Ontogeny and function of the circadian clock in intestinal organoids.

Circadian rhythms regulate diverse aspects of gastrointestinal physiology ranging from the composition of microbiota to motility. However, development of the intestinal circadian clock and detailed mechanisms regulating circadian physiology of the intestine remain largely unknown. In this report, we show that both pluripotent stem cell-derived human intestinal organoids engrafted into mice and patient-derived human intestinal enteroids possess circadian rhythms and demonstrate circadian phase-dependent necrotic cell death responses to Clostridium difficile toxin B (TcdB). Intriguingly, mouse and human enteroids demonstrate anti-phasic necrotic cell death responses to TcdB. RNA-Seq analysis shows that ~3-10% of the detectable transcripts are rhythmically expressed in mouse and human enteroids. Remarkably, we observe anti-phasic gene expression of Rac1, a small GTPase directly inactivated by TcdB, between mouse and human enteroids, and disruption of Rac1 abolishes clock-dependent necrotic cell death responses. Our findings uncover robust functions of circadian rhythms regulating clock-controlled genes in both mouse and human enteroids governing organism-specific, circadian phase-dependent necrotic cell death responses, and lay a foundation for human organ- and disease-specific investigation of clock functions using human organoids for translational applications.

Intercellular Coupling of the Cell Cycle and Circadian Clock in Adult Stem Cell Culture.

Circadian clock-gated cell division cycles are observed from cyanobacteria to mammals via intracellular molecular connections between these two oscillators. Here we demonstrate WNT-mediated intercellular coupling between the cell cycle and circadian clock in 3D murine intestinal organoids (enteroids). The circadian clock gates a population of cells with heterogeneous cell-cycle times that emerge as 12-hr synchronized cell division cycles. Remarkably, we observe reduced-amplitude oscillations of circadian rhythms in intestinal stem cells and progenitor cells, indicating an intercellular signal arising from differentiated cells governing circadian clock-dependent synchronized cell division cycles. Stochastic simulations and experimental validations reveal Paneth cell-secreted WNT as the key intercellular coupling component linking the circadian clock and cell cycle in enteroids.

Histone Deacetylase Inhibition by Gut Microbe-Generated Short-Chain Fatty Acids Entrains Intestinal Epithelial Circadian Rhythms.

BACKGROUND & AIMS: The circadian clock orchestrates ∼24-hour oscillations of gastrointestinal epithelial structure and function that drive diurnal rhythms in gut microbiota. Here, we use experimental and computational approaches in intestinal organoids to reveal reciprocal effects of gut microbial metabolites on epithelial timekeeping by an epigenetic mechanism. METHODS: We cultured enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 and Bmal1 were measured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolomics to identify phase-shifting metabolites. RESULTS: Sterile filtrates from 3 of 7 ASF species (ASF360 Lactobacillus intestinalis, ASF361 Ligilactobacillus murinus, and ASF502 Clostridium species) induced minimal alterations in circadian rhythms, whereas filtrates from 4 ASF species (ASF356 Clostridium species, ASF492 Eubacterium plexicaudatum, ASF500 Pseudoflavonifactor species, and ASF519 Parabacteroides goldsteinii) induced profound, concentration-dependent phase shifts. Random forest classification identified short-chain fatty acid (SCFA) (butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of ASF "shifters." Experiments with SCFAs confirmed machine learning predictions, with a median phase shift of 6.2 hours in murine enteroids. Pharmacologic or botanical histone deacetylase (HDAC) inhibitors yielded similar findings. Further, mithramycin A, an inhibitor of HDAC inhibition, reduced SCFA-induced phase shifts by 20% (P < .05) and conditional knockout of HDAC3 in enteroids abrogated butyrate effects on Per2 expression. Key findings were reproducible in human Bmal1-luciferase enteroids, colonoids, and Per2-luciferase Caco-2 cells. CONCLUSIONS: Gut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Mucosal Genomics Implicate Lymphocyte Activation and Lipid Metabolism in Refractory Environmental Enteric Dysfunction.

BACKGROUND & AIMS: Environmental enteric dysfunction (EED) limits the Sustainable Development Goals of improved childhood growth and survival. We applied mucosal genomics to advance our understanding of EED. METHODS: The Study of Environmental Enteropathy and Malnutrition (SEEM) followed 416 children from birth to 24 months in a rural district in Pakistan. Biomarkers were measured at 9 months and tested for association with growth at 24 months. The duodenal methylome and transcriptome were determined in 52 undernourished SEEM participants and 42 North American controls and patients with celiac disease. RESULTS: After accounting for growth at study entry, circulating insulin-like growth factor-1 (IGF-1) and ferritin predicted linear growth, whereas leptin correlated with future weight gain. The EED transcriptome exhibited suppression of antioxidant, detoxification, and lipid metabolism genes, and induction of anti-microbial response, interferon, and lymphocyte activation genes. Relative to celiac disease, suppression of antioxidant and detoxification genes and induction of antimicrobial response genes were EED-specific. At the epigenetic level, EED showed hyper-methylation of epithelial metabolism and barrier function genes, and hypo-methylation of immune response and cell proliferation genes. Duodenal coexpression modules showed association between lymphocyte proliferation and epithelial metabolic genes and histologic severity, fecal energy loss, and wasting (weight-for-length/height Z < -2.0). Leptin was associated with expression of epithelial carbohydrate metabolism and stem cell renewal genes. Immune response genes were attenuated by giardia colonization. CONCLUSIONS: Children with reduced circulating IGF-1 are more likely to experience stunting. Leptin and a gene signature for lymphocyte activation and dysregulated lipid metabolism are implicated in wasting, suggesting new approaches for EED refractory to nutritional intervention. ClinicalTrials.gov, Number: NCT03588013. (https://clinicaltrials.gov/ct2/show/NCT03588013).

Perinatal Outcomes of Asynchronous Influenza Vaccination, Ceará, Brazil, 2013-2018.

In Ceará, Brazil, seasonal influenza transmission begins before national annual vaccination campaigns commence. To assess the perinatal consequences of this misalignment, we tracked severe acute respiratory infection (SARI), influenza, and influenza immunizations during 2013-2018. Among 3,297 SARI cases, 145 (4.4%) occurred in pregnant women. Statewide vaccination coverage was >80%; however, national vaccination campaigns began during or after peak influenza season. Thirty to forty weeks after peak influenza season, birthweights decreased by 40 g, and rates of prematurity increased from 10.7% to 15.5%. We identified 61 children born to mothers with SARI during pregnancy; they weighed 10% less at birth and were more likely to be premature than 122 newborn controls. Mistiming of influenza vaccination campaigns adversely effects perinatal outcomes in Ceará. Because Ceará is the presumptive starting point for north-to-south seasonal influenza transmission in Brazil, earlier national immunization campaigns would provide greater protection for pregnant women and their fetuses in Ceará and beyond.

Bile Acid Profiling Reveals Distinct Signatures in Undernourished Children with Environmental Enteric Dysfunction.

BACKGROUND: Intestinal inflammation and malabsorption in environmental enteric dysfunction (EED) are associated with early childhood growth faltering in impoverished settings worldwide. OBJECTIVES: The goal of this study was to identify candidate biomarkers associated with inflammation, EED histology, and as predictors of later growth outcomes by focusing on the liver-gut axis by investigating the bile acid metabolome. METHODS: Undernourished rural Pakistani infants (n = 365) with weight-for-height Z score (WHZ) < -2 were followed up to the age of 24 mo and monitored for growth, infections, and EED. Well-nourished local children (n = 51) were controls, based on consistent WHZ > 0 and height-for-age Z score (HAZ) > -1 on 2 consecutive visits at 3 and 6 mo. Serum bile acid (sBA) profiles were measured by tandem MS at the ages of 3-6 and 9 mo and before nutritional intervention. Biopsies and duodenal aspirates were obtained following upper gastrointestinal endoscopy from a subset of children (n = 63) that responded poorly to nutritional intervention. BA composition in paired plasma and duodenal aspirates was compared based on the severity of EED histopathological scores and correlated to clinical and growth outcomes. RESULTS: Remarkably, >70% of undernourished Pakistani infants displayed elevated sBA concentrations consistent with subclinical cholestasis. Serum glycocholic acid (GCA) correlated with linear growth faltering (HAZ, r = -0.252 and -0.295 at the age of 3-6 and 9 mo, respectively, P <0.001) and biomarkers of inflammation. The proportion of GCA positively correlated with EED severity for both plasma (rs = 0.324 P = 0.02) and duodenal aspirates (rs = 0.307 P = 0.06) in children with refractory wasting that underwent endoscopy, and the proportion of secondary BA was low in both undernourished and EED children. CONCLUSIONS: Dysregulated bile acid metabolism is associated with growth faltering and EED severity in undernourished children. Restoration of intestinal BA homeostasis may offer a novel therapeutic target for undernutrition in children with EED. This trial was registered at clinicaltrials.gov as NCT03588013.

Artificial Intelligence-based Analytics for Diagnosis of Small Bowel Enteropathies and Black Box Feature Detection.

OBJECTIVES: Striking histopathological overlap between distinct but related conditions poses a disease diagnostic challenge. There is a major clinical need to develop computational methods enabling clinicians to translate heterogeneous biomedical images into accurate and quantitative diagnostics. This need is particularly salient with small bowel enteropathies; environmental enteropathy (EE) and celiac disease (CD). We built upon our preliminary analysis by developing an artificial intelligence (AI)-based image analysis platform utilizing deep learning convolutional neural networks (CNNs) for these enteropathies. METHODS: Data for the secondary analysis was obtained from three primary studies at different sites. The image analysis platform for EE and CD was developed using CNNs including one with multizoom architecture. Gradient-weighted class activation mappings (Grad-CAMs) were used to visualize the models' decision-making process for classifying each disease. A team of medical experts simultaneously reviewed the stain color normalized images done for bias reduction and Grad-CAMs to confirm structural preservation and biomedical relevance, respectively. RESULTS: Four hundred and sixty-one high-resolution biopsy images from 150 children were acquired. Median age (interquartile range) was 37.5 (19.0-121.5) months with a roughly equal sex distribution; 77 males (51.3%). ResNet50 and shallow CNN demonstrated 98% and 96% case-detection accuracy, respectively, which increased to 98.3% with an ensemble. Grad-CAMs demonstrated models' ability to learn different microscopic morphological features for EE, CD, and controls. CONCLUSIONS: Our AI-based image analysis platform demonstrated high classification accuracy for small bowel enteropathies which was capable of identifying biologically relevant microscopic features and emulating human pathologist decision-making process. Grad-CAMs illuminated the otherwise "black box" of deep learning in medicine, allowing for increased physician confidence in adopting these new technologies in clinical practice.

Artificial Intelligence Applied to Gastrointestinal Diagnostics: A Review.

Artificial intelligence (AI), a discipline encompassed by data science, has seen recent rapid growth in its application to healthcare and beyond, and is now an integral part of daily life. Uses of AI in gastroenterology include the automated detection of disease and differentiation of pathology subtypes and disease severity. Although a majority of AI research in gastroenterology focuses on adult applications, there are a number of pediatric pathologies that could benefit from more research. As new and improved diagnostic tools become available and more information is retrieved from them, AI could provide physicians a method to distill enormous amounts of data into enhanced decision-making and cost saving for children with digestive disorders. This review provides a broad overview of AI and examples of its possible applications in pediatric gastroenterology.

Gram-negative Microbiota Blooms in Premature Twins Discordant for Parenteral Nutrition-associated Cholestasis.

Parenteral nutrition-associated cholestasis (PNAC) causes serious morbidity in the neonatal intensive care unit. Infection with gut-associated bacteria is associated with cholestasis, but the role of intestinal microbiota in PNAC is poorly understood. We examined the composition of stool microbiota from premature twins discordant for PNAC as a strategy to reduce confounding from variables associated with both microbiota and cholestasis. Eighty-four serial stool samples were included from 4 twin sets discordant for PNAC. Random Forests was utilized to determine genera most discriminatory in classifying samples from infants with and without PNAC. In infants with PNAC, we detected a significant increase in the relative abundance of Klebsiella, Veillonella, Enterobacter, and Enterococcus (P < 0.05). Bray-Curtis dissimilarities in infants with PNAC were significantly different (P < 0.05) from infants without PNAC. Our findings warrant further exploration in larger cohorts and experimental models of PNAC to determine if a microbiota signature predicts PNAC, as a basis for future interventions to mitigate liver injury.

Intervention and Mechanisms of Alanyl-glutamine for Inflammation, Nutrition, and Enteropathy: A Randomized Controlled Trial.

OBJECTIVE: Determine the minimum dosage of alanyl-glutamine (Ala-Gln) required to improve gut integrity and growth in children at risk of environmental enteropathy (EE). METHODS: This was a double-blinded randomized placebo-controlled dose-response trial. We enrolled 140 children residing in a low-income community in Fortaleza, Brazil. Participants were 2 to 60 months old and had weight-for-age (WAZ), height-for-age (HAZ), or weight-for-height (WHZ) z-scores less than -1. We randomized children to 10 days of nutritional supplementation: Ala-Gln at 3 g/day, Ala-Gln at 6 g/day, Ala-Gln at 12 g/day, or an isonitrogenous dose of glycine (Gly) placebo at 12.5 g/day. Our primary outcome was urinary lactulose-mannitol excretion testing. Secondary outcomes were anthropometry, fecal markers of inflammation, urine metabolic profiles, and malabsorption (spot fecal energy). RESULTS: Of 140 children, 103 completed 120 days of follow-up (24% dropout). In the group receiving the highest dose of Ala-Gln, we detected a modest improvement in urinary lactulose excretion from 0.19% on day 1 to 0.17% on day 10 (P = 0.05). We observed significant but transient improvements in WHZ at day 10 in 2 Ala-Gln groups, and in WHZ and WAZ in all Ala-Gln groups at day 30. We detected no effects on fecal inflammatory markers, diarrheal morbidity, or urine metabolic profiles; but did observe modest reductions in fecal energy and fecal lactoferrin in participants receiving Ala-Gln. CONCLUSIONS: Intermediate dose Ala-Gln promotes short-term improvement in gut integrity and ponderal growth in children at risk of EE. Lower doses produced improvements in ponderal growth in the absence of enhanced gut integrity.

Study of Environmental Enteropathy and Malnutrition (SEEM) in Pakistan: protocols for biopsy based biomarker discovery and validation.

BACKGROUND: Environmental Enteropathy (EE), characterized by alterations in intestinal structure, function, and immune activation, is believed to be an important contributor to childhood undernutrition and its associated morbidities, including stunting. Half of all global deaths in children < 5 years are attributable to under-nutrition, making the study of EE an area of critical priority. METHODS: Community based intervention study, divided into two sub-studies, 1) Longitudinal analyses and 2) Biopsy studies for identification of EE features via omics analyses. Birth cohorts in Matiari, Pakistan established: moderately or severely malnourished (weight for height Z score (WHZ) < - 2) children, and well-nourished (WHZ > 0) children. Blood, urine, and fecal samples, for evaluation of potential biomarkers, will be collected at various time points from all participants (longitudinal analyses). Participants will receive appropriate educational and nutritional interventions; non-responders will undergo further evaluation to determine eligibility for further workup, including upper gastrointestinal endoscopy. Histopathological changes in duodenal biopsies will be compared with duodenal biopsies obtained from USA controls who have celiac disease, Crohn's disease, or who were found to have normal histopathology. RNA-Seq will be employed to characterize mucosal gene expression across groups. Duodenal biopsies, luminal aspirates from the duodenum, and fecal samples will be analyzed to define microbial community composition (omic analyses). The relationship between histopathology, mucosal gene expression, and community configuration will be assessed using a variety of bioinformatic tools to gain better understanding of disease pathogenesis and to identify mechanism-based biomarkers. Ethical review committees at all collaborating institutions have approved this study. All results will be made available to the scientific community. DISCUSSION: Operational and ethical constraints for safely obtaining intestinal biopsies from children in resource-poor settings have led to a paucity of human tissue-based investigations to understand and reverse EE in vulnerable populations. Furthermore, EE biomarkers have rarely been correlated with gold standard histopathological confirmation. The Study of Environmental Enteropathy and Malnutrition (SEEM) is designed to better understand the pathophysiology, predictors, biomarkers, and potential management strategies of EE to inform strategies to eradicate this debilitating pathology and accelerate progress towards the 2030 Sustainable Development Goals. TRIAL REGISTRATION: Retrospectively registered; clinicaltrials.gov ID NCT03588013 .

Rhythm and bugs: circadian clocks, gut microbiota, and enteric infections.

PURPOSE OF REVIEW: To highlight recent developments in understanding the dynamic relationship between circadian rhythms, the gut microbiome, and gastrointestinal infections. RECENT FINDINGS: In humans and mice, the composition and functions of the intestinal microbiome display diurnal rhythms orchestrated by feeding behaviors and host circadian gene expression. Jet lag, or circadian disruption, perturbs these rhythms to produce gut dysbiosis. When mice are orally infected with Salmonella typhimurium in the morning (the beginning of their rest period) they show higher levels of colonization and gut inflammation vs. infection at other times of day. At the cellular level, recent studies highlight circadian regulation of innate and adaptive gut immunity in coordination with the microbiome, as well as intestinal stem cell growth and regeneration. SUMMARY: Taken together, these reports support a key role for circadian rhythms in regulating the gut microbiome and host responses to gastrointestinal pathogens. Further research is needed to translate these findings to improving outcomes for patients with gastrointestinal infections by guiding the right interventions for the right patients at the right time.

Glutamine and alanyl-glutamine promote crypt expansion and mTOR signaling in murine enteroids.

L-glutamine (Gln) is a key metabolic fuel for intestinal epithelial cell proliferation and survival and may be conditionally essential for gut homeostasis during catabolic states. We show that L-alanyl-L-glutamine (Ala-Gln), a stable Gln dipeptide, protects mice against jejunal crypt depletion in the setting of dietary protein and fat deficiency. Separately, we show that murine crypt cultures (enteroids) derived from the jejunum require Gln or Ala-Gln for maximal expansion. Once expanded, enteroids deprived of Gln display a gradual atrophy of cryptlike domains, with decreased epithelial proliferation, but stable proportions of Paneth and goblet cell differentiation, at 24 h. Replenishment of enteroid medium with Gln selectively activates mammalian target of rapamycin (mTOR) signaling pathways, rescues proliferation, and promotes crypt regeneration. Gln deprivation beyond 48 h leads to destabilization of enteroids but persistence of EGFP-Lgr5-positive intestinal stem cells with the capacity to regenerate enteroids upon Gln rescue. Collectively, these findings indicate that Gln deprivation induces a reversible quiescence of intestinal stem cells and provides new insights into nutritional regulation of intestinal epithelial homeostasis.

Zinc treatment ameliorates diarrhea and intestinal inflammation in undernourished rats.

BACKGROUND: WHO guidelines recommend zinc supplementation as a key adjunct therapy for childhood diarrhea in developing countries, however zinc's anti-diarrheal effects remain only partially understood. Recently, it has been recognized that low-grade inflammation may influence stunting. In this study, we examined whether oral zinc supplementation could improve weight, intestinal inflammation, and diarrhea in undernourished weanling rats. METHODS: Rats were undernourished using a northeastern Brazil regional diet (RBD) for two weeks, followed by oral gavage with a saturated lactose solution (30 g/kg) in the last 7 days to induce osmotic diarrhea. Animals were checked for diarrhea daily after lactose intake. Blood was drawn in order to measure serum zinc levels by atomic absorption spectroscopy. Rats were euthanized to harvest jejunal tissue for histology and cytokine profiles by ELISA. In a subset of animals, spleen samples were harvested under aseptic conditions to quantify bacterial translocation. RESULTS: Oral zinc supplementation increased serum zinc levels following lactose-induced osmotic diarrhea. In undernourished rats, zinc improved weight gain following osmotic diarrhea and significantly reduced diarrheal scores by the third day of lactose intake (p < 0.05), with improved jejunum histology (p < 0.0001). Zinc supplementation diminished bacterial translocation only in lactose-challenged undernourished rats (p = 0.03) compared with the untreated challenged controls and reduced intestinal IL-1ß and TNF-α cytokines to control levels. CONCLUSION: Altogether our findings provide novel mechanisms of zinc action in the setting of diarrhea and undernutrition and support the use of zinc to prevent the vicious cycle of malnutrition and diarrhea.

A novel method for culturing enteric neurons generates neurospheres containing functional myenteric neuronal subtypes.

BACKGROUND: The enteric nervous system (ENS) is comprised of neurons, glia, and neural progenitor cells that regulate essential gastrointestinal functions. Advances in high-efficiency enteric neuron culture would facilitate discoveries surrounding ENS regulatory processes, pathophysiology, and therapeutics. NEW METHOD: Development of a simple, robust, one-step method to culture murine enteric neurospheres in a 3D matrix that supports neural growth and differentiation. RESULTS: Myenteric plexus cells isolated from the entire length of adult murine small intestine formed ≥3000 neurospheres within 7 days. Matrigel-embedded neurospheres exhibited abundant neural stem and progenitor cells expressing Sox2, Sox10 and Msi1 by day 4. By day 5, neural progenitor cell marker Nestin appeared in the periphery of neurospheres prior to differentiation. Neurospheres produced extensive neurons and neurites, confirmed by Tubulin beta III, PGP9.5, HuD/C, and NeuN immunofluorescence, including neural subtypes Calretinin, ChAT, and nNOS following 8 days of differentiation. Individual neurons within and external to neurospheres generated depolarization induced action potentials which were inhibited in the presence of sodium channel blocker, Tetrodotoxin. Differentiated neurospheres also contained a limited number of glia and endothelial cells. COMPARISON WITH EXISTING METHODS: This novel one-step neurosphere growth and differentiation culture system, in 3D format (in the presence of GDNF, EGF, and FGF2), allows for ∼2-fold increase in neurosphere count in the derivation of enteric neurons with measurable action potentials. CONCLUSION: Our method describes a novel, robust 3D culture of electrophysiologically active enteric neurons from adult myenteric neural stem and progenitor cells.

The Environmental Enteric Dysfunction Biopsy Initiative (EEDBI) Consortium: mucosal investigations of environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is an asymptomatic acquired disorder characterized by upper small bowel inflammation, villus blunting, and gut permeability. It is a major contributor to poor growth in childhood as well as other highly consequential outcomes such as delayed neuorcognitive development. After decades of intermittent interest in this entity, we are now seeing a resurgence in the field of EED. However, recent studies have been hampered by a lack of investigation of the target tissue-the upper small bowel. In 2016, the EEDBI (Environmental Enteric Dysfunction Biopsy Initiative) Consortium was established as a common scientific platform across 3 independent EED biopsy cohort studies in Bangladesh, Pakistan, and Zambia. Two centers in the United States recruited comparison groups of children undergoing endoscopy for clinical indications. The EEDBI Consortium goal was to augment the contributions of the individual centers and answer high-level questions amenable to analysis and interpretation across the studies. Here, we describe the Consortium and its cohorts and recruitment procedures across studies. We also offer details applicable to all papers in this supplement, which describe EED mucosal histology, morphometry, immunohistochemistry, and transcriptomics as well as histology relationship to pathogens and biomarkers.

Anthropometry relationship with duodenal histologic features of children with environmental enteric dysfunction: a multicenter cross-sectional study.

BACKGROUND: Environmental enteric dysfunction (EED) is a precursor of growth faltering in children living in impoverished conditions who are frequently exposed to environmental toxins and enteropathogens, leading to small bowel inflammatory, malabsorptive, and permeability derangements and low-grade chronic systemic inflammation. OBJECTIVES: We explored the association between anthropometrics and duodenal histologic features of EED among children from 3 lower middle-income country centers. METHODS: In this cross-sectional study, Pakistani children (n = 63) with wasting, Bangladesh children (n = 116) with stunting or at risk for stunting (height-for-age Z score [HAZ] <-1 but ≥-2), and Zambian children (n = 108) with wasting or stunting received nutritional intervention. Children with anthropometric status refractory to intervention underwent endoscopy. Linear regression models included anthropometric around endoscopy, scores of histology parameters, and a global index score of EED-the total score percent-5 (TSP-5). Multivariable models were adjusted for center, age, sex, and histology slide quality. RESULTS: Intersite variation was observed while exploring the association between anthropometrics and the TSP-5; for example, Pakistani children had the worst HAZ, yet their median TSP-5 score was lower than that of the other 2 centers. Even within each site, no overall pattern of higher TSP-5 score was observed with worsening HAZ. During univariate analysis, TSP-5 (coefficient: 0.01; 95% confidence interval [CI]: 0, 0.02), goblet cell depletion (coefficient: 0.22; 95% CI: 0.06, 0.37), and Paneth cell depletion (coefficient: 0.14; 95% CI: 0.01, 0.27) were associated with HAZ scores; however, they lost statistical significance in the multivariable models, with study center most strongly confounding the relationships seen in univariate models between anthropometry and histology. CONCLUSIONS: This study contributes a crucial negative finding that duodenal morphological features did not associate with anthropometric phenotypes; hence, anthropometric measurements may not be a suitable outcome measure for use in EED trials. Trial outcomes may need to be defined by combining the functional and structural elements of the gut to monitor EED.

Duodenal transcriptomics demonstrates signatures of tissue inflammation and immune cell infiltration in children with environmental enteric dysfunction across global centers.

BACKGROUND: Environmental enteric dysfunction (EED) is an inflammatory condition of the small intestine that is prevalent in children residing in low- and middle-income countries. EED is accompanied by profound histopathologic changes in the small bowel, loss of absorptive capacity, increased intestinal permeability, increased microbial translocation, and nutrient loss. OBJECTIVES: We sought to identify dysregulated genes and pathways that might underlie pediatric EED. METHODS: RNA-sequencing libraries were generated from endoscopically obtained duodenal tissue from undernourished children with EED from 3 prospective cohorts of children with EED. The EED transcriptome was defined in comparison to North American children without EED. Weighted gene coexpression network analysis (WGCNA) was tested for gene modules associated with EED and its histologic features. RESULTS: The 1784 upregulated genes in EED were highly enriched for immune and inflammatory processes, including IL-17 and JAK-STAT signaling, and cytokine-cytokine receptor interactions. The 1388 downregulated genes included genes corresponding to xenobiotic metabolism, detoxification, and antioxidant capacities. A gene coexpression module enriched for antimicrobial responses and chemokine activity was significantly associated with villous blunting, goblet cell depletion, and overall histologic severity of EED. CONCLUSIONS: The transcriptome signatures of EED include specific innate and adaptive immune responses that are consistently elevated across study centers, coupled with reduced detoxification and antioxidant capacities. These data may have implications for targeted interventions to improve EED outcomes.

Multiplexed immunohistochemical evaluation of small bowel inflammatory and epithelial parameters in environmental enteric dysfunction.

BACKGROUND: Environmental enteric dysfunction (EED) is characterized by reduced absorptive capacity and barrier function of the small intestine, leading to poor ponderal and linear childhood growth. OBJECTIVES: To further define gene expression patterns that are associated with EED to uncover new pathophysiology of this disorder. METHODS: Duodenal biopsies from cohorts of children with EED from Bangladesh, Pakistan and Zambia were analyzed by immunohistochemistry (IHC) to interrogate gene products that distinguished differentiation and various biochemical pathways in immune and epithelial cells, some identified by prior bulk RNA sequence analyses. Immunohistochemical staining was digitally quantified from scanned images and compared to cohorts of North American children with celiac disease (gluten-sensitive enteropathy) or with no known enteric disease and no pathologic abnormality (NPA) detected in their clinical biopsies. RESULTS: After multivariable statistical analysis, we identified statistically significant (P < 0.05, 2-tailed t-test) elevated signals representing cluster of differentiation 45 (80%; 95% confidence interval [CI]: 24%, 127%), lipocalin 2 (659%; 95% CI: 198%, 1838%), and regenerating family 1 beta (221%; 95% CI: 47%, 600%) and lower signals corresponding to granzyme B (-74%; 95% CI: -82%, -62%), and sucrase isomaltase (-58%; 95% CI: -75%, -29%) in EED biopsies compared with NPA biopsies. Computerized algorithms also detected statistically significant elevation in intraepithelial lymphocytes (49%; 95% CI: 9%, 105%) and proliferation of leukocytes (267%; 95% CI: 92%, 601%) in EED biopsies compared with NPA biopsies. CONCLUSIONS: Our results support a model of chronic epithelial stress that decreases epithelial differentiation and absorptive function. The close association of several IHC parameters with manual histologic scoring suggests that automated digital quantification of IHC panels complements traditional histomorphologic assessment in EED.