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

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

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

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

Enhanced survival following oral and systemic Salmonella enterica serovar Typhimurium infection in polymeric immunoglobulin receptor knockout mice.

BACKGROUND: Polymeric immunoglobulin receptor (pIgR) transport of secretory immunoglobulin A (SIgA) to mucosal surfaces is thought to promote gut integrity and immunity to Salmonella enterica serovar Typhimurium (S. Typhimurium), an invasive pathogen in mice. To elucidate potential mechanisms, we assessed intestinal barrier function and both oral and systemic S. Typhimurium virulence in pIgR knockout (KO) and wildtype (WT) mice. METHODS: In uninfected animals, we harvested jejunal segments for Ussing chamber analyses of transepithelial resistance (TER); mesenteric lymph nodes (mLN) for bacterial culture; and serum and stool for IgA. Separately, we infected mice either orally or intravenously (IV) with S. Typhimurium to compare colonization, tissue dynamics, and inflammation between KOs and WTs. RESULTS: Uninfected KOs displayed decreased TER and dramatically increased serum IgA and decreased fecal IgA vs. WT; however, KO mLNs yielded fewer bacterial counts. Remarkably, WTs challenged orally with S. Typhimurium exhibited increased splenomegaly, tissue colonization, and pro-inflammatory cytokines vs. pIgR KOs, which showed increased survival following either oral or IV infection. CONCLUSIONS: Absence of pIgR compromises gut integrity but does not exacerbate bacterial translocation nor S. Typhimurium infection. These findings raise the possibility that immune adaptation to increased gut permeability and elevated serum IgA in the setting of SIgA deficiency provides compensatory protection against invasive gut pathogens.

Familial Association of Granulocyte-Macrophage Colony-Stimulating Factor Autoantibodies in Inflammatory Bowel Disease.

OBJECTIVES: Elevated granulocyte-macrophage colony-stimulating factor auto-antibodies (GM-CSF Ab) are associated with increased intestinal permeability and stricturing behavior in Crohn disease (CD). We tested for familial association of serum GM-CSF Ab level in CD and ulcerative colitis (UC) families. METHODS: Serum GM-CSF Ab concentration was determined in 230 pediatric CD probands and 404 of their unaffected parents and siblings, and 45 UC probands and 71 of their unaffected parents and siblings. A linear mixed effects model was used to test for familial association. The intra-class correlation coefficient (ICC) was used to determine the degree of association of the serum GM-CSF Ab level within families in comparison with the degree of association among families. RESULTS: The median (IQR) serum GM-CSF Ab concentration was higher in CD probands than in UC probands (1.5 [0.5,5.4] µg/mL vs 0.7 [0.3, 1.6] µg/mL, P = 0.0002). The frequency of elevated serum GM-CSF Ab concentration ≥1.6 µg/mL was increased in unaffected siblings of CD probands with elevated GM-CSF Ab, compared with unaffected siblings of CD probands without elevated GM-CSF Ab (33% vs 13%, respectively, P = 0.04). A similar result was observed within UC families. In families of CD patients, the mean (95th CI) ICC was equal to 0.153 (0.036, 0.275), P = 0.001, whereas in families of UC patients, the mean (95th CI) ICC was equal to 0.27 (0.24, 0.31), P = 0.047. CONCLUSIONS: These data confirmed familial association of serum GM-CSF Ab levels. This could be accounted for by either genetic or environmental factors shared within the family.

Biomarkers of Environmental Enteropathy, Inflammation, Stunting, and Impaired Growth in Children in Northeast Brazil.

Critical to the design and assessment of interventions for enteropathy and its developmental consequences in children living in impoverished conditions are non-invasive biomarkers that can detect intestinal damage and predict its effects on growth and development. We therefore assessed fecal, urinary and systemic biomarkers of enteropathy and growth predictors in 375 6-26 month-old children with varying degrees of malnutrition (stunting or wasting) in Northeast Brazil. 301 of these children returned for followup anthropometry after 2-6m. Biomarkers that correlated with stunting included plasma IgA anti-LPS and anti-FliC, zonulin (if >12m old), and intestinal FABP (I-FABP, suggesting prior barrier disruption); and with citrulline, tryptophan and with lower serum amyloid A (SAA) (suggesting impaired defenses). In contrast, subsequent growth was predicted in those with higher fecal MPO or A1AT and also by higher L/M, plasma LPS, I-FABP and SAA (showing intestinal barrier disruption and inflammation). Better growth was predicted in girls with higher plasma citrulline and in boys with higher plasma tryptophan. Interactions were also seen with fecal MPO and neopterin in predicting subsequent growth impairment. Biomarkers clustered into markers of 1) functional intestinal barrier disruption and translocation, 2) structural intestinal barrier disruption and inflammation and 3) systemic inflammation. Principle components pathway analyses also showed that L/M with %L, I-FABP and MPO associate with impaired growth, while also (like MPO) associating with a systemic inflammation cluster of kynurenine, LBP, sCD14, SAA and K/T. Systemic evidence of LPS translocation associated with stunting, while markers of barrier disruption or repair (A1AT and Reg1 with low zonulin) associated with fecal MPO and neopterin. We conclude that key noninvasive biomarkers of intestinal barrier disruption, LPS translocation and of intestinal and systemic inflammation can help elucidate how we recognize, understand, and assess effective interventions for enteropathy and its growth and developmental consequences in children in impoverished settings.

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

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

Protein-energy malnutrition alters IgA responses to rotavirus vaccination and infection but does not impair vaccine efficacy in mice.

BACKGROUND: Conflicting evidence links malnutrition to the reduced efficacy of rotavirus vaccines in developing countries, where diarrhea and undernutrition remain leading causes of child deaths. Here, we adapted mouse models of rotavirus vaccination (rhesus rotavirus, RRV), rotavirus infection (EDIM), and protein-energy malnutrition (PEM) to test the hypothesis that undernutrition reduces rotavirus vaccine immunogenicity and efficacy. METHODS: We randomized wild type Balb/C dams with 3-day-old pups to a control diet (CD) or an isocaloric, multideficient regional basic diet (RBD) that produces PEM. At 3 weeks of age, we weaned CD and RBD pups to their dams' diet and subrandomized weanlings to receive a single dose of either live oral rotavirus vaccine (RRV) or PBS. At 6 weeks of age, we orally challenged all groups with murine rotavirus (EDIM). Serum and stool specimens were collected before and after RRV and EDIM administration to measure viral shedding and antibody responses by ELISA. RESULTS: RBD pups and weanlings exhibited significant failure to thrive compared to age-matched CD mice (P<.0001). RRV vaccination induced higher levels of serum anti-RV IgA responses in RBD vs. CD mice (P<.0001). Vaccination protected CD and RBD mice equally against EDIM infection, as measured by viral shedding. In unvaccinated RBD mice, EDIM shedding peaked 1 day earlier (P<.05), however we detected no effects of undernutrition on viral clearance nor of infection on bodyweight. EDIM infection provoked higher anti-RV serum IgA levels in RBD vs. CD mice, regardless of vaccination (P<.0001). Last, RRV vaccination mitigated stool IgA responses to EDIM more in CD vs. RBD mice (P<.0001). CONCLUSIONS: Despite modulated IgA responses to vaccination and infection, undernutrition does not impair rotavirus vaccine efficacy nor exacerbate infection in this mouse model of protein-energy malnutrition. Alternative models are needed to elucidate host-pathogen factors undermining rotavirus vaccine effectiveness in high-risk global settings.

Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells.

Engineered skin substitutes (ESS) have been used successfully to treat life-threatening burns, but lack cutaneous appendages. To address this deficiency, dermal constructs were prepared using collagen-glycosaminoglycan scaffolds populated with murine dermal papilla cells expressing green fluorescent protein (mDPC-GFP), human dermal papilla cells (hDPC) and/or human fibroblasts (hF). Subsequently, human epidermal keratinocytes (hK) or hK genetically modified to overexpress stabilized ß-catenin (hK') were used to prepare ESS epithelium. After 10 days incubation at air-liquid interface, ESS were grafted to athymic mice and were evaluated for 6 weeks. Neofollicles were observed in ESS containing mDPC-GFP, but not hDPC or hF, independent of whether or not the hK were genetically modified. Based on detection of GFP fluorescence, mDPC were localized to the dermal papillae of the well-defined follicular structures of grafted ESS. In addition, statistically significant increases in LEF1, WNT10A and WNT10B were found in ESS with neofollicles. These results demonstrate a model for generation of chimeric hair in ESS.

Alanyl-glutamine promotes intestinal epithelial cell homeostasis in vitro and in a murine model of weanling undernutrition.

Alanyl-glutamine (Ala-Gln) has recently been shown to enhance catch-up growth and gut integrity in undernourished children from Northeast Brazil. We hypothesized that the intestinal epithelial effects of Ala-Gln in malnourished weanling mice and mouse small intestinal epithelial (MSIE) cells would include modulation of barrier function, proliferation, and apoptosis. Dams of 10-day-old suckling C57BL/6 pups were randomized to a standard diet or an isocaloric Northeast Brazil "regional basic diet," moderately deficient in protein, fat, and minerals. Upon weaning to their dam's diet on day of life 21, pups were randomized to Ala-Gln solution or water. At 6 wk of age, mice were killed, and jejunal tissue was collected for morphology, immunohistochemistry, and Ussing chamber analysis of transmucosal resistance and permeability. Proliferation of MSIE cells in the presence or absence of Ala-Gln was measured by MTS and bromodeoxyuridine assays. MSIE apoptosis was assessed by annexin and 7-amino-actinomycin D staining. Pups of regional basic diet-fed dams exhibited failure to thrive. Jejunal specimens from undernourished weanlings showed decreased villous height and crypt depth, decreased transmucosal resistance, increased permeability to FITC-dextran, increased claudin-3 expression, and decreased epithelial proliferation and increased epithelial apoptosis (as measured by bromodeoxyuridine and cleaved caspase-3 staining, respectively). Undernourished weanlings supplemented with Ala-Gln showed improvements in weight velocity, villous height, crypt depth, transmucosal resistance, and epithelial proliferation/apoptosis compared with unsupplemented controls. Similarly, Ala-Gln increased proliferation and reduced apoptosis in MSIE cells. In summary, Ala-Gln promotes intestinal epithelial homeostasis in a mouse model of malnutrition-associated enteropathy, mimicking key features of the human disease.

Onecut-2 knockout mice fail to thrive during early postnatal period and have altered patterns of gene expression in small intestine.

Ablation of the mouse genes for Onecut-2 and Onecut-3 was reported previously, but characterization of the resulting knockout mice was focused on in utero development, principally embryonic development of liver and pancreas. Here we examined postnatal development of these Onecut knockout mice, especially the critical period before weaning. Onecut-3 knockout mice develop normally during this period. However, Onecut-2 knockout mice fail to thrive, lagging behind their littermates in size and weight. By postnatal day (d)19, they are consistently 25-30% smaller. Onecut-2 knockout mice also have a much higher level of mortality before weaning, with only approximately 70% survival. Interestingly, Onecut-2 knockout mice that are heterozygous for the Onecut-3 knockout allele are diminished even further in their ability to thrive. They are approximately 50-60% as large as their normal-sized littermates at d19, and less than half of these mice survive to weaning. As reported previously, the Onecut-2/Onecut-3 double knockout is a perinatal lethal. Microarray technology was used to determine the effect of Onecut-2 ablation on gene expression in duodenum, whose epithelium has among the highest levels of Onecut-2. A subset of intestinally expressed genes showed dramatically altered patterns of expression. Many of these genes encode proteins associated with the epithelial membrane, including many involved in transport and metabolism. Previously, we reported that Onecut-2 was critical to temporal regulation of the adenosine deaminase gene in duodenum. Many of the genes with altered patterns of expression in Onecut-2 knockout mouse duodenum displayed changes in the timing of gene expression.

Temporal regulation of enhancer function in intestinal epithelium: a role for Onecut factors.

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2-3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.

Cdx binding determines the timing of enhancer activation in postnatal duodenum.

In mammalian intestine, adenosine deaminase (ADA) is expressed at high levels only along the villi of the duodenal epithelium. A duodenum-specific enhancer identified in the second intron of the human ADA gene controls this pattern of expression. This enhancer faithfully recapitulates this expression pattern in transgenic mice, when included in CAT reporter gene constructions. Multiple binding sites for PDX-1 and GATA factors were previously identified within the approximately 300-bp region that encompasses the enhancer. Mutation analyses demonstrated that binding of PDX-1 and of GATA-4 was absolutely essential for enhancer function. In the present study, we have identified additional enhancer binding sites for Cdx factors, for YY1, and for NFI family members. Detailed EMSA studies were used to confirm binding at these sites. This brings the number of confirmed binding sites within the enhancer to thirteen, with five different factors or family of factors contributing to the putative enhanceosome complex. Mutation analysis was utilized to examine the specific roles of the newly identified sites. Two sites were identified that bound both Cdx1 and Cdx2. Mutations were identified in these two sites that completely and specifically eliminated Cdx binding. In transgenic mice, these enhancer mutations dramatically changed the developmental timing of enhancer activation (delaying it by 2-3 weeks) without affecting other aspects of enhancer function. In the chromatin context of certain transgenic insertion sites, mutation of the two YY1 sites to specifically ablate binding caused a delay in enhancer activation similar to that observed with the Cdx mutations. No overt changes were observed from mutation of the NFI site.

Environmental cadmium levels increase phytochelatin and glutathione in lettuce grown in a chelator-buffered nutrient solution.

Phytochelatins are enzymatically synthesized peptides involved in metal detoxification and have been measured in plants grown at very high Cd concentrations, but few studies have examined the response of plants at lower environmentally relevant Cd concentrations. Using an ethylenediaminetetraacetic acid (EDTA)-buffered nutrient medium, we have varied Cd exposure and measured phytochelatin and glutathione concentrations in romaine lettuce (Lactuca sativa L. var. longifolia Lam. var. Parris Island) grown in a flow-through hydroponic (FTH) system. Very low free ionic Cd (10(-9.6) M) increased average phytochelatin concentrations above those of controls, and increasing Cd resulted in increased phytochelatin production, though increases were tissue dependent. Glutathione concentrations also increased with increasing Cd. In other standard hydroponic experiments, the media were manipulated to vary total Cd concentration while the ionic Cd was fixed. We found that the total amount of Cd (primarily EDTA bound) in the medium altered thiol production in roots, whereas thiols in leaves remained constant. The Cd uptake into roots and translocation to old leaves was also influenced by the total concentration in the medium. Cadmium in all tissues was lower and in some tissues thiol concentrations were higher than in FTH-grown plants grown in identical medium, suggesting that nutrient delivery technique is also an important variable. Though phytochelatin and glutathione production can be sensitive to changes in bioavailable Cd, thiol concentrations will not necessarily reflect the Cd content of the plant tissues.