Machine-learning-based integrative -'omics analyses reveal immunologic and metabolic dysregulation in environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a subclinical enteropathy challenging to diagnose due to an overlap of tissue features with other inflammatory enteropathies. EED subjects (n = 52) from Pakistan, controls (n = 25), and a validation EED cohort (n = 30) from Zambia were used to develop a machine-learning-based image analysis classification model. We extracted histologic feature representations from the Pakistan EED model and correlated them to transcriptomics and clinical biomarkers. In-silico metabolic network modeling was used to characterize alterations in metabolic flux between EED and controls and validated using untargeted lipidomics. Genes encoding beta-ureidopropionase, CYP4F3, and epoxide hydrolase 1 correlated to numerous tissue feature representations. Fatty acid and glycerophospholipid metabolism-related reactions showed altered flux. Increased phosphatidylcholine, lysophosphatidylcholine (LPC), and ether-linked LPCs, and decreased ester-linked LPCs were observed in the duodenal lipidome of Pakistan EED subjects, while plasma levels of glycine-conjugated bile acids were significantly increased. Together, these findings elucidate a multi-omic signature of EED.

A shared group of bacterial taxa in the duodenal microbiota of undernourished Pakistani children with environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a subclinical syndrome of altered small intestinal function postulated to be an important contributor to childhood undernutrition. The role of small intestinal bacterial communities in the pathophysiology of EED is poorly defined due to a paucity of studies where there has been a direct collection of small intestinal samples from undernourished children. Sixty-three members of a Pakistani cohort identified as being acutely malnourished between 3 and 6 months of age and whose wasting (weight-for-length Z-score [WLZ]) failed to improve after a 2-month nutritional intervention underwent esophagogastroduodenoscopy (EGD). Paired duodenal luminal aspirates and duodenal mucosal biopsies were obtained from 43 children. Duodenal microbiota composition was characterized by sequencing bacterial 16S rRNA gene amplicons. Levels of bacterial taxa (amplicon sequence variants [ASVs]) were referenced to anthropometric indices, histopathologic severity in biopsies, expression of selected genes in the duodenal mucosa, and fecal levels of an immunoinflammatory biomarker (lipocalin-2). A "core" group of eight bacterial ASVs was present in the duodenal samples of 69% of participants. Streptococcus anginosus was the most prevalent, followed by Streptococcus sp., Gemella haemolysans, Streptococcus australis, Granulicatella elegans, Granulicatella adiacens, and Abiotrophia defectiva. At the time of EGD, none of the core taxa were significantly correlated with WLZ. Statistically significant correlations were documented between the abundances of Granulicatella elegans and Granulicatella adiacens and the expression of duodenal mucosal genes involved in immune responses (dual oxidase maturation factor 2, serum amyloid A, and granzyme H). These results suggest that a potential role for members of the oral microbiota in pathogenesis, notably Streptococcus, Gemella, and Granulicatella species, warrants further investigation.IMPORTANCEUndernutrition among women and children is a pressing global health problem. Environmental enteric dysfunction (EED) is a disease of the small intestine (SI) associated with impaired gut mucosal barrier function and reduced capacity for nutrient absorption. The cause of EED is ill-defined. One emerging hypothesis is that alterations in the SI microbiota contribute to EED. We performed a culture-independent analysis of the SI microbiota of a cohort of Pakistani children with undernutrition who had failed a standard nutritional intervention, underwent upper gastrointestinal tract endoscopy, and had histologic evidence of EED in their duodenal mucosal biopsies. The results revealed a shared group of bacterial taxa in their duodenums whose absolute abundances were correlated with levels of the expression of genes in the duodenal mucosa that are involved in inflammatory responses. A number of these bacterial taxa are more typically found in the oral microbiota, a finding that has potential physiologic and therapeutic implications.

Duodenal quantitative mucosal morphometry in children with environmental enteric dysfunction: a cross-sectional multi-country analysis.

BACKGROUND: Environmental enteric dysfunction (EED), a chronic inflammatory condition of the small intestine, is an important driver of childhood malnutrition globally. Quantifying intestinal morphology in EED allows for exploration of its association with functional and disease outcomes. OBJECTIVE: We sought to define morphometric characteristics of childhood EED and determine whether morphology features were associated with disease pathophysiology. METHODS: Morphometric measurements and histology were assessed on duodenal biopsy slides for this cross-sectional study from children with EED in Bangladesh, Pakistan, and Zambia (n=69), and those with no pathologic abnormality (NPA; n=8) or celiac disease (n=18) in North America. Immunohistochemistry was also conducted on 46, 8, and 18 biopsy slides, respectively. Linear mixed-effects regression models were used to reveal morphometric differences between EED compared to NPA or celiac disease, and identify associations between morphometry and histology or immunohistochemistry amongst children with EED. RESULTS: In duodenal biopsies, median EED villus height (248 µm), crypt depth (299 µm), and villus:crypt (V:C) ratio (0.9) values ranged between those of NPA (396 µm villus height; 246 µm crypt depth; 1.6 V:C ratio) and celiac disease (208 µm villus height; 365 µm crypt depth; 0.5 V:C ratio). Among EED biopsy slides, morphometric assessments were not associated with histologic parameters or immunohistochemical markers, other than pathologist determined subjective semi-quantitative villus architecture. CONCLUSIONS: Morphometric analysis of duodenal biopsy slides across geographies identified morphologic features of EED, specifically short villi, elongated crypts, and a smaller V:C ratio relative to NPA slides; although not as severe as in celiac slides. Morphometry did not explain other EED features, suggesting that EED histopathologic processes may be operating independently of morphology. While acknowledging the challenges with obtaining relevant tissue, these data form the basis for further assessments of the role of morphometry in EED.

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.

Impact of diarrhoea and acute respiratory infection on environmental enteric dysfunction and growth of malnourished children in Pakistan: a longitudinal cohort study.

Background: Diarrhoea and acute respiratory infections (ARI) are assumed to be major drivers of growth and likely contribute to environmental enteric dysfunction (EED), which is a precursor to childhood malnutrition. In the present study, we checked the correlation between diarrhoeal/ARI burden and EED using a novel duodenal histological index. Methods: Between November 2017 and July 2019, a total of 365 infants with weight-for-height Z scores (WHZ score) of <-2 were enrolled, and 51 infants with WHZ scores of >0 and height-for-age Z scores (HAZ scores) of >-1 were selected as age-matched healthy controls. Morbidity was assessed weekly and categorised as the total number of days with diarrhoea and acute respiratory infection (ARI) from enrolment until two years of age and was further divided into four quartiles in ascending order. Findings: The HAZ declined until two years of age regardless of morbidity burden, and WHZ and weight-for-age Z scores (WAZ scores) were at their lowest at six months. Sixty-three subjects who had a WHZ score <-2 and failed to respond to nutritional and educational interventions were further selected at 15 months to investigate their EED histological scores with endoscopy further. EED histological scores of the subjects were higher with increasing diarrhoeal frequency yet remained statistically insignificant (p = 0.810). Interpretation: There was not a clear correlation between diarrhoea and ARI frequency with growth faltering, however, children with the highest frequency of diarrhoea had the highest EED histological scores and growth faltering. Funding: Bill and Melinda Gates Foundation and The National Institutes of Health.

Standard RUTF vs. locally-made RUSF for acutely malnourished children: A quasi-experimental comparison of the impact on growth and compliance in a rural community of Pakistan.

BACKGROUND: The reduction in severe and moderate acute malnutrition (SAM and MAM) rates in Pakistan has been sub-optimal compared to other low-and middle-income countries (LMICs). Specially-formulated products have been designed globally to manage SAM and MAM, such as ready-to-use therapeutic food (RUTF) and ready-to-use supplementary food (RUSF), with variable efficacies. RUTF is primarily produced and patented in industrialized countries, raising supply challenges in resource-constrained regions with a high burden of acute malnutrition. RUSF minimizes costs by using locally-available ingredients while providing similar nutritional value. In this study, we compared the efficacy, side effects, and compliance of two months of supplementation with either RUTF or RUSF. METHODS: Children aged nine months in the rural district of Matiari, Pakistan, with a weight-for-height z-score (WHZ) <-2 received either RUTF (500 kcal sachet) for two months in 2015 or RUSF (520 kcal sachet) for two months in 2018. RESULTS: The RUSF group had a higher height gain and mid-upper arm circumferences (MUAC) score. Higher compliance was noted with lower side effects in the RUSF group. A higher compliance rate did correlate with the growth parameters in respective groups. CONCLUSION: Our study found that both RUTF and RUSF partially improve the anthropometric status of acutely malnourished children, with neither being superior to the other.

Mucosal transcriptomics highlight lncRNAs implicated in ulcerative colitis, Crohn's disease, and celiac disease.

Ulcerative colitis (UC), Crohn's disease (CD), and celiac disease are prevalent intestinal inflammatory disorders with nonsatisfactory therapeutic interventions. Analyzing patient data-driven cohorts can highlight disease pathways and new targets for interventions. Long noncoding RNAs (lncRNAs) are attractive candidates, since they are readily targetable by RNA therapeutics, show relative cell-specific expression, and play key cellular functions. Uniformly analyzing gut mucosal transcriptomics from 696 subjects, we have highlighted lncRNA expression along the gastrointestinal (GI) tract, demonstrating that, in control samples, lncRNAs have a more location-specific expression in comparison with protein-coding genes. We defined dysregulation of lncRNAs in treatment-naive UC, CD, and celiac diseases using independent test and validation cohorts. Using the Predicting Response to Standardized Pediatric Colitis Therapy (PROTECT) inception UC cohort, we defined and prioritized lncRNA linked with UC severity and prospective outcomes, and we highlighted lncRNAs linked with gut microbes previously implicated in mucosal homeostasis. HNF1A-AS1 lncRNA was reduced in all 3 conditions and was further reduced in more severe UC form. Similarly, the reduction of HNF1A-AS1 ortholog in mice gut epithelia showed higher sensitivity to dextran sodium sulfate-induced colitis, which was coupled with alteration in the gut microbial community. These analyses highlight prioritized dysregulated lncRNAs that can guide future preclinical studies for testing them as potential targets.

Essential Fatty Acid Deficiency Associates with Growth Faltering and Environmental Enteric Dysfunction in Children.

Environmental enteric dysfunction (EED) is characterized by intestinal inflammation, malabsorption and growth-faltering in children with heightened exposure to gut pathogens. The aim of this study was to characterize serum non-esterified fatty acids (NEFA), in association with childhood undernutrition and EED, as potential biomarkers to predict growth outcomes. The study comprised a cohort of undernourished rural Pakistani infants (n = 365) and age-matched controls followed prospectively up to 24 months of age. Serum NEFA were quantified at ages 3-6 and 9 months and correlated with growth outcomes, serum bile acids and EED histopathological biomarkers. Serum NEFA correlated with linear growth-faltering and systemic and gut biomarkers of EED. Undernourished children exhibited essential fatty acid deficiency (EFAD), with low levels of linoleic acid and total n-6 polyunsaturated fatty acids, compensated by increased levels of oleic acid and increased elongase and desaturase activities. EFAD correlated with reduced anthropometric Z scores at 3-6 and 9 months of age. Serum NEFA also correlated with elevated BA and liver dysfunction. Essential fatty acid depletion and altered NEFA metabolism were highly prevalent and associated with acute and chronic growth-faltering in EED. The finding suggests that targeting early interventions to correct EFAD and promote FA absorption in children with EED may facilitate childhood growth in high-risk settings.

GATA6-AS1 Regulates Intestinal Epithelial Mitochondrial Functions, and its Reduced Expression is Linked to Intestinal Inflammation and Less Favourable Disease Course in Ulcerative Colitis.

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 ulcerative colitis [UC]. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not 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 the GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2 cells 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's disease [CD] ileum and UC rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to a more severe UC form, and to a less favourable UC course. The GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in coeliac disease that is induced in UC, CD and coeliac disease, in contrast to GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with a reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in a reduction of metabolites linked to 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 coeliac disease, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 levels.

Environmental enteric dysfunction: gut and microbiota adaptation in pregnancy and infancy.

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.

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.

Nutritional deficiency in an intestine-on-a-chip recapitulates injury hallmarks associated with environmental enteric dysfunction.

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.

Association of Anti-Rotavirus IgA Seroconversion with Growth, Environmental Enteric Dysfunction and Enteropathogens in Rural Pakistani Infants.

BACKGROUND: The underperformance of oral vaccines in children of low- and middle-income countries is partly attributable to underlying environmental enteric dysfunction (EED). METHODOLOGY: 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. RESULTS: 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. CONCLUSION: 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. TRIAL REGISTRATION: Clinical Trials ID: NCT03588013.

Fecal sphingolipids predict parenteral nutrition-associated cholestasis in the neonatal intensive care unit.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

Impact of enteropathogens on faltering growth in a resource-limited setting.

Introduction: Environmental 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. Methods: A 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. Results: Giardia, 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). Conclusion: Protozoal 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.

Distance from Healthcare Facilities Is Associated with Increased Morbidity of Acute Infection in Pediatric Patients in Matiari, Pakistan.

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 < 0.010) and diarrhea (r = 0.228, p < 0.001) as well as participants living closer to Maternal Health Centers (MHCs): ARI (r = 0.185, p < 0.002) and diarrhea (r = 0.223, p < 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.

Self-Attentive Adversarial Stain Normalization.

Hematoxylin and Eosin (H&E) stained Whole Slide Images (WSIs) are utilized for biopsy visualization-based diagnostic and prognostic assessment of diseases. Variation in the H&E staining process across different lab sites can lead to significant variations in biopsy image appearance. These variations introduce an undesirable bias when the slides are examined by pathologists or used for training deep learning models. Traditionally proposed stain normalization and color augmentation strategies can handle the human level bias. But deep learning models can easily disentangle the linear transformation used in these approaches, resulting in undesirable bias and lack of generalization. To handle these limitations, we propose a Self-Attentive Adversarial Stain Normalization (SAASN) approach for the normalization of multiple stain appearances to a common domain. This unsupervised generative adversarial approach includes self-attention mechanism for synthesizing images with finer detail while preserving the structural consistency of the biopsy features during translation. SAASN demonstrates consistent and superior performance compared to other popular stain normalization techniques on H&E stained duodenal biopsy image data.

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.