Studies of human infectious diseases have been limited by the paucity of functional models that mimic normal human physiology and pathophysiology. Recent advances in the development of multicellular, ...physiologically active organotypic cultures produced from embryonic and pluripotent stem cells, as well as from stem cells isolated from biopsies and surgical specimens are allowing unprecedented new studies and discoveries about host–microbe interactions. Here, we summarize recent developments in the use of organoids for studying human viral pathogens, including intestinal infections with human rotavirus, norovirus, enteroviruses and adenoviruses (intestinal organoids and enteroids), neuronal infections with Zika virus (cerebral organoids) and respiratory infections with respiratory syncytial virus in (lung bud organoids). Biologic discovery of host-specific genetic and epigenetic factors affecting infection, and responses to infection that lead to disease are possible with the use of organoid cultures. Continued development to increase the complexity of these cultures by including components of the normal host tissue microenvironment such as immune cells, blood vessels and microbiome, will facilitate studies on human viral pathogenesis, and advance the development of platforms for pre-clinical evaluation of vaccines, antivirals and therapeutics.
The major barrier to research and development of effective interventions for human noroviruses (HuNoVs) has been the lack of a robust and reproducible in vitro cultivation system. HuNoVs are the ...leading cause of gastroenteritis worldwide. We report the successful cultivation of multiple HuNoV strains in enterocytes in stem cell-derived, nontransformed human intestinal enteroid monolayer cultures. Bile, a critical factor of the intestinal milieu, is required for straindependent HuNoV replication. Lack of appropriate histoblood group antigen expression in intestinal cells restricts virus replication, and infectivity is abrogated by inactivation (e.g., irradiation, heating) and serum neutralization. This culture system recapitulates the human intestinal epithelium, permits human host-pathogen studies of previously noncultivatable pathogens, and allows the assessment of methods to prevent and treat HuNoV infections.
Human gastrointestinal tract research is limited by the paucity of in vitro intestinal cell models that recapitulate the cellular diversity and complex functions of human physiology and disease ...pathology. Human intestinal enteroid (HIE) cultures contain multiple intestinal epithelial cell types that comprise the intestinal epithelium (enterocytes and goblet, enteroendocrine, and Paneth cells) and are physiologically active based on responses to agonists. We evaluated these nontransformed, three-dimensional HIE cultures as models for pathogenic infections in the small intestine by examining whether HIEs from different regions of the small intestine from different patients are susceptible to human rotavirus (HRV) infection. Little is known about HRVs, as they generally replicate poorly in transformed cell lines, and host range restriction prevents their replication in many animal models, whereas many animal rotaviruses (ARVs) exhibit a broader host range and replicate in mice. Using HRVs, including the Rotarix RV1 vaccine strain, and ARVs, we evaluated host susceptibility, virus production, and cellular responses of HIEs. HRVs infect at higher rates and grow to higher titers than do ARVs. HRVs infect differentiated enterocytes and enteroendocrine cells, and viroplasms and lipid droplets are induced. Heterogeneity in replication was seen in HIEs from different patients. HRV infection and RV enterotoxin treatment of HIEs caused physiological lumenal expansion detected by time-lapse microscopy, recapitulating one of the hallmarks of rotavirus-induced diarrhea. These results demonstrate that HIEs are a novel pathophysiological model that will allow the study of HRV biology, including host restriction, cell type restriction, and virus-induced fluid secretion.
Our research establishes HIEs as nontransformed cell culture models to understand human intestinal physiology and pathophysiology and the epithelial response, including host restriction of gastrointestinal infections such as HRV infection. HRVs remain a major worldwide cause of diarrhea-associated morbidity and mortality in children ≤5 years of age. Current in vitro models of rotavirus infection rely primarily on the use of animal rotaviruses because HRV growth is limited in most transformed cell lines and animal models. We demonstrate that HIEs are novel, cellularly diverse, and physiologically relevant epithelial cell cultures that recapitulate in vivo properties of HRV infection. HIEs will allow the study of HRV biology, including human host-pathogen and live, attenuated vaccine interactions; host and cell type restriction; virus-induced fluid secretion; cell-cell communication within the epithelium; and the epithelial response to infection in cultures from genetically diverse individuals. Finally, drug therapies to prevent/treat diarrheal disease can be tested in these physiologically active cultures.
Effect-based methods including cell-based bioassays, reporter gene assays and whole-organism assays have been applied for decades in water quality monitoring and testing of enriched solid-phase ...extracts. There is no common EU-wide agreement on what level of bioassay response in water extracts is acceptable. At present, bioassay results are only benchmarked against each other but not against a consented measure of chemical water quality. The EU environmental quality standards (EQS) differentiate between acceptable and unacceptable surface water concentrations for individual chemicals but cannot capture the thousands of chemicals in water and their biological action as mixtures. We developed a method that reads across from existing EQS and includes additional mixture considerations with the goal that the derived effect-based trigger values (EBT) indicate acceptable risk for complex mixtures as they occur in surface water. Advantages and limitations of various approaches to read across from EQS are discussed and distilled to an algorithm that translates EQS into their corresponding bioanalytical equivalent concentrations (BEQ). The proposed EBT derivation method was applied to 48 in vitro bioassays with 32 of them having sufficient information to yield preliminary EBTs. To assess the practicability and robustness of the proposed approach, we compared the tentative EBTs with observed environmental effects. The proposed method only gives guidance on how to derive EBTs but does not propose final EBTs for implementation. The EBTs for some bioassays such as those for estrogenicity are already mature and could be implemented into regulation in the near future, while for others it will still take a few iterations until we can be confident of the power of the proposed EBTs to differentiate good from poor water quality with respect to chemical contamination.
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•Effect-based triggers (EBTs) for bioassays discriminate good from poor water quality.•EBTs can be derived by read across from existing water quality guideline values.•Mixture factor warranted for bioassays responding to many different chemicals.•EBT derivation method applicable to every bioassay subject to data availability•Here we derived preliminary EBTs for 32 bioassays and discuss many more.
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•Coastal styrofoams bound the greatest DLCs.•Aging effect could increase DLCs on microplastics.•Leaching of DLCs could induce dioxin-like effects in marine organisms.•Dioxin-like ...effects would be affected by styrene oligomers.
Microplastics have become one of the most pervasive emerging pollutants in the marine environment because of their wide occurrence and high sorption ability for hydrophobic organic contaminants (HOCs). Among the associated HOCs, dioxin-like chemicals (DLCs) can pose severe health risks; however, information on effects of microplastics bound DLCs is lacking. To fill this knowledge gap, this study integrated chemical analysis and in vitro bioassays to elucidate the potential dioxin-like effects of microplastics bound DLCs. Chemical analysis results demonstrated that styrofoams possessed significantly greater DLCs than other coastal or open ocean plastic particles. This was probably due to the presence of additives and greater sorption ability of expanded polystyrene. However, styrofoams did not show as strong dioxin-like effects as predicted by the bioanalysis equivalent model in bioassays. This could be attributed to the decreased DLC bioavailability and increased competition with the presence of styrene oligomers. Besides, bioassay results also demonstrated that aging increased the associated DLC concentrations, since extra sorption from surrounding environment occurred during prolonged retention periods. Finally, it was estimated that the leaching of DLCs could induce dioxin-like effects in marine organisms under 100% (11/11) and 18% (2/11) scenarios for aged pellets and styrofoams through aqueous or dietary exposures.
Uranium (U) can enter aquatic environments from natural and anthropogenic processes, accumulating in sediments to concentrations that could, if bioavailable, adversely affect benthic organisms. To ...better predict the sorption and mobility of U in aquatic ecosystems, we investigated the sediment-solution partition coefficients (Kd) of U for nine uncontaminated freshwater sediments with a wide range of physicochemical characteristics over an environmentally relevant pH range. Test solutions were reconstituted to mimic water quality conditions and U(VI) concentrations (0.023–2.3 mg U/L) found downstream of Canadian U mines. Adsorption of U(VI) to each sediment was greatest at pH 6 and 7, and significantly reduced at pH 8. There were significant differences in pH-dependent sorption among sediments with different physicochemical properties, with sorption increasing up until thresholds of 12% total organic carbon, 37% fine fraction (≤50 μm), and 29 g/kg of iron content. The Kd values for U(VI) were predicted using the Windermere Humic Aqueous Model (WHAM) using total U(VI) concentrations, and water and sediment physicochemical parameters. Predicted Kd-U values were generally within a factor of three of the observed values. These results improve the understanding and assessment of U sorption to field sediment, and quantify the relationship with sediment properties that may influence the bioavailability and ecological risk of U-contaminated sediments.
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•Kd-U values were pH-dependent, with greater sorption at pH 6 and 7, compared to pH 8.•A positive correlation was observed between sediment Fe content and adsorption of U.•Sorption of U was predicted by WHAM7 within a factor of 3 from the observed values.
The sorption of uranium(VI) was significantly influenced by solution pH and physicochemical properties of sediment, and was modeled well with the geochemical speciation model, WHAM7.
Human rotavirus (HRV) and human norovirus (HuNoV) infections are recognized as the most common causes of epidemic and sporadic cases of gastroenteritis worldwide. The study of these two human ...gastrointestinal viruses is important for understanding basic virus-host interactions and mechanisms of pathogenesis and to establish models to evaluate vaccines and treatments. Despite the introduction of live-attenuated vaccines to prevent life-threatening HRV-induced disease, the burden of HRV illness remains significant in low-income and less-industrialized countries, and small animal models or ex vivo models to study HRV infections efficiently are lacking. Similarly, HuNoVs remained non-cultivatable until recently. With the advent of non-transformed human intestinal enteroid (HIE) cultures, we are now able to culture and study both clinically relevant HRV and HuNoV in a biologically relevant human system. Methods described here will allow investigators to use these new culture techniques to grow HRV and HuNoV and analyze new aspects of virus replication and pathogenesis.
Human noroviruses (HuNoVs) are the leading cause of viral gastroenteritis worldwide; yet currently, no vaccines or FDA-approved antiviral drugs are available to counter these pathogens. To understand ...HuNoV biology and the epithelial response to infection, we performed transcriptomic analyses, RT-qPCR, CRISPR-Cas9 modification of human intestinal enteroid (HIE) cultures, and functional studies with two virus strains (a pandemic GII.4 and a bile acid-dependent GII.3 strain). We identified a predominant type III interferon (IFN)-mediated innate response to HuNoV infection. Replication of both strains is sensitive to exogenous addition of IFNs, suggesting the potential of IFNs as therapeutics. To obtain insight into IFN pathway genes that play a role in the antiviral response to HuNoVs, we developed knockout (KO) HIE lines for IFN alpha and lambda receptors and the signaling molecules, MAVS, STAT1, and STAT2. An unexpected differential response of enhanced replication and virus spread was observed for GII.3, but not the globally dominant GII.4 HuNoV in STAT1-knockout HIEs compared to parental HIEs. These results indicate cellular IFN responses restrict GII.3 but not GII.4 replication. The strain-specific sensitivities of innate responses against HuNoV replication provide one explanation for why GII.4 infections are more wide-spread and highlight strain specificity as an important factor in HuNoV biology. Genetically modified HIEs for innate immune genes are useful tools for studying immune responses to viral or microbial pathogens.
The intestinal epithelium can limit enteric pathogens by producing antiviral cytokines, such as IFNs. Type I IFN (IFN-α/β) and type III IFN (IFN-λ) function at the epithelial level, and their ...respective efficacies depend on the specific pathogen and site of infection. However, the roles of type I and type III IFN in restricting human enteric viruses are poorly characterized as a result of the difficulties in cultivating these viruses in vitro and directly obtaining control and infected small intestinal human tissue. We infected nontransformed human intestinal enteroid cultures frommultiple individuals with human rotavirus (HRV) and assessed the host epithelial response by using RNA-sequencing and functional assays. The dominant transcriptional pathway induced by HRV infection is a type III IFN-regulated response. Early after HRV infection, low levels of type III IFN protein activate IFN-stimulated genes. However, this endogenous response does not restrict HRV replication because replication-competent HRV antagonizes the type III IFN response at pre- and posttranscriptional levels. In contrast, exogenous IFN treatment restricts HRV replication, with type I IFN being more potent than type III IFN, suggesting that extraepithelial sources of type I IFN may be the critical IFN for limiting enteric virus replication in the human intestine.
Currently, 9 out of 10 experimental drugs fail in clinical studies. This has caused a 40% plunge in the number of drugs approved by the US Food and Drug Administration (FDA) since 2005. It has been ...suggested that the mechanistic differences between human diseases modeled in animals (mostly rodents) and the pathophysiology of human diseases might be one of the critical factors that contribute to drug failure in clinical trials. Rapid progress in the field of human stem cell technology has allowed the in-vitro recreation of human tissue that should complement and expand upon the limitations of cell and animal models currently used to study human diseases and drug toxicity. Recent success in the identification and isolation of human intestinal epithelial stem cells (Lgr5(+)) from the small intestine and colon has led to culture of functional intestinal epithelial units termed organoids or enteroids. Intestinal enteroids are comprised of all four types of normal epithelial cells and develop a crypt-villus differentiation axis. They demonstrate major intestinal physiologic functions, including Na(+) absorption and Cl(-) secretion. This review discusses the recent progress in establishing human enteroids as a model of infectious diarrheal diseases such as cholera, rotavirus, and enterohemorrhagic Escherichia coli, and use of the enteroids to determine ways to correct the diarrhea-induced ion transport abnormalities via drug therapy.
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