Development of an in vitro drug sensitivity assay for Trichuris muris first-stage larvae

Trichuriasis represents a major public health problem in the developing world and is regarded as a neglected disease. Albendazole and mebendazole, the two drugs of choice against trichuriasis display only moderate cure rates, hence alternative drugs are needed. To identify candidate compounds, in vitro drug sensitivity testing currently relies on the adult Trichuris muris motility assay. The objective of the present study was to develop a simple and cost-effective drug sensitivity assay using Trichuris muris first-stage larvae (L1).; Several potential triggers that induce hatching of T. muris were studied, including gastrointestinal enzymes, acidic environment and intestinal microflora. Next, optimal culture conditions for T. muris L1 were determined assessing a wide range of culture media. T. muris L1 were incubated in the presence of mebendazole, ivermectin, nitazoxanide, levamisole or oxantel pamoate at 37°C. The viability of the parasites was evaluated microscopically after 24 hours. The usefulness of fluorescent markers (resazurin, calcein AM, ethidium homodimer-1 or fluorescein-conjugated albumin) in drug sensitivity testing was also assessed.; The established L1 motility assay provided accurate and reproducible drug effect data in vitro. IC50 values for oxantel pamoate, levamisole and nitazoxanide were 0.05, 1.75 and 4.43 μg/mL, respectively. Mebendazole and ivermectin failed to show any trichuricidal effect on L1. No correlation was found between data from the four fluorescent markers and the comparative motility assay.; The motility assay based on L1 was found suitable for drug sensitivity screening. It is rather simple, cost-effective, time-saving and sustains medium-throughput testing. Furthermore, it greatly reduces the need for the animal host and is therefore more ethical. None of the viability markers assessed in this study were found to be satisfactory

Comparison of novel and existing tools for studying drug sensitivity against the hookworm Ancylostoma ceylanicum in vitro

The motility assay is the current gold standard for evaluating drug effects on hookworm larvae and adults, however, among other drawbacks the assay is time consuming, and prone to individual subjectivity. We evaluated six alternative in vitro assays, namely the feeding inhibition assay, the colourimetric AlamarBlue®, MTT formazan and acid phosphatase activity assays, as well as isothermal calorimetry and the xCELLigence System using Ancylostoma ceylanicum third-stage larvae, stimulated third-stage larvae and adults. The performances of the assays were compared to the motility assay using three standard drugs: albendazole, levamisole and ivermectin (100-1 μg/ml). None of the assays investigated offered an advantage over the motility assay, because they were all inapplicable to third-stage larvae, which were presumably metabolically and physically too inactive. Among all assays tested the xCELLigence System performed best on adult worms as the test was accurate, simple, required a minimal number of worms and offered the possibility for conducting a medium-throughput screenin

How to train your myeloid cells: a way forward for helminth vaccines?

Soil-transmitted helminths affect approximately 1.5 billion people worldwide. However, as no vaccine is currently available for humans, the current strategy for elimination as a public health problem relies on preventive chemotherapy. Despite more than 20 years of intense research effort, the development of human helminth vaccines (HHVs) has not yet come to fruition. Current vaccine development focuses on peptide antigens that trigger strong humoral immunity, with the goal of generating neutralizing antibodies against key parasite molecules. Notably, this approach aims to reduce the pathology of infection, not worm burden, with only partial protection observed in laboratory models. In addition to the typical translational hurdles that vaccines struggle to overcome, HHVs face several challenges (1): helminth infections have been associated with poor vaccine responses in endemic countries, probably due to the strong immunomodulation caused by these parasites, and (2) the target population displays pre-existing type 2 immune responses to helminth products, increasing the likelihood of adverse events such as allergy or anaphylaxis. We argue that such traditional vaccines are unlikely to be successful on their own and that, based on laboratory models, mucosal and cellular-based vaccines could be a way to move forward in the fight against helminth infection. Here, we review the evidence for the role of innate immune cells, specifically the myeloid compartment, in controlling helminth infections. We explore how the parasite may reprogram myeloid cells to avoid killing, notably using excretory/secretory (ES) proteins and extracellular vesicles (EVs). Finally, learning from the field of tuberculosis, we will discuss how anti-helminth innate memory could be harnessed in a mucosal-trained immunity-based vaccine

Secreted microRNA data from the parasitic filarial nematode Acanthocheilonema viteae

microRNAs (miRNAs) are an abundant class of non-coding RNA species with important regulatory roles in gene expression at the posttranscriptional level. The helminth; Acanthocheilonema viteae; serves as model organism for research on parasitic filarial nematodes. Total RNA secreted or excreted; in vitro; by 1500 adult female and male; A. viteae; over 3 weeks was isolated from culture media previously processed by differential ultracentrifugation. miRNA sequencing revealed the presence of 360 unique miRNA candidates released by adult; A. viteae in vitro; . Among them, 74 high-confidence unique miRNAs; ,; as well as several potential novel miRNA candidates were discovered. A large proportion of the sequenced miRNA candidates appeared differentially expressed between the male and female samples based on normalized copy count. The presence of extracellular vesicles, often rich in miRNAs, could not be confirmed unambiguously by transmission electron microscopy

Nematode microRNAs can Individually Regulate Interferon Regulatory Factor 4 and mTOR in Differentiating T Helper 2 Lymphocytes and Modulate Cytokine Production in Macrophages

Parasitic nematodes are masterful immunomodulators. This class of pathogens has evolved a spectrum of sophisticated strategies to regulate and evade host immune responses, mediated through the release of various molecules. In this context, the release of microRNAs (miRNAs), short post-transcriptional regulators of gene expression, has been of particular interest in the host-parasite interplay. Evidence that parasite-derived miRNAs modulate host innate and adaptive immune responses has become increasingly compelling. However, since miRNAs are usually contained in extracellular vesicles containing other mediators, it is difficult to assign an observed effect on host cells to miRNAs specifically. Here, the effects of some abundantly secreted miRNAs by nematodes used as models of gastrointestinal infections (Heligmosomoides polygyrus bakeri, Trichuris muris and Ascaris suum) were evaluated, addressing the potential of parasite miRNAs to impair in vitro differentiation of two important types of immune cells in the context of helminth infections, Th2 lymphocytes and macrophages. Mimicking a continuous exposure to low concentrations of nematode miRNAs, the interferon gamma signaling, the IL-2/STAT5 signaling, and the mTOR signaling pathways were identified as downregulated by Hpo-miR-71-5p. Interferon regulatory factor 4 (Irf4) was validated as a target of Hpo-miR-71-5p, while Mtor is targeted by Asu-miR-791-3p, abundant in the T. muris secretions. By trend, Hpo-miR-71-5p impacts mildly but consistently on the amounts of inflammatory cytokines in unpolarized macrophages but leads to slightly increased IL-10 level in alternatively activated cells. In addition, our data suggests that transfected miRNAs remain for days in recipient cells, and that Hpo-miR-71-5p can incorporate into mouse Argonaute protein complexes. Nematode miRNAs can impair both innate and adaptive arms of host immunity. Hpo-miR-71-5p in particular, absent in mammals, interacts with host genes and pathways with crucial involvement in anthelmintic immune responses. This report brings new insights into the dynamics of miRNA-driven immunomodulation and highlights putative targeted pathways. Although the absolute repression is subtle, it is expected that the dozens of different miRNAs released by nematodes may have a synergistic effect on surrounding host cells

ATUAÇÃO DE ASSISTENTES SOCIAIS EM NÚCLEO DE PRÁTICA JURÍDICA: A EXPERIÊNCIA NA REESTRUTURAÇÃO DO CENTRO UNIVERSITÁRIO UNIBRASIL

Resumo Este artigo tem como finalidade, apresentar o processo de reconstrução do setor de Serviço Social durante a reestruturação do Núcleo de Prática Jurídica (NPJ) Desembargador Cordeiro Clève do Centro Universitário Autônomo do Brasil – Unibrasil, localizado na cidade de Curitiba/Paraná. Entende-se que, por ser um processo, é fundamental constante revisitação em seu projeto de intervenção inicial. Para essa discussão, o presente trabalho divide-se em três partes: no primeiro momento explicita-se brevemente a atuação do referido setor no NPJ e a ligação com o Curso de Serviço Social do UNIBRASIL. Aponta-se a construção e sistematização da prática profissional e posteriormente são relatadas as demandas e atendimentos realizados no NPJ, especificamente, a atuação do Serviço Social. Por fim, destacam-se as considerações finais e as primeiras impressões sobre a atuação da categoria no NPJ

The Effect of In Vitro Cultivation on the Transcriptome of Adult Brugia malayi

Infections with filarial worms cause serious physical impairment and affect tens of millions of people in tropical and subtropical countries. To better understand the biology and phar- macology of these parasites, Brugia malayi is often used as a model. This parasite can be maintained in the laboratory in Mongolian jirds, enabling researchers to test drugs in vivoand in vitro, among other studies. The effects of removing worms from their hosts and cul- turing them may affect many aspects of their physiology, including response to drugs, but the extent to which the worms undergo changes during culture has remained unknown. Using deep RNA sequencing and bioinformatics tools, we examined the global transcriptomic profile of B. malayi females at four different time points over 5 days in culture. Focusing on genes that are differentially expressed at various time points, we observed a general perturbation of the expression profile between dissection from the host and receipt after shipment. The expression of several genes remained changed at the end of the experi- ment, after 5 days under controlled conditions; in particular, genes encoding cuticle colla- gens were prominently represented and strongly overexpressed

The Effects of Ivermectin on Brugia malayi Females In Vitro: A Transcriptomic Approach

Lymphatic filariasis and onchocerciasis are tropical diseases caused by infections with parasitic nematodes. Resulting chronic diseases can be strongly blinding and disfiguring, and contribute to an entrenched cycle of poverty in affected populations. Ivermectin is one of the pivotal drugs used to control these infections. The mechanism of antifilarial action of the drug is incompletely resolved. It kills circulating larval stages (microfilariae), but only reversibly sterilizes adult worms without killing them. Our limited understanding of the involved mechanisms hampers treatment optimization and sustainability of the efficacy of the drug, and investigations into its pharmacology are of paramount importance. Working with Brugia malayi adult females, we employed RNA sequencing and bioinformatics analyses to identify genes for which expression levels changed as a result of exposure to the drug in vitro. Ivermectin exposure altered the expression of genes that are likely to func- tion in the B. malayi female reproductive system even at the lowest concentration tested. Through several biological pathways, genes involved in meiosis were particularly affected. These findings provide some insight into the mechanisms involved in ivermectin-induced reduction in microfilaria output and impaired fertility, embryogenesis, and larval development

In Vitro and In Vivo Efficacy of Monepantel (AAD 1566) against Laboratory Models of Human Intestinal Nematode Infections

Soil-transmitted helminthiases affect more than one billion people among the most vulnerable populations in developing countries. Currently, control of these infections primarily relies on chemotherapy. Only five drugs are available, all of which have been in use for decades. None of the drugs are efficacious using single doses against all soil-transmitted helminths (STH) species and show low efficacy observed against Trichuris trichiura. In addition, the limited availability of current drug treatments poses a precarious situation should drug resistance occur. Therefore, there is great interest to develop novel drugs against infections with STH. Monepantel, which belongs to a new class of veterinary anthelmintics, the amino-acetonitrile derivatives, might be a potential drug candidate in humans. It has been extensively tested against livestock nematodes, and was found highly efficacious and safe for animals. Here we describe the in vitro and in vivo effect of monepantel, on Ancylostoma ceylanicum, Necator americanus, Trichuris muris, Strongyloides ratti, and Ascaris suum, five parasite-rodent models of relevance to human STH. Since we observed that monepantel showed only high activity on one of the hookworm species and lacked activity on the other parasites tested we cannot recommend the drug as a development candidate for human soil-transmitted helminthiases

Investigations on potential drug candidates and metabonomics-based diagnostic biomarker discovery for human soil-transmitted helminthiases

Soil-transmitted helminthiases (STHs) are diseases caused by nematode worms. The most common species affecting humans are Ascaris lumbricoides, Necator americanus Ancylostoma duodenale, Trichuris trichiura and Strongyloides stercoralis. More than 1 billion people are infected globally. Most at risk are the 3 billion poorest people of the world, particularly children. Heavy infections cause iron-deficiency anemia to growth stunting and intellectual retardation. STHs occur often concomitantly with other infections such as malaria. Currently, STHs morbidity control relies on only five drugs (albendazole, mebendazole, levamisole, pyrantel pamoate and ivermectin). These present limited efficacies, especially when administered in single dose against T. trichiura and hookworm infections. Although anthelmintic drug resistance has not yet appeared as a major public health problem, emergence of drug resistance may be inevitable. The situation is precarious and new drugs are urgently needed. In addition, existing tools for in vitro drug sensitivity testing are based on a viability assessment assay which lacks convenience and hinders high-throughput screening rates. Also, the lack of accurate diagnostic tools to detect STHs and malaria infections hampers an optimal management of these diseases. This work aimed first to set up nematode-rodent models at the Swiss Tropical and Public Health Institute (Swiss TPH), improve drug screening assays and evaluate potential new treatments for human STHs. Prior to this thesis, monepantel (AAD 1566), tribendimidine, nitazoxanide and oxantel pamoate had been identified as potential drug candidates for STHs. Secondly, we aimed to strengthen our understanding of the impact of a murine malaria and hookworm co-infection on the host’s metabolism and explore the potential of metabolic profiling as multiplexing diagnostic tool. Once the animal models corresponding to human helminthiases have been established, (Ancylostoma ceylanicum, N. americanus and Trichuris muris), the Alamar Blue, the MTT and the acid phosphatase assays, as well as the xCELLigence System, isothermal microcalorimetry, and the feeding-inhibition assay (A. ceylanicum only) were tested and compared to the current assay of choice, the motility assay. For T. muris, the Alamar Blue assay compared most favorably to the motility assay since it is precise and cost-effective. For A. ceylanicum, no alternative assay was found better than the motility assay for testing on L3, whereas the xCELLigence System was found accurate and convenient for adult worms. The potential of monepantel was assessed against A. ceylanicum, N. americanus, T. muris, Ascaris suum and Strongyloides ratti. In vivo, the veterinary drug showed good and moderate activities respectively, against A. ceylanicum (10 mg/kg: 100% worm burden reduction) and N. americanus (10 mg/kg: 58.3% worm burden reduction), but failed to show sufficient anthelmintic properties in the other three models. Tribendimidine, a Chinese anthelmintic, and its metabolites dADT and AdADT were tested using the hookworm models A. ceylanicum and Heligmosomoides bakeri. In A. ceylanicum-infected hamsters, a single oral dose of 10 mg/kg resulted in 74.8% worm burden reduction. In the H. bakeri model, a single oral dose of 2 mg/kg achieved a worm burden reduction of 100%. The metabolite AdADT showed moderate activity against both parasites. The combination tribendimidine-levamisole displayed an additive to synergistic behavior in the A. ceylanicum model in vivo. Nitazoxanide, an anti-protozoal drug was evaluated against A. ceylanicum and T. muris. In vitro, it had a marked effect on A. ceylanicum adult worms (IC50 = 0.74 µg/ml) and on T. muris L3 and adult worms (IC50s = 0.27 and 12.87 µg/ml, respectively). However, the drug lacked efficacy in both models in vivo. The “old” anthelmintic oxantel pamoate was studied in the A. ceylanicum, N. americanus and T. muris models. The drug lacked anti-hookworm activity in vivo (10 mg/kg), but showed promising trichuricidal properties in vitro and in vivo (ED50 = 4.7 mg/kg). Moreover, the combination oxantel pamoate-mebendazole revealed highly synergistic properties. Murine H. bakeri and Plasmodium berghei single and co-infection (delayed and simultaneous) models were established for metabolic analysis. Urine and plasma samples were subjected to 1H nuclear magnetic resonance (NMR) spectroscopy and subsequent multivariate analysis in order to identify infection-discriminating metabolic fingerprints. Characteristic metabolic fingerprints have been found for each of the infection scenarios. We detected two unknown metabolites and confirmed the accumulation of urinary pipecolic acid in P. berghei-infected mice. Pipecolic acid may therefore represent a candidate for human malaria diagnostics. 1H NMR spectroscopy was found powerful for detecting metabolic changes in the co-infection model, but still presents some drawbacks as diagnostic tool in its actual form