Detection of Epithelial‐Cell Injury, and Quantification of Infection, in the HCT‐8 Organoid Model of Cryptosporidiosis

Warren, Cirle A.; Destura, Raul V.; Sevilleja, Jesus Emmanuel; Barroso, Luis F.; Carvalho, Humberto M.; Barrett, Leah J.; O'Brien, Alison D.; Guerrant, Richard L.

doi:10.1086/588819

Cited by 67 publications

(39 citation statements)

References 30 publications

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“…Cryptosporidium parvum infection in humans is associated with villus blunting and crypt hyperplasia (Orenstein, 1997; Alcantara et al, 2008); hence, we examined whether there would be similar changes in the excysted oocyst–challenged mouse. On day 3 following oocyst challenge, ileal segments were examined for villous height and crypt depth.…”

Section: Resultsmentioning

confidence: 99%

Cryptosporidium-Malnutrition Interactions: Mucosal Disruption, Cytokines, and TLR Signaling In A Weaned Murine Model

Costa¹,

JohnBull²,

Reeves³

et al. 2011

Journal of Parasitology

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Cryptosporidiosis is a leading cause of persistent diarrhea in children in impoverished and developing countries and has both a short- and long-term impact on the growth and development of affected children. An animal model of cryptosporidial infection that mirrors closely the complex interaction between nutritional status and infection in children, particularly in vulnerable settings such as post-weaning and malnourishment, is needed to permit exploration of the pathogenic mechanisms involved. Weaned C57BL/6 mice received a protein-deficient (2%) diet for 3–12 days, then were infected with 5 × 107 excysted C. parvum oocyts, and followed for rate of growth, parasite stool shedding, and intestinal invasion/morphometry. Mice had about 20% reduction in weight gain over 12 days of malnutrition and an additional 20% weight loss after C. parvum challenge. Further, a significantly higher fecal C. parvum shedding was detected in malnourished infected mice compared to the nourished infected mice. Also, higher oocyst counts were found in ileum and colon tissue samples from malnourished infected mice, as well as a significant reduction in the villous height–crypt depth ratio in the ileum. Tissue Th1 cytokine concentrations in the ileum were significantly diminished by malnutrition and infection. mRNA for toll-like receptors 2 and 4 were diminished in malnourished infected mice. Treatment with nitazoxanide did not prevent weight loss or parasite stool shedding. These findings indicate that, in the weaned animal, malnutrition intensifies cryptosporidial infection, while cryptosporidial infection further impairs normal growth. Depressed TLR2 and 4 signaling and Th1 cytokine response may be important in the mechanisms underlying the vicious cycle of malnutrition and enteric infection.

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Section: Resultsmentioning

confidence: 99%

Cryptosporidium-Malnutrition Interactions: Mucosal Disruption, Cytokines, and TLR Signaling In A Weaned Murine Model

Costa¹,

JohnBull²,

Reeves³

et al. 2011

Journal of Parasitology

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“…An organoid-type culture of HCT-8 cells in a low-shear microgravity environment was shown to support C. parvum infection (34). A culture system using primary intestinal epithelial cells isolated from jejunal tissue obtained during gastric bypass surgery was reported to support C. parvum infection for 5 days, with the production of oocyst-like structures being detected in the culture supernatant (23).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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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

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“…We constructed a proto-oncogene Mdm2 promoter-linked SEAP reporter plasmid (pMdm2-SEAP4.14h) that was used to stably transfect HCT-8 human intestinal cancer cells (Figure 1A). HCT-8 cells serve as a model for diverse mucosal diseases [32, 33]. Moreover, the proximal part of the gastrointestinal tract from which HCT-8 cells are derived is the first target organ of exposure to the majority of ingested mycotoxins [34, 35].…”

Section: Resultsmentioning

confidence: 99%

Interference with mutagenic aflatoxin B1-induced checkpoints through antagonistic action of ochratoxin A in intestinal cancer cells: a molecular explanation on potential risk of crosstalk between carcinogens

et al. 2016

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Foodborne aflatoxin B1 (AFB1) and ochratoxin A (OTA) cause genotoxic injury and subsequent tumor formation. As a biomarker of oncogenic stimulation by genotoxic mycotoxins, p53-triggered Mdm2 was assessed in intestinal cancer cells. AFB1 increased Mdm2 reporter expression in a dose-dependent manner. However, this was strongly antagonized by OTA treatment. As a positive transcription factor of Mdm2 expression, p53 levels were also increased by AFB1 alone and reduced by OTA. With marginal cell death responses, AFB1 induced p53-mediated S phase arrest and cell cycle-regulating target genes, which was completely suppressed by OTA. Although enterocyte-dominant CYP3A5 counteracted AFB1-induced DNA damage, expression of CYP3A5 was decreased by OTA or AFB1. Instead, OTA enhanced expression of another metabolic inactivating enzyme CYP3A4, attenuation of formation of AFB1-DNA adduct and p53-mediated cell cycle checking responses to the mutagens. Finally, the growth of intestinal cancer cells exposed to the mycotoxin mixture significantly exceeded the expected growth calculated from that of cells treated with each mycotoxin. Although AFB1-induced mutagen formation was decreased by OTA, interference with checkpoints through antagonistic action of OTA may contribute to the survival of tumor cells with deleterious mutations by genotoxic mycotoxins, potently increasing the risk of carcinogenesis.

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Detection of Epithelial‐Cell Injury, and Quantification of Infection, in the HCT‐8 Organoid Model of Cryptosporidiosis

Cited by 67 publications

References 30 publications

Cryptosporidium-Malnutrition Interactions: Mucosal Disruption, Cytokines, and TLR Signaling In A Weaned Murine Model

Cryptosporidium-Malnutrition Interactions: Mucosal Disruption, Cytokines, and TLR Signaling In A Weaned Murine Model

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

Interference with mutagenic aflatoxin B1-induced checkpoints through antagonistic action of ochratoxin A in intestinal cancer cells: a molecular explanation on potential risk of crosstalk between carcinogens

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