The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica.

BACKGROUND: Two platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control. METHODOLOGY/ PRINCIPLE FINDINGS: Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD50 = 19.1 µM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 µM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss. CONCLUSIONS/ SIGNIFICANCE: 7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.

Sleep disordered breathing and chronic respiratory failure in patients with chronic pain on long term opioid therapy.

STUDY OBJECTIVES: The use of opioid medication for chronic pain has been increasing. The main aim of this study was to assess how many patients on opioids for chronic pain had sleep disordered breathing (SDB) and the type of SDB. The impact of these medications on daytime arterial blood gas (ABG) measurements and psychomotor vigilance was also studied. METHODS: Twenty-four patients (aged 18-75 years) on long-term opioids were prospectively recruited. Patients underwent home polysomnogram (PSG), psychomotor vigilance testing (PVT), and awake daytime ABG. Overnight PSG findings were compared to those of patients matched for age, sex, and BMI referred to our sleep service for evaluation of SDB. PVT results in the patient cohort were compared to PVT in healthy controls. RESULTS: Forty-six percent of opioid patients had severe SDB as defined by an apnea hypopnea index (AHI) > 30/h. The severity of SDB was similar in opioid-treated pain clinic patients and sleep clinic patients (mean ± SD AHI: Opioid-treated patients 32.7 ± 25.6; Sleep Study comparator group 28.9 ± 24.6, p = 0.6). Opioid patients had a higher frequency of central apneas and a lower arousal index (CAI: 3.9 ± 8.3 vs. 0.3 ± 0.5 events/h; p = 0.004, AI 8.0 ± 4.1 vs. 20.1 ± 13.8, p < 0.001). Pain clinic patients had impaired gas exchange during sleep and wakefulness. Nine of 20 (45%) had daytime hypercapnia, indicating a surprising number were in chronic respiratory failure. Morphine equivalent doses correlated with the severity of SDB. PVT was impaired when compared to a healthy PVT comparator group (RT: Opioid-treated patients 0.43 ± 0.27: Healthy PVT comparator group 0.28 ± 0.03 sec; p < 0.001). CONCLUSIONS: Patients on long-term opioids frequently have severe SDB, which in part is central in origin. PVT was markedly impaired. Half of the patients studied have evidence of chronic ventilatory failure. COMMENTARY: A commentary on this article appears in this issue on page 853

Cross-disciplinary approaches for measuring parasitic helminth viability and phenotype.

Parasitic worms (helminths) within the Phyla Nematoda and Platyhelminthes are responsible for some of the most debilitating and chronic infectious diseases of human and animal populations across the globe. As no subunit vaccine for any parasitic helminth is close to being developed, the frontline strategy for intervention is administration of therapeutic, anthelmintic drugs. Worryingly, and unsurprising due to co-evolutionary mechanisms, many of these worms are developing resistance to the limited compound classes currently being used. This unfortunate reality has led to a renaissance in next generation anthelmintic discovery within both academic and industrial sectors. However, a major bottleneck in this process is the lack of quantitative methods for screening large numbers of small molecules for their effects on the whole organism. Development of methodologies that can objectively and rapidly distinguish helminth viability or phenotype would be an invaluable tool in the anthelmintic discovery pipeline. Towards this end, we describe how several basic techniques currently used to assess single cell eukaryote viability have been successfully applied to parasitic helminths. We additionally demonstrate how some of these methodologies have been adopted for high-throughput use and further modified for assessing worm phenotype. Continued development in this area is aimed at increasing the rate by which novel anthelmintics are identified and subsequently translated into everyday, practical applications.

Cross-disciplinary approaches for measuring parasitic helminth viability and phenotype

Parasitic worms (helminths) within the Phyla Nematoda and Platyhelminthes are responsible for some of the most debilitating and chronic infectious diseases of human and animal populations across the globe. As no subunit vaccine for any parasitic helminth is close to being developed, the frontline strategy for intervention is administration of therapeutic, anthelmintic drugs. Worryingly, and unsurprising due to co-evolutionary mechanisms, many of these worms are developing resistance to the limited compound classes currently being used. This unfortunate reality has led to a renaissance in next generation anthelmintic discovery within both academic and industrial sectors. However, a major bottleneck in this process is the lack of quantitative methods for screening large numbers of small molecules for their effects on the whole organism. Development of methodologies that can objectively and rapidly distinguish helminth viability or phenotype would be an invaluable tool in the anthelmintic discovery pipeline. Towards this end, we describe how several basic techniques currently used to assess single cell eukaryote viability have been successfully applied to parasitic helminths. We additionally demonstrate how some of these methodologies have been adopted for high-throughput use and further modified for assessing worm phenotype. Continued development in this area is aimed at increasing the rate by which novel anthelmintics are identified and subsequently translated into everyday, practical applications.

Vermes parasíticos (helmintos) dos filos Nematoda e Platelmintos são responsáveis por algumas das doenças infecciosas crônicas e mais debilitantes das populações humana e animal em todo o globo. Já que nenhuma vacina está prestes a ser desenvolvida para nenhum parasita helmíntico, a frente estratégica de intervenção é a administração de drogas terapêuticas anti-helmínticas. De maneira preocupante, e não surpreendente devido a mecanismos coevolutivos, muitos destes vermes estão desenvolvendo resistência às limitadas classes de compostos que têm sido usados no momento. Esta infeliz realidade levou a um renascimento na descoberta de uma nova geração de anti-helmínticos tanto no setor acadêmico quanto no industrial. Contudo, um importante gargalo neste processo é a falta de métodos quantitativos para testar um grande número de pequenas moléculas em relação aos efeitos sobre o organismo inteiro. O desenvolvimento de metodologias que possam distinguir objetiva e rapidamente a viabilidade dos helmintos ou o fenótipo seria uma ferramenta valiosa para canalizar a descoberta de anti-helmínticos. Para este fim, descrevemos aqui como muitas técnicas básicas, correntemente usadas para avaliar a viabilidade de células únicas de eucariotos, têm sido aplicadas com sucesso para helmintos parasíticos. Adicionalmente demonstramos como algumas destas metodologias foram adotadas para uso em larga escala e além disso modificadas para avaliar o fenótipo de vermes. O desenvolvimento contínuo nesta área está voltado para aumentar a taxa com que novos anti-helmínticos são identificados e subsequentemente traduzidos em aplicações práticas cotidianas.

Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability.

BACKGROUND: Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive 'genome to drug' lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies. METHODOLOGY/PRINCIPAL FINDINGS: We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate) by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase), praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide) or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa) anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed), relevant to industrial (384-well microtiter plate compatibility) and academic (96-well microtiter plate compatibility) settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative. CONCLUSIONS/SIGNIFICANCE: The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species further offers an opportunity for great strides to be made against additional neglected tropical diseases of biomedical and veterinary importance.

Anti-schistosomal intervention targets identified by lifecycle transcriptomic analyses.

BACKGROUND: Novel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis. METHODOLOGY/PRINCIPAL FINDINGS: Utilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and/or adult transcripts with no known sequence similarity outside the Platyhelminthes), which were undetectable by the statistical comparisons. CONCLUSIONS/SIGNIFICANCE: Collectively, statistical and network-based exploratory analyses of transcriptomic datasets have led to a thorough characterisation of schistosome development. Information obtained from these experiments highlighted key transcriptional programs associated with lifecycle progression and identified numerous anti-schistosomal candidate molecules including G-protein coupled receptors, tetraspanins, Dyp-type peroxidases, fucosyltransferases, leishmanolysins and the netrin/netrin receptor complex.