In Vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds

Yarlett, Nigel; Morada, Mary; Gobin, Mohini; Voorhis, Wesley Van; Arnold, Samuel

doi:10.1007/978-1-4939-9748-0_19

Cited by 15 publications

(14 citation statements)

References 12 publications

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“…The authors were able to observe all developmental stages via immunofluorescent staining and obtain large numbers of infective C. parvum oocysts (10 8 per milliliter) distinct from seeded oocysts as far out as 18 months [110]. The potential manipulations of the environment and cultures allow for pharmacokinetic (PK) and pharmacodynamic (PD) explorations [111], though this method is cumbersome, expensive, and not easily scalable for multiple drugs and replicates.…”

Section: Advances In In Vitro Culturing Methodsmentioning

confidence: 99%

Phenotypic screening techniques forCryptosporidiumdrug discovery

Love

McNamara

2020

Expert Opinion on Drug Discovery

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Introduction: Two landmark epidemiological studies identified Cryptosporidium spp. as a significant cause of diarrheal disease in pediatric populations in resource-limited countries. Notably, nitazoxanide is the only approved drug for treatment of cryptosporidiosis but shows limited efficacy. As a result, many drug discovery efforts have commenced to find improved treatments. The unique biology of Cryptosporidium presents challenges for traditional drug discovery methods, which has inspired new assay platforms to study parasite biology and drug screening. Areas covered: The authors review historical advancements in phenotypic-based assays and techniques for Cryptosporidium drug discovery, as well as recent advances that will define future drug discovery. The reliance on phenotypic-based screens and repositioning of phenotypic hits from other pathogens has quickly created a robust pipeline of potential cryptosporidiosis therapeutics. The latest advances involve new in vitro culture methods for oocyst generation, continuous culturing capabilities, and more physiologically relevant assays for testing compounds. Expert opinion: Previous phenotypic screening techniques have laid the groundwork for recent cryptosporidiosis drug discovery efforts. The resulting improved methodologies characterize compound activity, identify, and validate drug targets, and prioritize new compounds for drug development. The most recent improvements in phenotypic assays are poised to help advance compounds into clinical development.

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Section: Advances In In Vitro Culturing Methodsmentioning

confidence: 99%

Phenotypic screening techniques forCryptosporidiumdrug discovery

Love

McNamara

2020

Expert Opinion on Drug Discovery

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“…More recently, complete life cycle development of C. parvum was obtained using a stem-cell-derived platform, which represents a progress milestone in cryptosporidium research (Wilke et al 2019). Another notable development is the continuous 3D cultivation of Cryptosporidium in hollow fiber bioreactors (Morada et al 2016;Yarlett et al 2020), and a few bioengineered intestinal models based on silk-protein scaffold, colon explants or lung/small intestine organoids cultured with parasites over weeks (Baydoun et al 2017;DeCicco Re-Pass et al 2017;Heo et al 2018). A more comprehensive review on these organoids and bioengineered intestinal models was reported by Gunasekera et al (2020).…”

Section: Lack Of Continuous In Vitro Cultivation Platform For Routine Usementioning

confidence: 99%

Current status and challenges in drug discovery against the globally important zoonotic cryptosporidiosis

2021

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The zoonotic cryptosporidiosis is globally distributed, one of the major diarrheal diseases in humans and animals. Cryptosporidium oocysts are also one of the major environmental concerns, making it a pathogen that fits well into the One Health concept. Despite its importance, fully effective drugs are not yet available. Anti-cryptosporidial drug discovery has historically faced many unusual challenges attributed to unique parasite biology and technical burdens. While significant progresses have been made recently, anti-cryptosporidial drug discovery still faces a major obstacle: identification of systemic drugs that can be absorbed by patients experiencing watery diarrhea and effectively pass through electron-dense (ED) band at the parasite-host cell interface to act on the epicellular parasite. There may be a need to develop an in vitro assay to effectively screen hits/leads for their capability to cross ED band. In the meantime, non-systemic drugs with strong mucoadhesive properties for extended gastrointestinal exposure may represent another direction in developing anti-cryptosporidial therapeutics. For developing both systemic and non-systemic drugs, a non-ruminant animal model exhibiting diarrheal symptoms suitable for routine evaluation of drug absorption and anti-cryptosporidial efficacy may be very helpful.

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“…Recently, there have been several breakthroughs including genetic manipulation (Vinayak, Pawlowic et al 2015, Sateriale, Pawlowic et al 2020) and lifecycle completion. Several promising approaches have been developed including using a cancer cell line as host (Miller, Josse et al 2018), biphasic and three-dimensional (organoid) culture systems, (Morada, Lee et al 2016, DeCicco RePass, Chen et al 2017, Heo, Dutta et al 2018, Cardenas, Bhalchandra et al 2020, hollow fiber technology (Yarlett, Morada et al 2020), and air-liquid interface (ALI) cultivation system (Wilke, Funkhouser-Jones et al 2019, Wilke, Wang et al 2020. These breakthroughs are enabling better, much needed, studies of the parasite's full life cycle.…”

Section: Introductionmentioning

confidence: 99%

“…Instead, an expanded family of apatela-related transcription factors, the AP2 family of proteins (ApiAP2), appear to be the predominant transcription factors in this phylum, including Cryptosporidium (Oberstaller, Pumpalova et al 2014, Jeninga, Quinn et al 2019. AP2 domains in C. parvum are reported to have reduced binding diversity relative to the malaria parasite Plasmodium falciparum and proposed to possess less dominancy in transcriptional regulation in C. parvum (Campbell, De Silva et al 2010, Yarlett, Morada et al 2020. It has been proposed that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors, which are present in Cryptosporidium while absent in other studied apicomplexans (Templeton, Iyer et al 2004, Yarlett, Morada et al 2020.…”

Section: Introductionmentioning

confidence: 99%

“…AP2 domains in C. parvum are reported to have reduced binding diversity relative to the malaria parasite Plasmodium falciparum and proposed to possess less dominancy in transcriptional regulation in C. parvum (Campbell, De Silva et al 2010, Yarlett, Morada et al 2020. It has been proposed that C. parvum is less reliant on ApiAP2 regulators in part because it utilizes E2F/DP1 transcription factors, which are present in Cryptosporidium while absent in other studied apicomplexans (Templeton, Iyer et al 2004, Yarlett, Morada et al 2020. Based on the similarity of gene expression profiles，it has been suggested that the number of co-expressed gene clusters in C. parvum is somewhere between 150 and 200, and putative ApiAP2 and E2F/DP1cis-regulatory elements were successfully detected in the upstream region of many co-expressed gene clusters (Oberstaller, Joseph et al 2013).…”

Section: Introductionmentioning

confidence: 99%

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Stage-Specific Long Non-coding RNAs inCryptosporidium parvumas Revealed by Stranded RNA-Seq

Li¹,

Baptista²,

Sateriale

et al. 2020

Preprint

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Cryptosporidium is a protist parasite that has been identified as the second leading cause of moderate to severe diarrhea in children younger than two and a significant cause of mortality worldwide. Cryptosporidium has a complex, obligate, intracellular but extra cytoplasmic lifecycle in a single host. How genes are regulated in this parasite remains largely unknown. Long non-coding RNAs (lncRNAs) play critical regulatory roles, including gene expression across a broad range of organisms. Cryptosporidium lncRNAs have been reported to enter the host cell nucleus and affect the host response. However, no systematic study of lncRNAs in Cryptosporidium has been conducted to identify additional lncRNAs. In this study, we analyzed a C. parvum in vitro strand-specific RNA-seq developmental time series covering both asexual and sexual stages to identify lncRNAs associated with parasite development. In total, we identified 396 novel lncRNAs 86% of which are differentially expressed. Nearly 10% of annotated mRNAs have an antisense lncRNA. lncRNAs also appear to occur most often at the 3’ end of their corresponding sense mRNA. Putative lncRNA regulatory regions were identified and many appear to encode bidirectional promoters. A positive correlation trend between lncRNA and the upstream mRNA expression was observed. Evolutionary conservation and expression of lncRNA candidates was observed between C. parvum, C. hominis and C. baileyi. Ten C. parvum protein-encoding genes with antisense transcripts have P. falciparum orthologs that also have antisense transcripts. Three C. parvum lncRNAs with exceptional properties (e.g., intron splicing) were experimentally validated using RT-PCR and RT-qPCR. We provide an initial characterization of the C. parvum non-coding transcriptome to facilitate further investigations into the roles of lncRNAs in parasite development and host-pathogen interactions.

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In Vitro Culture of Cryptosporidium parvum Using Hollow Fiber Bioreactor: Applications for Simultaneous Pharmacokinetic and Pharmacodynamic Evaluation of Test Compounds

Cited by 15 publications

References 12 publications

Phenotypic screening techniques forCryptosporidiumdrug discovery

Phenotypic screening techniques forCryptosporidiumdrug discovery

Current status and challenges in drug discovery against the globally important zoonotic cryptosporidiosis

Stage-Specific Long Non-coding RNAs inCryptosporidium parvumas Revealed by Stranded RNA-Seq

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