First-in-Human Assessment of Gut Permeability in Crohn's Disease Patients Using Fluorophore Technology.

Background and Aims: The dual sugar absorption test as a classic measure of human intestinal permeability has limited clinical utility due to lengthy and cumbersome urine collection, assay variability, and long turnaround. We aimed to determine if the orally administered fluorophore MB-102 (relmapirazin) (molecular weight [MW] = 372) compares to lactulose (L) (MW = 342) and rhamnose (R) (MW = 164)-based dual sugar absorption test as a measure of gut permeability in people with a spectrum of permeability including those with Crohn's disease (CD). Methods: We performed a single-center, randomized, open-label, crossover study comparing orally administered MB-102 (1.5 or 3.0 mg/kg) to L (1000 mg) and R (200 mg). Adults with active small bowel CD on magnetic resonance enterography (cases) and healthy adults (controls) were randomized to receive either MB-102 or L and R on study day 1, and the other tracer 3 to 7 days later. Urine was collected at baseline and 1, 2, 4, 6, 8, 10, and 12 hours after tracer ingestion to calculate the cumulative urinary percent excretion of MB-102 and L and R. Results: Nine cases and 10 controls completed the study without serious adverse events. Urinary recovery of administered MB-102 correlated with recovery of lactulose (r-squared = 0.83) for all participants. MB-102 urine recovery was also tracked with the L:R ratio urine recovery (r-squared = 0.57). In controls, the percentages of L and MB-102 recovered were similar within a narrow range, unlike in CD patients. Conclusion: This first-in-human study of an orally administered fluorophore to quantify gastrointestinal permeability in adults with CD demonstrates that MB-102 is well tolerated, and its recovery in urine mirrors that of percent L and the L:R ratio.

Noninvasive Markers of Inflammation and Protein Loss Augment Diagnosis of Pediatric Celiac Disease.

INTRODUCTION: Circulating tissue transglutaminase immunoglobulin A concentration is a sensitive and specific indicator of celiac disease, but discrepancies between serologic and histologic findings occur. We hypothesized that fecal markers of inflammation and protein loss would be greater in patients with untreated celiac disease than in healthy controls. Our study aims to evaluate multiple fecal and plasma markers in celiac disease and correlate these findings with serologic and histologic findings as noninvasive means of evaluating disease activity. METHODS: Participants with positive celiac serologies and controls with negative celiac serologies were prospectively enrolled before upper endoscopy. Blood, stool, and duodenal biopsies were collected. Concentrations of fecal lipocalin-2, calprotectin, and alpha-1-antitrypsin and plasma lipocalin-2 were determined. Biopsies underwent modified Marsh scoring. Significance was tested between cases and controls, modified Marsh score and tissue transglutaminase immunoglobulin A concentration. RESULTS: Lipocalin-2 was significantly elevated in the stool ( P = 0.006) but not the plasma of participants with positive celiac serologies. There was no significant difference in fecal calprotectin or alpha-1 antitrypsin between participants with positive celiac serologies and controls. Fecal alpha-1 antitrypsin >100 mg/dL was specific, but not sensitive for biopsy-proven celiac disease. DISCUSSION: Lipocalin-2 is elevated in the stool but not the plasma of patients with celiac disease suggesting a role of local inflammatory response. Calprotectin was not a useful marker in the diagnosis of celiac disease. While random fecal alpha-1 antitrypsin was not significantly elevated in cases compared with controls, an elevation of greater than 100 mg/dL was 90% specific for biopsy-proven celiac disease.

Quantification of Enteric Dysfunction in Cystic Fibrosis: Inter- and Intraindividual Variability.

OBJECTIVES: To test the utility of various biomarkers as indicators of gut dysfunction in cystic fibrosis (CF) and determine whether intraindividual variations in these measures are repeatable over short intervals and whether interindividual variations correlate with clinical outcomes. STUDY DESIGN: We performed a cross-sectional, limited longitudinal study of children with CF aged 1-21 years who provided blood and stool samples at 2 or 3 visits, 2 weeks and 3 months apart, which were assayed for markers of intestinal inflammation (fecal calprotectin [fCal], lipocalin-2 [fLcn2], neopterin), and permeability (plasma lipopolysaccharide [LPS] antibodies, LPS-binding protein) by enzyme immunoassays. Control specimens were obtained from children without CF who had undergone esophagogastroduodenoscopy and had no evidence of gut inflammation. RESULTS: Twenty-six of 29 participants with CF completed the study. Sixty-nine stools (57 case/12 control) and 76 plasmas (60 case/16 control) were analyzed. LPS antibody had reliable intraindividual stability. fCal, fLcn2, and neopterin were significantly greater in CF than in control samples. fCal was negatively correlated with 3-month interval change (Δ) in weight-for-age z-score, body mass index/weight-for-length z-score, and forced expiratory volume in 1 second. fLcn2 was negatively correlated with FEV1 but not with anthropometrics. No marker correlated with Δbody mass index/weight-for-length z-score or ΔFEV1. CONCLUSIONS: fLcn2 is elevated in people with CF and might predict worse interval pulmonary function. Expanded studies are warranted to test if fLcn2 correlates with changes in additional outcomes.

Non-Invasive markers of inflammation and protein loss augment diagnosis of celiac disease.

Background: Circulating tissue transglutaminase IgA (TTG IgA) concentrations are sensitive and specific indicators of celiac disease, but discrepancies between serologic and histologic findings still occur. We hypothesized that fecal markers of inflammation and protein loss would be greater in patients with untreated celiac disease than in healthy controls. Our study aims to evaluate multiple fecal and plasma markers in celiac disease and correlate these findings with serologic and histologic findings as non-invasive means of evaluating disease activity. Methods: Participants with positive celiac serologies and controls with negative celiac serologies were enrolled at the time of upper endoscopy. Blood, stool and duodenal biopsies were collected. Concentrations of fecal lipocalin-2, calprotectin and alpha-1-antitrypsin and plasma lipcalin-2 were determined. Biopsies underwent modified Marsh scoring. Significance was tested between cases and controls, modified Marsh score and TTG IgA concentration. Results: Lipocalin-2 was significantly elevated in the stool ( p =0.007) but not the plasma of participants with positive celiac serologies compared to controls. There was no significant difference in fecal calprotectin or alpha-1 antitrypsin between participants with positive celiac serologies and controls. Fecal alpha-1 antitrypsin >100mg/dL was specific, but not sensitive for biopsy proven celiac disease. Conclusions: Lipocalin-2 is elevated in the stool but not the plasma of patients with celiac disease suggesting a role in the local inflammatory response. Calprotectin was not a useful marker in the diagnosis of celiac disease and did not correlate with degree of histologic changes on biopsy. While random fecal alpha-1 antitrypsin was not significantly elevated in cases compared to controls, an elevation of greater than 100mg/dL was 90% specific for biopsy proven celiac disease.

Gut virome in early life: origins and implications.

The human body is colonized by a multitude of bacteria, fungi, and viruses, which play important roles in health and disease. Microbial colonization during early life is thought to be a particularly important period with lasting consequences for health. Viral populations in the gut are particularly dynamic in early life before they stabilize in adulthood. The composition of the early-life virome is increasingly recognized as a determinant of disease later in life. Here, we review the development of the virome in healthy infants, as well as the role of the early-life virome in the development of disease states including diarrhea, malnutrition, and autoimmune diseases.

Longitudinal gut virome analysis identifies specific viral signatures that precede necrotizing enterocolitis onset in preterm infants.

Necrotizing enterocolitis (NEC) is a serious consequence of preterm birth and is often associated with gut bacterial microbiome alterations. However, little is known about the development of the gut virome in preterm infants, or its role in NEC. Here, using metagenomic sequencing, we characterized the DNA gut virome of 9 preterm infants who developed NEC and 14 gestational age-matched preterm infants who did not. Infants were sampled longitudinally before NEC onset over the first 11 weeks of life. We observed substantial interindividual variation in the gut virome between unrelated preterm infants, while intraindividual variation over time was significantly less. We identified viral and bacterial signatures in the gut that preceded NEC onset. Specifically, we observed a convergence towards reduced viral beta diversity over the 10 d before NEC onset, which was driven by specific viral signatures and accompanied by specific viral-bacterial interactions. Our results indicate that bacterial and viral perturbations precede the sudden onset of NEC. These findings suggest that early life virome signatures in preterm infants may be implicated in NEC.

Rhamnose Is Superior to Mannitol as a Monosaccharide in the Dual Sugar Absorption Test: A Prospective Randomized Study in Children With Treatment-Naïve Celiac Disease.

Background and Aim: We sought to correlate two different measures of gut permeability [lactulose:mannitol (L:M) and lactulose:rhamnose (L:R)] to the severity of duodenal histopathology in children with and without elevated antibodies to tissue transglutaminase (tTG). A secondary objective was to correlate gut permeability with celiac disease (CD) serology and indices of inflammation and bacterial product translocation. Methods: We prospectively randomized children undergoing endoscopy with abnormal (n = 54) and normal (n = 10) concentrations of circulating antibodies to tTG, to either L:M or L:R. Biopsies underwent modified Marsh scoring to measure mucosal injury. Circulating anticore Escherichia coli lipopolysaccharide (LPS) IgG, α-1 acid glycoprotein, LPS-binding protein, and C-reactive protein concentrations were measured by enzyme immunoassays. Results: Of the 54 cases with positive celiac serology, 31 and 69% had modified Marsh 0/1 scores or ≥3a, respectively. Circulating tTG IgA correlated with the modified Marsh score (p = 0.03). L:R, but not L:M or percent L excreted, differed according to modified Marsh scores (p = 0.01). There was no significant association between any systemic marker of inflammation or gut injury, and modified Marsh scores. Concerningly, most participants had evidence of urinary M before the challenge sugar was administered. Conclusions: L:R, but not L:M, is associated with modified Marsh scores in children undergoing small bowel biopsy for suspected CD. Despite increased intestinal permeability, we see scant evidence of systemic exposure to gut microbes in these children. Gut permeability testing with L:R may predict which patients with abnormal celiac serology will have biopsy evidence for celiac disease and reduce the proportion of such patients undergoing endoscopy whose Marsh scores are ≤1. M should not be used as a monosaccharide for permeability testing in children.

Viral prevalence by gestational age and season in a large neonatal cord blood cohort.

OBJECTIVE: To investigate viral prevalence in a large neonatal cohort and determine the impact on pregnancy and birth outcomes. STUDY DESIGN: We prospectively collected 1044 neonatal samples from remnant neonatal cord blood RPR samples. We performed qRT-PCR/qPCR reactions for: adenovirus, anellovirus (alphatorquevirus and betatorquevirus), cytomegalovirus (CMV), Epstein-Barr virus (EBV), enterovirus, human herpesvirus 6 (HHV6), parechovirus, and parvovirus B19. RESULT: Overall viral prevalence was 5.6% with 58 positive samples. Alphatorquevirus (2%) and HHV6 (1.2%) were the two most prevalent viruses detected. Viral detection was most common in samples collected in the fall (September-November) and least common in those collected in winter (December-February). There was no statistical difference detected in viral prevalence or viral load by gestational age, preterm delivery, pre-eclampsia or chorioamnionitis. CONCLUSION: While there is seasonal variation in viral prevalence in neonatal cord blood samples, individual virus presence does not seem to effect pregnancy or birth outcomes.

Progression of pediatric celiac disease from potential celiac disease to celiac disease: a retrospective cohort study.

BACKGROUND: A subset of patients with serology suggesting celiac disease have an initially negative biopsy but subsequently develop histopathologic celiac disease. Here we characterize patients with potential celiac disease who progress to celiac disease. METHODS: We performed a retrospective analysis of children (0-18 years of age) with biopsy-confirmed celiac disease seen at St. Louis Children's Hospital between 2013 and 2018. RESULTS: Three hundred sixteen of 327 (96%) children with biopsy-confirmed celiac disease were diagnosed on initial biopsy. The 11 children with potential celiac disease who progressed to celiac disease had lower anti-tissue transglutaminase (anti-TTG IgA) concentrations (2.4 (1.6-5) X upper limit of normal (ULN) vs. 6.41 (3.4-10.5) X ULN) at time of first biopsy. Their median anti-TTG IgA concentrations rose from 2.4 (1.6-5) X ULN to 3.6 (3.1-9.2) X ULN between biopsies. CONCLUSIONS: Four percent of biopsy confirmed celiac patients initially had a negative biopsy, but later developed histopathologic celiac disease. This is likely an underestimate as no surveillance algorithm was in place. We recommend repeat assessment in children whose serology suggests celiac disease despite normal small bowel biopsy.

Putting the Virome on the Map: The Influence of Host Geography and Ethnicity on the Gut Virome.

We know little about factors that ordain the composition of the diverse community of viruses (virome) in the human gut. In this issue of Cell Host & Microbe, Zuo et al. highlight the importance of geography, urbanization, ethnicity, and diet on the shape of the adult gut DNA virome.

Growth velocity in children with Environmental Enteric Dysfunction is associated with specific bacterial and viral taxa of the gastrointestinal tract in Malawian children.

Environmental enteric dysfunction (EED) is characterized by diffuse villous atrophy of the small bowel. EED is strongly associated with stunting, a major public health problem linked to increased childhood morbidity and mortality. EED and subsequent stunting of linear growth are surmised to have microbial origins. To interrogate this relationship, we defined the comprehensive virome (eukaryotic virus and bacteriophage) and bacterial microbiome of a longitudinal cohort of rural Malawian children with extensive metadata and intestinal permeability testing at each time point. We found thirty bacterial taxa differentially associated with linear growth. We detected many eukaryotic viruses. Neither the total number of eukaryotic families nor a specific viral family was statistically associated with improved linear growth. We identified 3 differentially abundant bacteriophage among growth velocities. Interestingly, there was a positive correlation between bacteria and bacteriophage richness in children with subsequent adequate/moderate growth which children with subsequent poor growth lacked. This suggests that a disruption in the equilibrium between bacteria and bacteriophage communities might be associated with subsequent poor growth. Future studies of EED and stunting should include the evaluation of viral communities in addition to bacterial microbiota to understand the complete microbial ecology of these poorly understood entities.

Prenatal food insecurity post Hurricane Maria is associated with decreased Veillonella in the infant gut.

BACKGROUND: Hurricane Maria struck Puerto Rico on 20 September 2017 causing catastrophic devastation. Prolonged shortage of food had been a substantial challenge to the residents after Maria. Experiencing food insecurity in utero has been associated with negative health outcomes later in life. We aim to examine whether there is any alteration in the infant gut microbiome that is associated with prenatal food insecurity. METHODS: We established a cohort of infants aged 2-6 months who were exposed in utero to Hurricane Maria near San Juan, Puerto Rico and examined the gut microbiota (n = 29) using 16S ribosomal RNA gene sequencing. RESULTS: Among the enrolled infants, 30% of their mothers experienced "post-Maria poor access to food" for at least 1 month during pregnancy. The relative abundance of gut Veillonella spp. is significantly decreased among infants who experienced prenatal food insecurity, compared to those who did not (adjusted p = 0.025). There is no significant difference observed by prenatal food insecurity at the microbial community level in this cohort. CONCLUSIONS: Our finding indicated that infants who experienced prenatal food insecurity post hurricane harbor microbial alternations of specific bacterial taxa, which may further influence the microbial maturation and place the individual at a high-risk health trajectory. IMPACT: We identified that in utero exposure to food insecurity post Hurricane Maria is associated with decreased abundance of Veillonella in the infant gut. Our findings indicated that infants who experienced prenatal food insecurity post hurricane may harbor alterations of specific bacterial taxa in their gut microbiota. This study showed the association between prenatal adverse exposure and alterations of gut microbiome early in life in the context of an extreme event. This study provided insights into the mechanisms underlying prenatal adverse exposure and increased disease risks later in life. Our findings will potentially raise awareness of the negative impact of extreme climate events on the unborn.

A novel histological index for evaluation of environmental enteric dysfunction identifies geographic-specific features of enteropathy among children with suboptimal growth.

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.

Discordant transmission of bacteria and viruses from mothers to babies at birth.

BACKGROUND: The earliest microbial colonizers of the human gut can have life-long consequences for their hosts. Precisely how the neonatal gut bacterial microbiome and virome are initially populated is not well understood. To better understand how the maternal gut microbiome influences acquisition of the infant gut microbiome, we studied the early life bacterial microbiomes and viromes of 28 infant twin pairs and their mothers. RESULTS: Infant bacterial and viral communities more closely resemble those of their related co-twin than unrelated infants. We found that 63% of an infant's bacterial microbiome can be traced to their mother's gut microbiota. In contrast, only 15% of their viral communities are acquired from their mother. Delivery route did not determine how much of the bacterial microbiome or virome was shared from mother to infant. However, bacteria-bacteriophage interactions were altered by delivery route. CONCLUSIONS: The maternal gut microbiome significantly influences infant gut microbiome acquisition. Vertical transmission of the bacterial microbiome is substantially higher compared to vertical transmission of the virome. However, the degree of similarity between the maternal and infant gut bacterial microbiome and virome did not vary by delivery route. The greater similarity of the bacterial microbiome and virome between twin pairs than unrelated twins may reflect a shared environmental exposure. Thus, differences of the inter-generation transmissibility at birth between the major kingdoms of microbes indicate that the foundation of these microbial communities are shaped by different rules. Video Abstract.

Correction to: Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community.

Following publication of the original article [1], the authors reported a typographic error in the type of master mix kit used, the text should read as: "The qPCR was performed using Fast SYBR Green Master Mix (Thermo Fisher)."

Reply Re: "Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community".

How and when a newborn is first colonized by microbes continues to be of great interest due to its broad implications on human health and disease. Payne et al. express their opinion about our recent study in which we characterized the virome and bacterial microbiota of amniotic fluid from 24 uncomplicated term pregnancies. We conducted additional validation studies and respond to their comments. We conclude that in amniotic fluid from healthy term pregnancies, the bacterial microbiota is indistinguishable from contamination controls, and there is no evidence of a core virome.

Inherited nongenetic influences on the gut microbiome and immune system.

The gut microbiome and the immune system codevelop around the time of birth, well after genetic information has been passed from the parents to the offspring. Each of these "organ systems" displays plasticity. The immune system can mount highly specific adaptive responses to newly encountered antigens, and the gut microbiota is affected by changes in the environment. Despite this plasticity, there is a growing appreciation that these organ systems, once established, are remarkably stable. In health, the immune system rapidly mounts responses to infections, and once cleared, resolves inflammatory responses to return to homeostasis. However, a skewed immune system, such as seen in allergy, does not easily return to homeostasis. Allergic responses are often seen to multiple antigens. Likewise, a dysbiotic gut microbiota is seen in multiple diseases. Attempts to reset the gut microbiota as a therapy for disease have met with varied success. Therefore, how these codeveloping "organ systems" become established is a central question relevant to our overall health. Recent observations suggest that maternal factors encountered both in utero and after birth can directly or indirectly impact the development of the offspring's gut microbiome and immune system. Here, we discuss how these nongenetic maternal influences can have long-term effects on the progeny's health.