological signatures of in utero HIV exposure. We identified 262 differentially expressed genes (DEGs) in HEU compared to HUU infants. Weighted gene co-expression network analysis (WGCNA) identified six modules that had significant associations with clinical traits. Functional enrichment analysis on both DEGs and the six significantly associated modules revealed an enrichment of G-protein coupled receptors and the immune system, specifically affecting neutrophil function and antibacterial responses. Additionally, malaria pathogenicity genes (thrombospondin 1-(THBS 1), interleukin 6 (IL6), and arginine decarboxylase 2 (ADC2)) were down-regulated. Of interest, the down-regulated immunity genes were positively correlated to the expression of epigenetic factors of the histone family and high-mobility group protein B2 (HMGB2), suggesting their role in the dysregulation of the HEU transcriptional landscape. Overall, we show that genes primarily associated with neutrophil mediated immunity were repressed in the HEU infants. Our results suggest that this could be a contributing factor to the increased susceptibility to bacterial infections associated with higher morbidity and mortality commonly reported in HEU infants.