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Environmental enteric dysfunction (EED) is a subclinical syndrome of intestinal inflammation, malabsorption and barrier disruption that is highly prevalent in low- and middle-income countries in which poverty, food insecurity and frequent exposure to enteric pathogens impair growth, immunity and neurodevelopment in children. In this Review, we discuss advances in our understanding of EED, intestinal adaptation and the gut microbiome over the 'first 1,000 days' of life, spanning pregnancy and early childhood. Data on maternal EED are emerging, and they mirror earlier findings of increased risks for preterm birth and fetal growth restriction in mothers with either active inflammatory bowel disease or coeliac disease. The intense metabolic demands of pregnancy and lactation drive gut adaptation, including dramatic changes in the composition, function and mother-to-child transmission of the gut microbiota. We urgently need to elucidate the mechanisms by which EED undermines these critical processes so that we can improve global strategies to prevent and reverse intergenerational cycles of undernutrition.

Parenteral nutrition-associated cholestasis (PNAC) in the neonatal intensive care unit (NICU) causes significant morbidity and associated healthcare costs. Laboratory detection of PNAC currently relies on elevated serum conjugated bilirubin levels in the aftermath of impaired bile flow. Here, we sought to identify fecal biomarkers, which when integrated with clinical data, would better predict risk for developing PNAC.Using untargeted metabolomics in 200 serial stool samples from 60 infants, we applied statistical and machine learning approaches to identify clinical features and metabolic biomarkers with the greatest associative potential for risk of developing PNAC. Stools were collected prospectively from infants receiving PN with soybean oil-based lipid emulsion at a level IV NICU.Low birth weight, extreme prematurity, longer duration of PN, and greater number of antibiotic courses were all risk factors for PNAC (P 0.05). We identified 78 stool biomarkers with early predictive potential (P 0.05). From these 78 biomarkers, we further identified 12 sphingomyelin lipids with high association for the development of PNAC in precholestasis stool samples when combined with birth anthropometry.We demonstrate the potential for stool metabolomics to enhance early identification of PNAC risk. Earlier detection of high-risk infants would empower proactive mitigation with alterations to PN for at-risk infants and optimization of energy nutrition with PN for infants at lower risk.

Background and Aims Widespread dysregulation of long non-coding RNAs (lncRNAs) including a reduction in GATA6-AS1 was noted in inflammatory bowel disease (IBD). We previously reported a prominent inhibition of epithelial mitochondrial functions in UC. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not yet defined. Methods Mucosal transcriptomics was used to conform GATA6-AS1 reduction in several treatment naïve independent human cohorts (n=673). RNA pull-down followed by mass-spectrometry was used to determine GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2, were used to elaborate on GATA6-AS1 functions. Results GATA6-AS1 showed predominant expression in gut epithelia using single cell datasets. GATA6-AS1 levels were reduced in Crohn disease (CD) ileum and in ulcerative colitis (UC) rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to more severe UC form, and to less favorable UC course. GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in celiac disease that is induced in UC, CD, and celiac, in contrast GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in reduction of metabolites linked with aerobic respiration relevant to mucosal inflammation. TGM2 knockdown in GATA6-AS1 deficient cells rescued mitochondrial respiration. Conclusions GATA6-AS1 levels are reduced in UC, CD, and celiac, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 levels.

IntroductionEnvironmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy.MethodsA community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3–6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens.ResultsGiardia, Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO).ConclusionProtozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering.

In Ceara, 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 Ceara. Because Ceara 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 Ceara and beyond.

Autologous fecal transplant (AFT, transfer of ileostomy feces to the colon) for diversion colitis (DC) has not yet been described in children or adolescents. We performed serial AFTs in a 16-year-old with Crohn’s disease and DC. As evidenced by endoscopic, histologic, laboratory, and multiomic profiling of the colon, the first AFT led to interval improvement in DC, whereas the second AFT reactivated Crohn’s colitis. These findings ultimately guided shared decision-making towards total colectomy.

Environmental enteric dysfunction (EED)—a chronic inflammatory condition of the intestine—is characterized by villus blunting, compromised intestinal barrier function and reduced nutrient absorption. Here we show that essential genotypic and phenotypic features of EED-associated intestinal injury can be reconstituted in a human intestine-on-a-chip lined by organoid-derived intestinal epithelial cells from patients with EED and cultured in nutrient-deficient medium lacking niacinamide and tryptophan. Exposure of the organ chip to such nutritional deficiencies resulted in congruent changes in six of the top ten upregulated genes that were comparable to changes seen in samples from patients with EED. Chips lined with healthy epithelium or with EED epithelium exposed to nutritional deficiencies resulted in severe villus blunting and barrier dysfunction, and in the impairment of fatty acid uptake and amino acid transport; and the chips with EED epithelium exhibited heightened secretion of inflammatory cytokines. The organ-chip model of EED‐associated intestinal injury may facilitate the analysis of the molecular, genetic and nutritional bases of the disease and the testing of candidate therapeutics for it.

The underperformance of oral vaccines in children of low- and middle-income countries is partly attributable to underlying environmental enteric dysfunction (EED).We conducted a longitudinal, community-based study to evaluate the association of oral rotavirus vaccine (Rotarix®) seroconversion with growth anthropometrics, EED biomarkers and intestinal enteropathogens in Pakistani infants. Children were enrolled between three to six months of their age based on their nutritional status. We measured serum anti-rotavirus immunoglobulin A (IgA) at enrollment and nine months of age with EED biomarkers and intestinal enteropathogens.A total of 391 infants received two doses of rotavirus (RV) vaccine. 331/391 provided paired blood samples. Of these 331 children, 45% seroconverted at 9 months of age, 35% did not seroconvert and 20% were seropositive at baseline. Non-seroconverted children were more likely to be stunted, wasted and underweight at enrollment. In univariate analysis, insulin-like growth factor (IGF) concentration at 6 months were higher in seroconverters, median (25th, 75th percentile): 26.3 (16.5, 43.5) ng/ml vs. 22.5 (13.6, 36.3) ng/ml for non-seroconverters, p-value = 0.024. At nine months, fecal myeloperoxidase (MPO) concentrations were significantly lower in seroconverters, 3050(1250, 7587) ng/ml vs. 4623.3 (2189, 11650) ng/ml in non-seroconverted children, p-value = 0.017. In multivariable logistic regression analysis, alpha-1 acid glycoprotein (AGP) and IGF-1 concentrations were positively associated with seroconversion at six months. The presence of sapovirus and rotavirus in fecal samples at the time of rotavirus administration, was associated with non-seroconversion and seroconversion, respectively.We detected high baseline RV seropositivity and impaired RV vaccine immunogenicity in this high-risk group of children. Healthy growth, serum IGF-1 and AGP, and fecal shedding of rotavirus were positively associated with RV IgA seroconversion following immunization, whereas the presence of sapovirus was more common in non-seroconverters.Clinical Trials ID: NCT03588013.

The relationship between environmental factors and child health is not well understood in rural Pakistan. This study characterized the environmental factors related to the morbidity of acute respiratory infections (ARIs), diarrhea, and growth using geographical information systems (GIS) technology. Anthropometric, address and disease prevalence data were collected through the SEEM (Study of Environmental Enteropathy and Malnutrition) study in Matiari, Pakistan. Publicly available map data were used to compile coordinates of healthcare facilities. A Pearson correlation coefficient (r) was used to calculate the correlation between distance from healthcare facilities and participant growth and morbidity. Other continuous variables influencing these outcomes were analyzed using a random forest regression model. In this study of 416 children, we found that participants living closer to secondary hospitals had a lower prevalence of ARI (r = 0.154, p &lt, 0.010) and diarrhea (r = 0.228, p &lt, 0.001) as well as participants living closer to Maternal Health Centers (MHCs): ARI (r = 0.185, p &lt, 0.002) and diarrhea (r = 0.223, p &lt, 0.001) compared to those living near primary facilities. Our random forest model showed that distance has high variable importance in the context of disease prevalence. Our results indicated that participants closer to more basic healthcare facilities reported a higher prevalence of both diarrhea and ARI than those near more urban facilities, highlighting potential public policy gaps in ameliorating rural health.

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., Mouse and human 3D in vitro models show tissue maturation to determine species‐specific circadian rhytms in the intestine.

Environmental Enteric Dysfunction (EED) is a chronic inflammatory condition of the intestine characterized by villus blunting, compromised intestinal barrier function, and reduced nutrient absorption. Here, we show that key genotypic and phenotypic features of EED-associated intestinal injury can be reconstituted in a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by organoid-derived intestinal epithelial cells from EED patients and cultured in niacinamide- and tryptophan-deficient (-N/-T) medium. Exposure of EED Intestine Chips to -N/-T deficiencies resulted in transcriptional changes similar to those seen in clinical EED patient samples including congruent changes in six of the top ten upregulated genes. Exposure of EED Intestine Chips or chips lined by healthy intestinal epithelium (healthy Intestine Chips) to -N/-T medium resulted in severe villus blunting and barrier dysfunction, as well as impairment of fatty acid uptake and amino acid transport. EED Intestine Chips exhibited reduced secretion of cytokines at baseline, but their production was significantly upregulated compared to healthy Intestine Chips when exposed to -N/-T deficiencies. The human Intestine Chip model of EED-associated intestinal injury may be useful for analyzing the molecular, genetic, and nutritional basis of this disease and can serve as a preclinical model for testing potential EED therapeutics.

Infectious disease is the leading cause of mortality in children under five. This study has investigated environmental factors related to the morbidity of acute respiratory infections (ARIs), diarrhea, and growth using geographical information systems (GIS) technology. Anthropometric, address and disease prevalence data were collected through the SEEM study in Matiari, Pakistan. Publicly available map data was used to compile coordinates of healthcare facilities. A Pearson correlation coefficient (r) was used to calculate the correlation between distance from healthcare facilities and participant growth and morbidity. Other continuous variables influencing these outcomes were analyzed using a random forest regression model. In this study of 416 children, we found participants living closer to secondary hospitals had lower prevalence of ARI (r=0.154, p

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.

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.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.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.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.

Environmental enteric dysfunction (EED) is a subclinical condition of intestinal inflammation, barrier dysfunction and malabsorption associated with growth faltering in children living in poverty. This study explores association of altered duodenal permeability (lactulose, rhamnose and their ratio) with higher burden of enteropathogen in the duodenal aspirate, altered histopathological findings and higher morbidity (diarrhea) that is collectively associated with linear growth faltering in children living in EED endemic setting. In a longitudinal birth cohort, 51 controls (WHZ > 0, HAZ > −1.0) and 63 cases (WHZ< -2.0, refractory to nutritional intervention) were recruited. Anthropometry and morbidity were recorded on monthly bases up to 24 months of age. Dual sugar assay of urine collected after oral administration of lactulose and rhamnose was assessed in 96 children from both the groups. Duodenal histopathology (n = 63) and enteropathogen analysis of aspirate via Taqman array card (n = 60) was assessed in only cases. Giardia was the most frequent pathogen and was associated with raised L:R ratio (p = 0.068). Gastric microscopy was more sensitive than duodenal aspirate in H. pylori detection. Microscopically confirmed H. pylori negatively correlated with HAZ at 24 months (r = −0.313, p = 0.013). Regarding histopathological parameters, goblet cell reduction significantly correlated with decline in dual sugar excretion (p< 0.05). Between cases and controls, there were no significant differences in the median (25th, 75th percentile) of urinary concentrations (μg/ml) of lactulose [27.0 (11.50, 59.50) for cases vs. 38.0 (12.0, 61.0) for controls], rhamnose [66.0 (28.0, 178.0) vs. 86.5 (29.5, 190.5)] and L:R ratio [0.47 (0.24, 0.90) vs. 0.51 (0.31, 0.71)] respectively. In multivariable regression model, 31% of variability in HAZ at 24 months of age among cases and controls was explained by final model including dual sugars. In conclusion, enteropathogen burden is associated with altered histopathological features and intestinal permeability. In cases and controls living in settings of endemic enteropathy, intestinal permeability test may predict linear growth. However, for adoption as a screening tool for EED, further validation is required due to its complex intestinal pathophysiology., Author summary EED is a subclinical condition of compromised gut integrity secondary to frequent and repeated exposure to enteropathogens in global settings with a high prevalence of undernutrition. In this study, we reported association of gut mucosal architecture with a dual sugar intestinal permeability assay (lactulose-rhamnose) in Pakistani children. In the presence of duodenal enteropathogens, features such as chronic inflammation, intra-epithelial lymphocytosis, enterocyte injury and Paneth cell reduction were consistently observed. When comparing undernourished cases and controls living in the same setting, we found urinary excretion of the sugars was similar among groups; however, variability in HAZ among children at 24 months was partially explained by a model that includes excretion values.

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

Background Stool metabolites provide essential insights into the function of the gut microbiome. The current gold standard for storage of stool samples for metabolomics is flash-freezing at − 80 °C which can be inconvenient and expensive. Ambient temperature storage of stool is more practical, however no available methodologies adequately preserve the metabolomic profile of stool. A novel sampling kit (OMNImet.GUT; DNA Genotek, Inc.) was introduced for ambient temperature storage and stabilization of feces for metabolomics; we aimed to test the performance of this kit vs. flash-freezing. To do this stool was collected from an infant’s diaper was divided into two aliquots: 1) flash-frozen and 2) stored in an OMNImet.GUT tube at ambient temperature for 3–4 days. Samples from the same infant were collected at 2 different time points to assess metabolite changes over time. Subsequently, all samples underwent metabolomic analysis by liquid chromatography – tandem mass spectrometry (LC-MS/MS). Results Paired fecal samples (flash-frozen and ambient temperature) from 16 infants were collected at 2 time points (32 individual samples, 64 aliquots). Similar numbers of metabolites were detected in both the frozen and ambient temperature samples (1126 in frozen, 1107 in ambient temperature, 1064 shared between sample types). Metabolite abundances were strongly correlated between storage methods (median Spearman correlation Rs = 0.785 across metabolites). Hierarchical clustering analysis and principal component analysis showed that samples from the same individuals at a given time point clustered closely, regardless of the storage method. Repeat samples from the same individual were compared by paired t-test, separately for the frozen and OMNImet.GUT. The number of metabolites in each biochemical class that significantly changed (p Conclusion Ambient temperature storage and stabilization of stool in the OMNImet.GUT device yielded comparable metabolomic results to flash freezing in terms of 1) the identity and abundance of detected biochemicals 2) the distinct metabolomic profiles of subjects and 3) changes in metabolites over time that are plausibly microbiota-induced. This method potentially provides a more convenient, less expensive home collection and storage option for stool metabolomic analysis.

ObjectiveDevelop a deep learning-based methodology using the foundations of systems pathology to generate highly accurate predictive tools for complex gastrointestinal diseases, using celiac disease (CD) as a prototype.DesignTo predict the severity of CD, defined by Marsh–Oberhüber classification, we used deep learning to develop a model based on histopathologic features.ResultsThe study was based on a pediatric cohort of 124 patients identified with different classes of CD severity. The model predicted CD with an overall 88.7% accuracy with the highest for Marsh IIIc (91.0%; 95% sensitivity; 91% specificity). The model identified EECs as a defining feature of children with Marsh IIIc CD and endocrinopathies which was confirmed using immunohistochemistry.ConclusionThis deep learning image analysis platform has broad applications in disease treatment, management, and prognostication and paves the way for precision medicine.SummaryWhat is already known about this subject?–Deep Learning has the potential to generate predictive models for complex gastrointestinal diseases.What are the new findings?–Our deep learning-based model used the foundations of systems pathology to generate a highly accurate predictive tool for complex gastrointestinal diseases, using a celiac disease (CD) pediatric cohort as a prototype.–The model predicated CD severity with high accuracy and identified enteroendocrine cells as a defining feature of children with severe CD and endocrinopathies.How might it impact on clinical practice in the foreseeable future?–Assessment of histopathological markers at the time of diagnosis that can predict risk of severity or complications can have broad applications in disease treatment, management, and prognostication and pave the way for precision medicine.

The role of enteric neurons in driving intestinal peristalsis has been known for over a century. However, in recent decades, scientists have begun to unravel additional complex interactions between this nerve plexus and other cell populations in the intestine. Investigations into these potential interactions are complicated by a paucity of tractable models of these cellular relationships. Here, we describe a novel technique for ex vivo coculture of enteroids, so called “mini-guts,” in juxtaposition to the longitudinal muscle myenteric plexus (LMMP). Key to this system, we developed a LMMP culture media that: (i) allows the LMMP to maintain ex vivo peristalsis for 2 weeks along with proliferation of neurons, glia, smooth muscle and fibroblast cells, and (ii) supports the proliferation and differentiation of the intestinal stem cells into enteroids complete with epithelial enterocytes, Paneth cells, goblet cells, and enteroendocrine cells. Importantly, this technique identifies a culture condition that supports both the metabolic needs of intestinal epithelium as well as neuronal elements, demonstrating the feasibility of maintaining these two populations in a single culture system. This sets the stage for experiments to better define the regulatory interactions of these two important intestinal cell populations.

The gastrointestinal (GI) tract is innervated by the enteric nervous system (ENS), an extensive neuronal network that traverses along its walls. Due to local reflex circuits, the ENS is capable of functioning with and without input from the central nervous system. The functions of the ENS range from the propulsion of food to nutrient handling, blood flow regulation, and immunological defense. Records of it first being studied emerged in the early 19thcentury when the submucosal and myenteric plexuses were discovered. This was followed by extensive research and further delineation of its development, anatomy, and function during the next two centuries. The morbidity and mortality associated with the underdevelopment, infection, or inflammation of the ENS highlight its importance and the need for us to completely understand its normal function. This review will provide a general overview of the ENS to date and connect specific GI diseases including short bowel syndrome with neuronal pathophysiology and current therapies. Exciting opportunities in which the ENS could be used as a therapeutic target for common GI diseases will also be highlighted, as the further unlocking of such mechanisms could open the door to more therapy-related advances and ultimately change our treatment approach.

Recent models indicate seasonal influenza transmission in Brazil begins annually in the semiarid state of Ceará (pop. 8.8M)—before vaccine campaigns begin. To assess the extent and maternal-child health consequences of this misalignment, we tracked severe acute respiratory infections (SARI), influenza, and influenza immunizations from 2013-2018. Of 3,297 SARI cases, 145 (4%) occurred in pregnancy. Vaccine coverage was >80%; however, campaigns often occurred during or after peak influenza. Birth weights nadired and prematurity increased 30-40 weeks following peak influenza, by a magnitude of 40g and 10.7% to 15.5%, respectively. We identified 61 babies of mothers with gestational SARI; they weighed 10% less at birth (P = 0·019) and were more often premature (OR: 2.944; 95% CI: 1.100 – 7.879) relative to controls (n=122). Mistiming of influenza vaccination adversely impacts pregnancy and birth outcomes in Ceará, with critical implications for influenza transmission dynamics nationally.

ObjectivesStriking histopathological overlap between distinct but related conditions poses a significant 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.MethodsData for secondary analysis was obtained from three primary studies at different sites. The image analysis platform for EE and CD was developed using convolutional neural networks (CNNs: ResNet and custom Shallow CNN). Gradient-weighted Class Activation Mappings (Grad-CAMs) were used to visualize the models’ decision making process. A team of medical experts simultaneously reviewed the stain color normalized images done for bias reduction and Grad-CAM visualizations to confirm structural preservation and biological relevance, respectively.Results461 high-resolution biopsy images from 150 children were acquired. Median age (interquartile range) was 37·5 (19·0 to 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 distinct microscopic morphological features.ConclusionOur AI-based image analysis platform demonstrated high classification accuracy for small bowel enteropathies which was capable of identifying biologically relevant microscopic features, emulating human pathologist decision making process, performing in the case of suboptimal computational environment, and being modified for improving disease classification accuracy. Grad-CAMs that were employed illuminated the otherwise ‘black box’ of deep learning in medicine, allowing for increased physician confidence in adopting these new technologies in clinical practice.What is knownStriking histopathological overlap exists between distinct but related conditions which poses a significant disease diagnostic challenge; such as for small bowel enteropathies including Environmental Enteropathy (EE) and Celiac Disease (CD).There is a major clinical need to develop computational [including Artificial Intelligence (AI) and deep learning] methods enabling clinicians to translate heterogeneous biomedical images into accurate and quantitative diagnostics.A major issue plaguing the use of AI in medicine is the so-called ‘black box’ of deep learning, an analogy which describes the lack of insight that humans have into how the models arrive at their decision-makingWhat is newAI-based image analysis platform demonstrated high classification accuracy for small bowel enteropathies (EE vs. CD vs. histologically normal controls).Gradient-weighted Class Activation Mappings (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.

Background and aimsThe circadian clock orchestrates ~24-hour oscillations of gastrointestinal (GI) epithelial structure and function that drive diurnal rhythms in the composition, localization, and metabolism of gut microbiota. Here, we use experimental and computational approaches in enteroids to reveal reciprocal effects of microbial metabolites on intestinal epithelial timekeeping by an epigenetic mechanism.MethodsWe cultured 3D PER2∷LUCIFERASE andBmal1-ELuciferasejejunal enteroids in media supplemented with sterile supernatants from the altered Schaedler Flora (ASF), a defined murine microbiota. Circadian oscillations of bioluminescent PER2 andBmal1were measured in enteroids cultured in the presence or absence of individual ASF supernatants. Separately, we applied machine learning to ASF metabolic profiles to identify phase-shifting metabolites.ResultsFiltrates from 3 of 7 ASF species (ASF360Lactobacillus intestinalis, ASF361Ligilactobacillus murinus, ASF502Clostridiumspp.) induced minimal alterations in circadian rhythms, whereas 4 ASF species (ASF356Clostridiumspp., ASF492Eubacterium plexicaudatum, ASF500Pseudoflavonifactorspp., ASF519Parabacteroides goldsteinii) induced profound, concentration-dependent phase delays. Random forest classification identified short chain fatty acids (SCFA: butyrate, propionate, acetate, and isovalerate) production as a discriminating feature of “shifters”, i.e., ASF taxa whose metabolites induce phase delay. Experiments with SCFAs confirmed machine learning predictions, with a median phase delay of 6.2 hours. Pharmacological or botanical HDAC inhibitors generated similar phase delays. Further, mithramycin A, an inhibitor of HDAC inhibition, abrogated SCFA-induced phase delays by 20% (PBmal1-luciferaseenteroids.ConclusionsGut microbe-generated SCFAs entrain intestinal epithelial circadian rhythms, in part, by an HDACi-dependent mechanism, with critical implications for understanding microbial and circadian network regulation of intestinal epithelial homeostasis.

Image classification is central to the big data revolution in medicine. Improved information processing methods for diagnosis and classification of digital medical images have shown to be successful via deep learning approaches. As this field is explored, there are limitations to the performance of traditional supervised classifiers. This paper outlines an approach that is different from the current medical image classification tasks that view the issue as multi-class classification. We performed a hierarchical classification using our Hierarchical Medical Image classification (HMIC) approach. HMIC uses stacks of deep learning models to give particular comprehension at each level of the clinical picture hierarchy. For testing our performance, we use biopsy of the small bowel images that contain three categories in the parent level (Celiac Disease, Environmental Enteropathy, and histologically normal controls). For the child level, Celiac Disease Severity is classified into 4 classes (I, IIIa, IIIb, and IIIC).

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.

Despite nutrition interventions, stunting thought to be secondary to underlying environmental enteropathy (EE) remains pervasive among infants residing in resource-poor countries and remains poorly characterized. From a birth cohort of 380 children, 65 malnourished infants received 12 weeks of nutritional supplementation with ready-to-use therapeutic food (RUTF). Eleven children with insufficient response to RUTF underwent upper endoscopy with duodenal biopsies, which were compared with U.S., age-matched specimens for healthy, celiac disease, non-celiac villous atrophy, non-celiac intraepithelial lymphocytosis, and graft-versus-host disease patients. Of the 11 children biopsied, EE was found in 10 (91%) with one subject with celiac disease. Morphometry demonstrated decreased villus-to-crypt (V:C) ratios in EE relative to healthy and non-celiac lymphocytosis patients. Environmental enteropathy villus volumes were significantly decreased relative to healthy controls. In EE, average CD3+ cells per 100 epithelial cells and per 1,000 µm2 of lamina propria and the number of lamina propria CD20+ B-cell aggregates were increased relative to all other groups. Our results indicate that V:C ratios are reduced in EE but are less severe than in celiac disease. Environmental enteropathy intraepithelial and lamina propria T lymphocytosis is of greater magnitude than that in celiac disease. The increases in lamina propria B and T lymphocytes suggest that non-cytolytic lymphocytic activation may be a more prominent feature of EE relative to celiac disease. These results provide new insights into shared yet distinct histological and immunological features of EE and celiac disease in children.

Background Crohn disease (CD), environmental enteric dysfunction (EED) and celiac disease are prevalent enteropathies linked to chronic gut inflammation. However, unlike celiac and Crohn that are prevalent in westernized countries, EED is seen in only in low- and middle-income countries of the developing world. Recent work has defined specific EED duodenal microbiota associated with stunting, and transmission of EED histologic changes and weight loss by microbial transfer to germ free mice. Methods We performed high throughput transcriptomics of 94 duodenal samples from the SEEM cohort:52 undernourished EED participants, 17 celiac and 25 healthy North American controls. We compared those to our previously reported transcriptomics of 248 newly diagnosed ileal CD from RISK. Comparison were performed between cases and controls within each cohort. Results Above 90% of the expressed protein coding genes are shared between duodenum samples within SEEM, and Crohn ileal within RISK enabling the anticipated comparison. A substantial number of the down-regulated genes in undernourished EED participants overlapped with celiac (212/481, 44%) and with Crohn Disease (249/481, 52%). Shared reduced signatures and functions included decrease in epithelial lipid metabolism, vitamins, and brush border transport signatures. Relative to celiac disease, suppression of antioxidant and detoxification genes were EED- and Crohn-specific, which include the reduction of metallothioneins (MT family) that buffer against toxic metals, and aldo-keto NADPH- genes (AKR1C family) involved in detoxification of environmental compounds. A smaller proportion of the EED up-regulated signal overlapped with celiac (85/781), and we noted a more significant overlap with Crohn disease (248/781, Chi squares p Conclusion We highlight important biologic signals widely overlapping between Crohn Disease (CD) and EED transcriptome in the small intestine, suggesting mechanisms involving altered gut microbiota. Inhibiting innate immune activation or reverting Crohn dysbiosis may be beneficial future therapy for Crohn Disease and EED.

Background The Cholera Hospital-Based Intervention for 7 Days (CHoBI7) mobile health (mHealth) program was a cluster-randomized controlled trial of diarrhea patient households conducted in Dhaka, Bangladesh. Methods Patients were block-randomized to 3 arms: standard message on oral rehydration solution use; health facility delivery of CHoBI7 plus mHealth (no home visits); and health facility delivery of CHoBI7 plus 2 home visits and mHealth. The primary outcome was reported diarrhea in the past 2 weeks collected monthly for 12 months. The secondary outcomes were stunting, underweight, and wasting at a 12-month follow-up. Analysis was intention-to-treat. Results Between 4 December 2016 and 26 April 2018, 2626 participants in 769 households were randomly allocated to 3 arms: 849 participants to the standard message arm, 886 to mHealth with no home visits arm, and 891 to the mHealth with 2 home visits. Children, In this trial, young children in diarrhea patient households receiving the CHoBI7 mobile health program had less stunting and diarrhea compared with those only receiving the standard recommendation on oral rehydration solution for dehydration.

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.

The incidence of gastroenteritis has greatly reduced due to improved hygiene conditions in developing countries and the use of rotavirus vaccine. Still thousands of children, however, die from gastroenteritis, most of them in poor countries. Yet gastroenteritis management is simple, inexpensive, and effective and is largely the same all over the world. Universal guidelines for gastroenteritis guide the management and include simple interventions put forward early in the course of the disease. Treatment includes rehydration, continuing oral feeding, and anti-infective drugs in selected clinical conditions related to the symptoms or to host-related risk, and possible additional drug treatment to reduce the duration and severity of symptoms. There may be minor geographical differences in the treatment applied due to health care organizations that do not substantially change the standard universal recommendations. Prevention is recommended with sanitation interventions and rotavirus universal immunization. Implementation of those interventions through educational initiatives and local programs in target areas are needed. A series of recommendations for interventions, education, and research priorities are included here with the aim of reducing the burden of gastroenteritis, to be pursued by scientists, physicians, policy makers, and stakeholders involved. They include the need of recommendations for the management of gastroenteritis in malnourished children, in those with chronic conditions, in neonates, and in emergency settings. A reference system to score dehydration, the definition of optimal composition of rehydration solution and the indications for anti-infective therapy are also included. Rotavirus immunization should be actively promoted, and evidence-based guidelines should be universally implemented. Research priorities are also indicated.

Celiac Disease (CD) and Environmental Enteropathy (EE) are common causes of malnutrition and adversely impact normal childhood development. CD is an autoimmune disorder that is prevalent worldwide and is caused by an increased sensitivity to gluten. Gluten exposure destructs the small intestinal epithelial barrier, resulting in nutrient mal-absorption and childhood under-nutrition. EE also results in barrier dysfunction but is thought to be caused by an increased vulnerability to infections. EE has been implicated as the predominant cause of under-nutrition, oral vaccine failure, and impaired cognitive development in low-and-middle-income countries. Both conditions require a tissue biopsy for diagnosis, and a major challenge of interpreting clinical biopsy images to differentiate between these gastrointestinal diseases is striking histopathologic overlap between them. In the current study, we propose a convolutional neural network (CNN) to classify duodenal biopsy images from subjects with CD, EE, and healthy controls. We evaluated the performance of our proposed model using a large cohort containing 1000 biopsy images. Our evaluations show that the proposed model achieves an area under ROC of 0.99, 1.00, and 0.97 for CD, EE, and healthy controls, respectively. These results demonstrate the discriminative power of the proposed model in duodenal biopsies classification.

Background A major limitation to understanding the etiopathogenesis of environmental enteric dysfunction (EED) is the lack of a comprehensive, reproducible histologic framework for characterizing the small bowel lesions. We hypothesized that the development of such a system will identify unique histology features for EED, and that some features might correlate with clinical severity. Methods Duodenal endoscopic biopsies from two cohorts where EED is prevalent (Pakistan, Zambia) and North American children with and without gluten sensitive enteropathy (GSE) were processed for routine hematoxylin & eosin (H&E) staining, and scanned to produce whole slide images (WSIs) which we shared among study pathologists via a secure web browser-based platform. A semi-quantitative scoring index composed of 11 parameters encompassing tissue injury and response patterns commonly observed in routine clinical practice was constructed by three gastrointestinal pathologists, with input from EED experts. The pathologists then read the WSIs using the EED histology index, and inter-observer reliability was assessed. The histology index was further used to identify within- and between-child variations as well as features common across and unique to each cohort, and those that correlated with host phenotype. Results Eight of the 11 histologic scoring parameters showed useful degrees of variation. The overall concordance across all parameters was 96% weighted agreement, kappa 0.70, and Gwet’s AC 0.93. Zambian and Pakistani tissues shared some histologic features with GSE, but most features were distinct, particularly abundance of intraepithelial lymphocytes in the Pakistani cohort, and marked villous destruction and loss of secretory cell lineages in the Zambian cohort. Conclusions We propose the first EED histology index for interpreting duodenal biopsies. This index should be useful in future clinical and translational studies of this widespread, poorly understood, and highly consequential disorder, which might be caused by multiple contributing processes, in different regions of the world., Author summary The study of EED has been limited by the lack of a rigorously tested, reproducible histology index that can provide insight to the pathogenesis of this entity. In this study we report the first duodenal histology index that was developed using an unbiased approach, with excellent inter-observer reproducibility, for the study of EED. The EED histology index readily identified histologic features that are common or unique to cohorts of distinct geographic locations. Incorporating the histology index into future clinical studies will provide useful insight into the pathogenesis and for intervention strategy development.

Machine learning and computer vision have found applications in medical science and, recently, pathology. In particular, deep learning methods for medical diagnostic imaging can reduce delays in diagnosis and give improved accuracy rates over other analysis techniques. This paper focuses on methods with applicability to automated diagnosis of images obtained from gastrointestinal biopsies. These deep learning techniques for biopsy images may help detect distinguishing features in tissues affected by enteropathies. Learning from different areas of an image, or looking for similar patterns in new images, allow for the development of potential classification or clustering models Techniques like these provide a cutting-edge solution to detecting anomalies. In this paper we explore state of the art deep learning architectures used for the visual recognition of natural images and assess their applicability in medical image analysis of digitized human gastrointestinal biopsy slides.

The Study of Environmental Enteropathy (EE) and Malnutrition in Pakistan (SEEM) is a community intervention trial designed to understand the pathophysiology of EE and its associated clinical phenotypes in children less than two years of age. Undernutrition is the underlying cause of three million deaths annually, accounting for 45% of all deaths among children under five years of age. Linear growth stunting affects a further 165 million. In Pakistan, where our study is focused, an estimated one in three children are underweight and nearly half are stunted. This manuscript summarizes the challenges faced by the field team and the solutions employed for achieving our research objectives at the rural field site, Matiari. Our study design involved a longitudinal collection of field data and biological samples which required constant engagement of field staff with the participating families. Challenges faced in SEEM included: 1) quality assurance parameters of anthropometry data; 2) 24-hour food recall data collection; 3) clinical management of acutely sick children enrolled in our study; 4) obtaining informed consent for upper gastrointestinal endoscopy (an invasive procedure); and 5) biological sample collection (blood, urine, stool) from malnourished children at the community level. We accomplished our goal of enrolling 350 malnourished children and 50 well-nourished children with a more than 95% success rate of collecting blood, urine and stool specimens. We collected 72% microbiome stool samples within 30 minutes of the child passing stool. We conducted an upper gastrointestinal biopsy in 63 children. Broadly, solutions to these challenges included good governance, the establishment of credibility, and the development of a longitudinal bidirectional relationship embedded in trust and effective communication with the caretakers of the children enrolled in our study. Continuous monitoring, regular training of staff, and effective teamwork were key factors that led to the successful execution of our study.

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), Graphical abstract

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.

Understanding the complex relationship between early childhood infectious diseases, nutritional status, poverty, and cognitive development is significantly hindered by the lack of studies that adequately address confounding between these variables. This study assesses the independent contributions of early childhood diarrhea (ECD) and malnutrition on cognitive impairment in later childhood. A cohort of 131 children from a shantytown community in northeast Brazil was monitored from birth to 24 months for diarrhea and anthropometric status. Cognitive assessments including Test of Nonverbal Intelligence (TONI), coding tasks (WISC-III), and verbal fluency (NEPSY) were completed when children were an average of 8.4 years of age (range = 5.6–12.7 years). Multivariate analysis of variance models were used to assess the individual as well as combined effects of ECD and stunting on later childhood cognitive performance. ECD, height for age (HAZ) at 24 months, and weight for age (WAZ) at 24 months were significant univariate predictors of the studies three cognitive outcomes: TONI, coding, and verbal performance (P < 0.05). Multivariate models showed that ECD remained a significant predictor, after adjusting for the effect of 24 months HAZ and WAZ, for both TONI (HAZ, P = 0.029 and WAZ, P = 0.006) and coding (HAZ, P = 0.025 and WAZ, P = 0.036) scores. WAZ and HAZ were also significant predictors after adjusting for ECD. ECD remained a significant predictor of coding (WISC III) after number of household income was considered (P = 0.006). This study provides evidence that ECD and stunting may have independent effects on children's intellectual function well into later childhood.