A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum

Varughese, Eunice A.; Bennett‐Stamper, Christina; Wymer, Larry; Yadav, J. S.

doi:10.1016/j.mimet.2014.07.017

Cited by 28 publications

(34 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The lower levels of infection obtained when the scaffolds were infected with isolated sporozoites versus oocysts is not surprising, given that this has been demonstrated previously, where a decrease in infectivity of 65% between infection with oocysts and infection with purified sporozoites was shown (24). It should also be noted that we analyzed only the luminal and scaffold contents and not the surrounding culture medium, which was changed every other day, for quantitative reverse transcription-PCR (qRT-PCR)-based quantification.…”

Section: Discussionsupporting

confidence: 73%

“…Additionally, the infectious dose of oocysts was restricted to small amounts, as a previous study showed that a larger inoculum resulted in poor infection levels and increased cell death (35). A non-carcinoma-derived human small intestinal cell line, FHs 74 Int, was also reported to support C. parvum infection at higher levels than other cell lines (24). However, these cells cannot be propagated for longer than 12 passages, and infection was carried out for only 48 h.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

et al. 2017

View full text Add to dashboard Cite

Cryptosporidium spp. are apicomplexan parasites of global importance that cause human diarrheal disease. In vitro culture models that may be used to study this parasite and that have physiological relevance to in vivo infection remain suboptimal. Thus, the pathogenesis of cryptosporidiosis remains poorly characterized, and interventions for the disease are limited. In this study, we evaluated the potential of a novel bioengineered three-dimensional (3D) human intestinal tissue model (which we developed previously) to support long-term infection by Cryptosporidium parvum. Infection was assessed by immunofluorescence assays and confocal and scanning electron microscopy and quantified by quantitative reverse transcription-PCR. We found that C. parvum infected and developed in this tissue model for at least 17 days, the extent of the study time used in the present study. Contents from infected scaffolds could be transferred to fresh scaffolds to establish new infections for at least three rounds. Asexual and sexual stages and the formation of new oocysts were observed during the course of infection. Additionally, we observed ablation, blunting, or distortion of microvilli in infected epithelial cells. Ultimately, a 3D model system capable of supporting continuous Cryptosporidium infection will be a useful tool for the study of hostparasite interactions, identification of putative drug targets, screening of potential interventions, and propagation of genetically modified parasites. KEYWORDS 3D model, Cryptosporidium, in vitro culture, intestinal epithelial cells C ryptosporidium spp. are apicomplexan parasites of global importance that cause diarrheal disease in humans and animals worldwide (1, 2). Though immunocompetent individuals experience self-limiting or asymptomatic infection, immunocompromised hosts, such as untreated patients with HIV infection or AIDS (3) and malnourished children (1, 2) in resource-limited settings, may experience severe diarrhea, wasting, and death. In a recent case-control study, Cryptosporidium was one of four pathogens responsible for moderate to severe diarrhea in children under the age of 5 years and was associated with an increased risk of death in children from 1 to 2 years of age in seven countries in sub-Saharan Africa and South Asia (4). The only FDA-approved drug for the treatment of cryptosporidiosis is nitazoxanide, a drug with broad antiparasitic properties (5). However, this drug is not effective in immunocompromised patients (6) and has not been widely tested in malnourished children in resource-limited countries. Though significant improvements in water purification methods have occurred (7) since the outbreak of waterborne cryptosporidiosis in Milwaukee, WI, in 1993 (8), industrialized countries are seeing increased numbers of cases, largely due to recreational water outbreaks (9).Unlike many other apicomplexan parasites which often require more than one host to complete their life cycles (10), Cryptosporidium completes its entire life cycle in a

show abstract

Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Most models of C. parvum infection in vitro employ transformed or immortalized adenocarcinoma-derived human IEC lines, such as Caco-2, HCT-8, and HT29 14, 15 . Primary human and bovine IEC models permit infections and Cryptosporidium hominis and C. parvum , respectively 16, 17 , and C. parvum infection in a non-cancer-derived IEC line, FHs 74 Int, has also been reported 18 . However, these cell lines support C. parvum infection for only a few days, do not permit completion of the life cycle or continuous propagation 12 , and can display variation in gene expression depending on culture conditions 19 .…”

Section: Novel Cell Culture Models Enable Propagation Of Cryptosporidmentioning

confidence: 99%

Recent Breakthroughs and Ongoing Limitations in Cryptosporidium Research

2018

View full text Add to dashboard Cite

The intestinal apicomplexan parasite Cryptosporidium is a major cause of diarrheal disease in humans worldwide. However, treatment options are severely limited. The search for novel interventions is imperative, yet there are several challenges to drug development, including intractability of the parasite and limited technical tools to study it. This review addresses recent, exciting breakthroughs in this field, including novel cell culture models, strategies for genetic manipulation, transcriptomics, and promising new drug candidates. These advances will stimulate the ongoing quest to understand Cryptosporidium and the pathogenesis of cryptosporidiosis and to develop new approaches to combat this disease.

show abstract

“…Previously, we demonstrated that FHs 74 Int cells showed the highest levels of infectivity as compared to the other available cell lines [ 19 ]. Using the FHs 74 Int cell type, this study for the first time implicates SHP-2 in the process of C .…”

Section: Introductionmentioning

confidence: 99%

SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells

et al. 2015

Self Cite

View full text Add to dashboard Cite

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.

show abstract

A new in vitro model using small intestinal epithelial cells to enhance infection of Cryptosporidium parvum

Cited by 28 publications

References 43 publications

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Novel Bioengineered Three-Dimensional Human Intestinal Model for Long-Term Infection of Cryptosporidium parvum

Recent Breakthroughs and Ongoing Limitations in Cryptosporidium Research

SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells

Contact Info

Product

Resources

About