BACKGROUND: Noroviruses are an important viral cause of chronic diarrhea in immunocompromised individuals.
METHODS: We collected norovirus-positive stool samples (n=448) from immunocompromised patients (n=88) at the National Institutes of Health Clinical Research Center, U.S. from 2010-2022. We assessed clinical characteristics of the cohort, norovirus molecular epidemiology, and infectivity of norovirus specimens in human intestinal enteroids (HIEs) monolayers.
RESULTS: Thirty-nine of the 88 patients had sequential stool samples that allowed documentation of chronic norovirus infection with shedding levels ranging from 104 to 1011 genome copies/g of stool. The majority with confirmed chronic norovirus infection in this cohort (32/39, 82%) had clinical evidence of an inborn error of immunity (13 identified monogenic diseases), most with combined immunodeficiency (15 of 32) or common variable immunodeficiency (11 of 32). Noroviruses detected in the cohort were genetically diverse: both Genogroup I (GI.2, GI.3, GI.5, and GI.6) and Genogroup II (GII.1-GII.4, GII.6, GII.7, GII.12, GII.14, and GII.17) genotypes were detected, with GII.4 variants (Osaka, Apeldoorn, Den Haag, New Orleans, and Sydney) predominant (51 of 88, 57.9%). Viruses belonging to the GII.4 Sydney variant group that replicated in HIEs (n=9) showed a higher fold-increase in RNA genome copies during infection compared to others that replicated.
CONCLUSIONS: Genetically and biologically diverse noroviruses established chronic infection in individuals with both inborn and acquired immunologic defects enrolled in an NIH surveillance study spanning 12 years, demonstrating the unique nature of each virus and host interaction.

After oral administration, the intestine is the first site of drug absorption, making it a key determinant of the bioavailability of a drug, and hence drug efficacy and safety. Existing non-clinical models of the intestinal barrier in vitro often fail to mimic the barrier and absorption of the human intestine. We explore if human enteroid monolayers are a suitable tool for intestinal absorption studies compared to primary tissue (Ussing chamber) and Caco-2 cells. Bidirectional drug transport was determined in enteroid monolayers, fresh tissue (Ussing chamber methodology) and Caco-2 cells. Apparent permeability (Papp) and efflux ratios for enalaprilat (paracellular), propranolol (transcellular), talinolol (P-glycoprotein (P-gp)) and rosuvastatin (Breast cancer resistance protein (BCRP)) were determined and compared between all three methodologies and across intestinal regions. Bulk RNA sequencing was performed to compare gene expression between enteroid monolayers and primary tissue. All three models showed functional efflux transport by P-gp and BCRP with higher basolateral to apical (B-to-A) transport compared to apical-to-basolateral (A-to-B). B-to-A Papp values were similar for talinolol and rosuvastatin in tissue and enteroids. Paracellular transport of enalaprilat was lower and transcellular transport of propranolol was higher in enteroids compared to tissue. Enteroids appeared show more region- specific gene expression compared to tissue. Fresh tissue and enteroid monolayers both show active efflux by P-gp and BCRP in jejunum and ileum. Hence, the use of enteroid monolayers represents a promising and versatile experimental platform to complement current in vitro models.

Shiga toxin (Stx) is produced by some pathogenic strains of Escherichia coli. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using in vitro and in vitro models.
OBJECTIVE: These studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to E. coli O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing E. coli-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.

Drug metabolism in the intestinal wall affects bioavailability of orally administered drugs and is influenced by age. Hence, it is important to fully understand the drug metabolizing capacity of the gut to predict systemic exposure. The aim of this study was to investigate the potential of enteroids as a tool to study CYP3A4/5 -mediated metabolism in both children and adults. Bioconversion of midazolam, a CYP3A4/5 model substrate, was studied using enteroid monolayers as well as tissue explants in the Ussing chamber, both derived from pediatric [median (range age): 54 weeks (2 days - 13 years), n = 21] and adult (n = 5) tissue. Caco-2 cellular monolayers were employed as controls. In addition, mRNA expression of CYP3A4 was determined in enteroid monolayers (n = 11), tissue (n = 23) and Caco-2 using RT-qPCR. Midazolam metabolism was successfully detected in all enteroid monolayers, as well as in all tissue explants studied in the Ussing chamber, whereas Caco-2 showed no significant metabolite formation. The extracted fraction of midazolam was similar between enteroid monolayers and tissue. The fraction of midazolam extracted increased with age in enteroid monolayers derived from 0 to 70 week old donors. No statistically significant correlation was observed in tissue likely due to high variability observed and the smaller donor numbers included in the study. At the level of gene expression, CYP3A4 increased with age in tissues (n = 32), while this was not reflected in enteroid monolayers (n = 16). Notably, asymmetric metabolite formation was observed in enteroids and tissue, with higher metabolite formation on the luminal side of the barrier. In summary, we demonstrated that enteroids can be used to measure CYP3A4/5 midazolam metabolism, which we show is similar as observed in fresh isolated tissue. This was the case both in children and adults, indicating the potential of enteroids to predict intestinal metabolism. This study provides promising data to further develop enteroids to study drug metabolism in vitro and potentially predict oral absorption for special populations as an alternative to using fresh tissue.

Infection of ruminants such as cattle with Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne\'s disease, a disease characterized by chronic inflammation of the small intestine and diarrhoea. Infection with MAP is acquired via the faecal-to-oral route and the pathogen initially invades the epithelial lining of the small intestine. In this study we used an in vitro 3D mouse enteroid model to determine the influence of M cells in infection of the gut epithelia by MAP, in comparison with another bacterial intestinal pathogen of veterinary importance, Salmonella enterica serovar Typhimurium. The differentiation of M cells in the enteroid cultures was induced by stimulation with the cytokine receptor activator of nuclear factor-κB ligand (RANKL), and the effects on MAP and Salmonella uptake and intracellular survival were determined. The presence of M cells in the cultures correlated with increased uptake and intracellular survival of Salmonella, but had no effect on MAP. Interestingly neither pathogen was observed to preferentially accumulate within GP2-positive M cells.

Human intestinal enteroids (HIEs) are gaining recognition as physiologically relevant models of the intestinal epithelium. While HIEs from adults are used extensively in biomedical research, few studies have used HIEs from infants. Considering the dramatic developmental changes that occur during infancy, it is important to establish models that represent infant intestinal characteristics and physiological responses. We established jejunal HIEs from infant surgical samples and performed comparisons to jejunal HIEs from adults using RNA sequencing (RNA-Seq) and morphologic analyses. We then validated differences in key pathways through functional studies and determined whether these cultures recapitulate known features of the infant intestinal epithelium. RNA-Seq analysis showed significant differences in the transcriptome of infant and adult HIEs, including differences in genes and pathways associated with cell differentiation and proliferation, tissue development, lipid metabolism, innate immunity, and biological adhesion. Validating these results, we observed a higher abundance of cells expressing specific enterocyte, goblet cell, and enteroendocrine cell markers in differentiated infant HIE monolayers, and greater numbers of proliferative cells in undifferentiated 3D cultures. Compared to adult HIEs, infant HIEs portray characteristics of an immature gastrointestinal epithelium including significantly shorter cell height, lower epithelial barrier integrity, and lower innate immune responses to infection with an oral poliovirus vaccine. HIEs established from infant intestinal tissues reflect characteristics of the infant gut and are distinct from adult cultures. Our data support the use of infant HIEs as an ex vivo model to advance studies of infant-specific diseases and drug discovery for this population.
OBJECTIVE: Tissue or biopsy stem cell-derived human intestinal enteroids are increasingly recognized as physiologically relevant models of the human gastrointestinal epithelium. While enteroids from adults and fetal tissues have been extensively used for studying many infectious and non-infectious diseases, there are few reports on enteroids from infants. We show that infant enteroids exhibit both transcriptomic and morphological differences compared to adult cultures. They also differ in functional responses to barrier disruption and innate immune responses to infection, suggesting that infant and adult enteroids are distinct model systems. Considering the dramatic changes in body composition and physiology that begin during infancy, tools that appropriately reflect intestinal development and diseases are critical. Infant enteroids exhibit key features of the infant gastrointestinal epithelium. This study is significant in establishing infant enteroids as age-appropriate models for infant intestinal physiology, infant-specific diseases, and responses to pathogens.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne\'s Disease, a chronic granulomatous enteritis of ruminants. MAP establishes an infection in the host via the small intestine. This requires the bacterium to adhere to, and be internalised by, cells of the intestinal tract. The effector molecules expressed by MAP for this purpose remain to be fully identified and understood. Mammalian cell entry (mce) proteins have been shown to enable other Mycobacterial species to attach to and invade host epithelial cells. Here, we have expressed Mce1A, Mce1D, Mce3C and Mce4A proteins derived from MAP on the surface of a non-invasive Escherichia coli to characterise their role in the initial interaction between MAP and the host. To this end, expression of mce1A was found to significantly increase the ability of the E. coli to attach and survive intracellularly in human monocyte-like THP-1 cells, whereas expression of mce1D was found to significantly increase attachment and invasion of E. coli to bovine epithelial cell-like MDBK cells, implying cell-type specificity. Furthermore, expression of Mce1A and Mce1D on the surface of a previously non-invasive E. coli enhanced the ability of the bacterium to infect 3D bovine basal-out enteroids. Together, our data contributes to our understanding of the effector molecules utilised by MAP in the initial interaction with the host, and may provide potential targets for therapeutic intervention.

There are several in vitro systems that enable evaluation of the absorption direction, but there are few quantitative systems that enable easy evaluation of the excretion direction. Enteroids, organoids derived from intestine, have been frozen and passaged for various research. But it is not clear how the freezing and passaging affect the expression and function of transporters. We investigated the effects of passage and cryopreservation of enteroids. We focused on P-gp (P-glycoprotein) and compared the transfer rates of rhodamine 123 (Rh123) into the lumen of enteroids with and without a P-gp inhibitor. mRNA expression levels did not change significantly before and after passage and cryopreservation. Accumulation of Rh123 in the lumen of enteroids was observed. With some P-gp inhibitors, excretion of Rh123 into the lumen of enteroids was inhibited and the nonexcreted Rh123 accumulated in enteroids epithelial cells. The transfer rate of Rh123 into the lumen of enteroids with a P-gp inhibitor was significantly decreased compared to that of without a P-gp inhibitor. Before and after passage and cryopreservation, the transfer rate was almost the same as that of primary cultured enteroids. We succeeded in easily evaluating whether a component is a substrate of P-gp using enteroids.

Human noroviruses (HuNoVs) are the leading cause of acute gastroenteritis worldwide. Currently, there are no targeted antivirals for the treatment of HuNoV infection. Histo-blood group antigens (HBGAs) on the intestinal epithelium are cellular attachment factors for HuNoVs; molecules that block the binding of HuNoVs to HBGAs thus have the potential to be developed as antivirals. Human milk oligosaccharides (HMOs) are glycans in human milk with structures analogous to HBGAs. HMOs have been shown to act as decoy receptors to prevent the attachment of multiple enteric pathogens to host cells. Previous X-ray crystallography studies have demonstrated the binding of HMO 2\'-fucosyllactose (2\'FL) in the same pocket as HBGAs for some HuNoV strains. We evaluated the effect of 2\'FL on the replication of a globally dominant GII.4 Sydney [P16] HuNoV strain using human intestinal enteroids (HIEs) from adults and children. A significant reduction in GII.4 Sydney [P16] replication was seen in duodenal and jejunal HIEs from multiple adult donors, all segments of the small intestine from an adult organ donor and in two pediatric duodenal HIEs. However, 2\'FL did not inhibit HuNoV replication in two infant jejunal HIEs that had significantly lower expression of α1-2-fucosylated glycans. 2\'FL can be synthesized in large scale, and safety and tolerance have been assessed previously. Our data suggest that 2\'FL has the potential to be developed as a therapeutic for HuNoV gastroenteritis.

Human norovirus (HuNoV) and human rotavirus (HRV) are the leading causes of gastrointestinal diarrhea. There are no approved antivirals and rotavirus vaccines are insufficient to cease HRV associated mortality. Furthermore, treatment of chronically infected immunocompromised patients is limited to off-label compassionate use of repurposed antivirals with limited efficacy, highlighting the urgent need of potent and specific antivirals for HuNoV and HRV. Recently, a major breakthrough in the in vitro cultivation of HuNoV and HRV derived from the use of human intestinal enteroids (HIEs). The replication of multiple circulating HuNoV and HRV genotypes can finally be studied and both in the same non-transformed and physiologically relevant model. Activity of previously described anti-norovirus or anti-rotavirus drugs, such as 2\'-C-methylcytidine (2CMC), 7-deaza-2\'-C-methyladenosine (7DMA), nitazoxanide, favipiravir and dasabuvir, was assessed against clinically relevant human genotypes using 3D-HIEs. 2CMC showed the best activity against HuNoV GII.4, while 7DMA was the most potent antiviral against HRV. We identified the anti-norovirus and -rotavirus activity of molnupiravir and its active metabolite, N4-hydroxycytidine (NHC), a broad-spectrum antiviral used to treat coronavirus disease 2019 (COVID-19). Molnupiravir and NHC inhibit HuNoV GII.4, HRV G1P[8], G2P[4] and G4P[6] in 3D-HIEs with high selectivity and show a potency comparable to 2CMC against HuNoV. Moreover, molnupiravir and NHC block HRV viroplasm formation, but do not alter its size or subcellular localization. Taken together, molnupiravir inhibits both HuNoV and HRV replication, suggesting that the drug could be a candidate for the treatment of patients chronically infected with either one of these diarrhea causing viruses.