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

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

Histopathology underlying environmental enteric dysfunction in a cohort study of undernourished children in Bangladesh, Pakistan, and Zambia compared with United States children.

BACKGROUND: Environmental enteric dysfunction (EED) is an asymptomatic intestinal disorder associated with growth impairment, delayed neurocognitive development, and impaired oral vaccine responses. OBJECTIVES: We set out to develop and validate a histopathologic scoring system on duodenal biopsies from a cohort study of children with growth failure in Bangladesh, Pakistan, and Zambia ("EED") with reference to biopsies from United States children with no clinically reported histologic pathology (referred to hereafter as "normal") or celiac disease. METHODS: Five gastrointestinal pathologists evaluated 745 hematoxylin and eosin slide images from 291 children with EED (mean age: 1.6 y) and 66 United States children (mean age: 6.8 y). Histomorphologic features (i.e., villus/crypt architecture, goblet cells, epithelial and lamina propria acute/chronic inflammation, Brunner's glands, Paneth cells, epithelial detachment, enterocyte injury, and foveolar metaplasia) were used to score each histopathologic slide. Generalized estimating equations were used to determine differences between EED, normal, and celiac disease, and receiver operating characteristic curves were used to assess predictive value. RESULTS: Biopsies from the duodenal bulb showed higher intramucosal Brunner's gland scores and lower intraepithelial lymphocyte scores than from the second or third parts of the duodenum (D2/3), so only D2/3 were included in the final analysis. Although 7 parameters differed significantly between EED and normal biopsies in regression models, only 5 (blunted villus architecture, increased intraepithelial lymphocytosis, goblet cell depletion, Paneth cell depletion, and reduced intramucosal Brunner's glands) were required to create a total score percentage (TSP-5) that correctly identified EED against normal biopsies (AUC: 0.992; 95% CI: 0.983, 0.998). Geographic comparisons showed more severe goblet cell depletion in Bangladesh and more marked intraepithelial lymphocytosis in Pakistan. CONCLUSIONS: This scoring system involving 5 histologic parameters demonstrates very high discrimination between EED and normal biopsies, indicating that this scoring system can be applied with confidence to studies of intestinal biopsies in EED.

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

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

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

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

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

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

Biomarker relationships with small bowel histopathology among malnourished children with environmental enteric dysfunction in a multicountry cohort study.

BACKGROUND: Validated biomarkers could catalyze environmental enteric dysfunction (EED) research. OBJECTIVES: Leveraging an EED histology scoring system, this multicountry analysis examined biomarker associations with duodenal histology features among children with EED. We also examined differences in 2-h compared with 1-h urine collections in the lactulose rhamnose (LR) dual sugar test. METHODS: Three cohorts of undernourished children unresponsive to nutrition intervention underwent esophagogastroduodenoscopy and duodenal biopsies. Histopathology scores were compared to fecal calprotectin (CAL), myeloperoxidase (MPO), neopterin (NEO), and urinary LR ratio and lactulose percentage recovery. Log-transformed biomarkers were used in linear regressions adjusted for age, center, and sample collection-biopsy time interval in multivariable models. RESULTS: Data on >1 biomarker were available for 120 Bangladeshi (CAL, MPO, NEO, and LR), 63 Pakistani (MPO, NEO, and LR), and 63 Zambian children (CAL). Median age at endoscopy was similar (19 mo) across centers. Median sample collection prior to endoscopy was consistent with each center's study design: 2 wk in Bangladesh (urine and stool) and Zambia (stool), and 6 (urine) and 11 (stool) mo in Pakistan. In multivariable models, intraepithelial lymphocytes were associated with CAL (exponentiated [exp.] coefficient: 1.19; 95% confidence interval [CI]: 1, 1.41), intramucosal Brunner's glands with MPO (exp. coefficient: 1.33; 95% CI: 1.05, 1.69) and NEO (exp. coefficient: 1.37; 95% CI: 1.1, 1.7), and chronic inflammation with NEO (exp. coefficient: 1.61; 95% CI: 1.17, 2.17). Intraepithelial lymphocytes were associated with lactulose % recovery (exp. coefficient: 1.22; 95% CI: 1.05, 1.41). LR recovery was substantially lower in 1-h collections than in 2-h collections. CONCLUSIONS: Four commonly used markers of enteric dysfunction were associated with specific histologic features. One-hour urine collection may be insufficient to reflect small bowel permeability in LR testing. While acknowledging the challenges with obtaining relevant tissue, these findings form the basis for further EED biomarker validation research.

Duodenal quantitative mucosal morphometry in children with environmental enteric dysfunction: a cross-sectional multicountry 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. OBJECTIVES: 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 with NPA or celiac disease and identify associations between morphometry and histology or immunohistochemistry among 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 semiquantitative 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. Although acknowledging the challenges with obtaining relevant tissue, these data form the basis for further assessments of the role of morphometry in EED.

Intestinal Epithelial Digestive, Transport, and Barrier Protein Expression Is Increased in Environmental Enteric Dysfunction.

Environmental enteric dysfunction (EED) is characterized by malabsorption and diarrhea that result in irreversible deficits in physical and intellectual growth. We sought to define the expression of transport and tight junction proteins by quantitative analysis of duodenal biopsies from patients with EED. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age-matched North American healthy controls, patients with celiac disease, and patients with nonceliac disease with villous atrophy or intraepithelial lymphocytosis. Expression of brush border digestive and transport proteins and paracellular (tight junction) proteins was assessed by quantitative multiplex immunofluorescence microscopy. EED was characterized by partial villous atrophy and marked intraepithelial lymphocytosis. Epithelial proliferation and enteroendocrine, tuft, and Paneth cell numbers were unchanged, but there was significant goblet cell expansion in EED biopsies. Expression of proteins involved in nutrient and water absorption and that of the basolateral Cl- transport protein NKCC1 were also increased in EED. Finally, the barrier-forming tight junction protein claudin-4 (CLDN4) was significantly upregulated in EED, particularly within villous enterocytes. In contrast, expression of CFTR, CLDN2, CLDN15, JAM-A, occludin, ZO-1, and E-cadherin was unchanged. Upregulation of a barrier-forming tight junction protein and brush border and basolateral membrane proteins that support nutrient and water transport in EED is paradoxical, as their increased expression would be expected to be correlated with increased intestinal barrier function and enhanced absorption, respectively. These data suggest that EED activates adaptive intestinal epithelial responses to enhance nutrient absorption but that these changes are insufficient to restore 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.

Machine learning model demonstrates stunting at birth and systemic inflammatory biomarkers as predictors of subsequent infant growth - a four-year prospective study.

BACKGROUND: Stunting affects up to one-third of the children in low-to-middle income countries (LMICs) and has been correlated with decline in cognitive capacity and vaccine immunogenicity. Early identification of infants at risk is critical for early intervention and prevention of morbidity. The aim of this study was to investigate patterns of growth in infants up through 48 months of age to assess whether the growth of infants with stunting eventually improved as well as the potential predictors of growth. METHODS: Height-for-age z-scores (HAZ) of children from Matiari (rural site, Pakistan) at birth, 18 months, and 48 months were obtained. Results of serum-based biomarkers collected at 6 and 9 months were recorded. A descriptive analysis of the population was followed by assessment of growth predictors via traditional machine learning random forest models. RESULTS: Of the 107 children who were followed up till 48 months of age, 51% were stunted (HAZ < - 2) at birth which increased to 54% by 48 months of age. Stunting status for the majority of children at 48 months was found to be the same as at 18 months. Most children with large gains started off stunted or severely stunted, while all of those with notably large losses were not stunted at birth. Random forest models identified HAZ at birth as the most important feature in predicting HAZ at 18 months. Of the biomarkers, AGP (Alpha- 1-acid Glycoprotein), CRP (C-Reactive Protein), and IL1 (interleukin-1) were identified as strong subsequent growth predictors across both the classification and regressor models. CONCLUSION: We demonstrated that children most children with stunting at birth remained stunted at 48 months of age. Value was added for predicting growth outcomes with the use of traditional machine learning random forest models. HAZ at birth was found to be a strong predictor of subsequent growth in infants up through 48 months of age. Biomarkers of systemic inflammation, AGP, CRP, IL1, were also strong predictors of growth outcomes. These findings provide support for continued focus on interventions prenatally, at birth, and early infancy in children at risk for stunting who live in resource-constrained regions of the world.

Artificial Intelligence Applied to Gastrointestinal Diagnostics: A Review.

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

Diagnosis of Celiac Disease and Environmental Enteropathy on Biopsy Images Using Color Balancing on Convolutional Neural Networks.

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.

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

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

Assessment of Machine Learning Detection of Environmental Enteropathy and Celiac Disease in Children.

Importance: Duodenal biopsies from children with enteropathies associated with undernutrition, such as environmental enteropathy (EE) and celiac disease (CD), display significant histopathological overlap. Objective: To develop a convolutional neural network (CNN) to enhance the detection of pathologic morphological features in diseased vs healthy duodenal tissue. Design, Setting, and Participants: In this prospective diagnostic study, a CNN consisting of 4 convolutions, 1 fully connected layer, and 1 softmax layer was trained on duodenal biopsy images. Data were provided by 3 sites: Aga Khan University Hospital, Karachi, Pakistan; University Teaching Hospital, Lusaka, Zambia; and University of Virginia, Charlottesville. Duodenal biopsy slides from 102 children (10 with EE from Aga Khan University Hospital, 16 with EE from University Teaching Hospital, 34 with CD from University of Virginia, and 42 with no disease from University of Virginia) were converted into 3118 images. The CNN was designed and analyzed at the University of Virginia. The data were collected, prepared, and analyzed between November 2017 and February 2018. Main Outcomes and Measures: Classification accuracy of the CNN per image and per case and incorrect classification rate identified by aggregated 10-fold cross-validation confusion/error matrices of CNN models. Results: Overall, 102 children participated in this study, with a median (interquartile range) age of 31.0 (20.3-75.5) months and a roughly equal sex distribution, with 53 boys (51.9%). The model demonstrated 93.4% case-detection accuracy and had a false-negative rate of 2.4%. Confusion metrics indicated most incorrect classifications were between patients with CD and healthy patients. Feature map activations were visualized and learned distinctive patterns, including microlevel features in duodenal tissues, such as alterations in secretory cell populations. Conclusions and Relevance: A machine learning-based histopathological analysis model demonstrating 93.4% classification accuracy was developed for identifying and differentiating between duodenal biopsies from children with EE and CD. The combination of the CNN with a deconvolutional network enabled feature recognition and highlighted secretory cells' role in the model's ability to differentiate between these histologically similar diseases.

Assessment and Validation of Syndromic Case Definitions for Respiratory Syncytial Virus Testing in a Low Resource Population.

Standardized case definitions are needed in decision-making regarding respiratory syncytial virus control strategies, including vaccine evaluation. A syndromic case definition comprising of "wheeze or apnea or cyanosis" could be useful for community-based surveillance of moderate respiratory syncytial virus infection among young infants particularly in resource-limited settings. However, this definition showed modest specificity (29.2%-49.6%), indicating that community-based surveillance may need augmentation with other data.

Impact of maternally derived pertussis antibody titers on infant whole-cell pertussis vaccine response in a low income setting.

BACKGROUND: Maternal vaccines against pertussis are not yet recommended in the developing world. Besides unclear burden estimates, another concern is that transplacental transfer of maternal pertussis antibodies could result in attenuation of the immune response to whole cell pertussis (DTwP) primary vaccination series in infants. This study was taken up to determine whether higher levels of maternal pertussis antibodies attenuate immune response of infants to DTwP vaccination series given at 6-10-14 weeks of age. METHODOLOGY: A total of 261 pregnant women and their infants from four low-income settlements in Karachi, Pakistan were enrolled in this study. The study endpoints were infant antibody titers for Pertussis toxin (PTx), Filamentous hemagglutinin antigen (FHA), Pertactin (PRN) and Fimbriae type 2/3 (FIM) - from birth through 18 weeks of age. Cord blood or pre-vaccine pertussis antibody titers indicate the concentration of maternal antibodies transferred to infants. Linear regression models were used to determine the association between higher maternal antibody titers and infant immune response to DTwP vaccine. Geometric Mean Ratio (GMR) was calculated as the ratio of infant antibody titers at specified time points against the maternal antibody titers at the time of delivery. RESULTS: At eighteen weeks of age, the adjusted ß regression coefficient for PTx was 0.06 (95% CI: -0.49-0.61), FHA 0.02 (95% CI: -0.26 -0.29), PRN 0.02 (95%CI -0.38- 0.43), and FIM 0.17 (95%CI: -0.21-0.54). Among infants who received at least two doses of DTwP vaccine, higher maternal antibody titers did not have any attenuating effect on infant post-immunization antibody titers against all four pertussis antigens. CONCLUSION: Maternal pertussis antibodies did not attenuate infant's immune response to pertussis antigens in DTwP primary vaccine given at 6-10-14 weeks of age.

Environmental Enteropathy in Undernourished Pakistani Children: Clinical and Histomorphometric Analyses.

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.

Serum anti-flagellin and anti-lipopolysaccharide immunoglobulins as predictors of linear growth faltering in Pakistani infants at risk for environmental enteric dysfunction.

BACKGROUND: Environmental Enteric Dysfunction (EED) in children from low-income countries has been linked to linear growth declines. There is a critical need to identify sensitive and early EED biomarkers. OBJECTIVE: Determine whether levels of antibodies against bacterial components flagellin (flic) and lipopolysaccharide (LPS) predict poor growth. DESIGN/METHODS: In a prospective birth cohort of 380 children in rural Pakistan blood and stool samples were obtained at ages 6 and 9 months. Linear mixed effects models were used to examine longitudinal associations between quartiles of anti-flic and anti-LPS antibodies and changes in LAZ, WAZ and WLZ scores. Spearman's correlations were measured between anti-flic and anti-LPS immunoglobulins with measures of systemic/enteric inflammation and intestinal regeneration. RESULTS: Anti-LPS IgA correlated significantly with CRP, AGP and Reg1 serum at 6mo and with MPO at 9mo. In multivariate analysis at 6mo of age, higher anti-LPS IgA levels predicted greater declines in LAZ scores over subsequent 18mo (comparing highest to lowest quartile, ß (SE) change in LAZ score/year = -0.313 (0.125), p-value = 0.013). Anti-flic Ig A in the two highest quartiles measured at 9mo of age had declines in LAZ of -0.269 (0.126), p = 0.033; and -0.306 (0.129), p = 0.018 respectively, during the subsequent 18mo of life, compared to those in the lowest quartile of anti-flic IgA. CONCLUSIONS AND RELEVANCE: Elevated anti-flic IgA and anti-LPS IgA antibodies at 6 and 9mo, predict declines in linear growth. Systemic and enteric inflammation correlated with anti-LPS IgA provides mechanistic considerations for potential future interventions.

High SMAD7 and p-SMAD2,3 expression is associated with environmental enteropathy in children.

Enteropathies such as Crohn's disease are associated with enteric inflammation characterized by impaired TGF-ß signaling, decreased expression of phosphorylated (p)-SMAD2,3 and increased expression of SMAD7 (an inhibitor of SMAD3 phosphorylation). Environmental enteropathy (EE) is an acquired inflammatory disease of the small intestine (SI), which is associated with linear growth disruption, cognitive deficits, and reduced oral vaccine responsiveness in children <5 y in resource-poor countries. We aimed to characterize EE inflammatory pathways by determining SMAD7 and p-SMAD2,3 levels (using Western blotting) in EE duodenal biopsies (N = 19 children, 7 from Pakistan, 12 from Zambia) and comparing these with healthy controls (Ctl) and celiac disease (CD) patients from Italy. Densitometric analysis of immunoblots showed that EE SI biopsies expressed higher levels of both SMAD7 (mean±SD in arbitrary units [a.u.], Ctl = 0.47±0.20 a.u., EE = 1.13±0.25 a.u., p-value = 0.03) and p-SMAD2,3 (mean±SD, Ctl = 0.38±0.14 a.u., EE = 0.60±0.10 a.u., p-value = 0.03). Immunohistochemistry showed that, in EE, SMAD7 is expressed in both the epithelium and in mononuclear cells of the lamina propria (LP). In contrast, p-SMAD3 in EE is expressed much more prominently in epithelial cells than in the LP. The high SMAD7 immunoreactivity and lack of p-SMAD3 expression in the LP suggests defective TGF-ß signaling in the LP in EE similar to a previously reported SMAD7-mediated inflammatory pathway in refractory CD and Crohn's disease. However, Western blot densitometry showed elevated p-SMAD2,3 levels in EE, possibly suggesting a different inflammatory pathway than Crohn's disease but more likely reflecting cumulative protein expression from across all compartments of the mucosa as opposed to the LP alone. Further studies are needed to substantiate these preliminary results and to illustrate the relationship between SMAD proteins, TGF-ß signaling, and inflammatory cytokine production, all of which may be potential therapeutic targets.

Epidemiology of Pertussis Among Young Pakistani Infants: A Community-Based Prospective Surveillance Study.

BACKGROUND: Pertussis remains a cause of morbidity and mortality among young infants. There are limited data on the pertussis disease burden in this age group from low- and lower-middle-income countries, including in South Asia. METHODS: We conducted an active community-based surveillance study from February 2015 to April 2016 among 2 cohorts of young infants in 4 low-income settlements in Karachi, Pakistan. Infants were enrolled either at birth (closed cohort) or at ages up to 10 weeks (open cohort) and followed until 18 weeks of age. Nasopharyngeal swab specimens were obtained from infants who met a standardized syndromic case definition and tested for Bordetella pertussis using real-time polymerase chain reaction. We determined the incidence of pertussis using a protocol-defined case definition, as well as the US Centers for Disease Control and Prevention (CDC) definitions for confirmed and probable pertussis. RESULTS: Of 2021 infants enrolled into the study, 8 infants met the protocol-defined pertussis case definition, for an incidence of 3.96 (95% confidence interval [CI], 1.84-7.50) cases per 1000 infants. Seven of the pertussis cases met the CDC pertussis case definition (5 confirmed, 2 probable), for incidences of CDC-defined confirmed pertussis of 2.47 (95% CI, .90-5.48) cases per 1000 infants, and probable pertussis of 0.99 (95% CI, .17-3.27) cases per 1000 infants. Three of the pertussis cases were severe according to the Modified Preziosi Scale score. CONCLUSIONS: In one of the first prospective surveillance studies of infant pertussis in a developing country, we identified a moderate burden of pertussis disease in early infancy in Pakistan.