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Background & Aims The use of antibiotics (ABs) is a common practice during the first months of life. ABs can perturb the intestinal microbiota, indirectly influencing the intestinal epithelial cells (IECs), but can also directly affect IECs independent of the microbiota. Previous studies have focused mostly on the impact of AB treatment during adulthood. However, the difference between the adult and neonatal intestine warrants careful investigation of AB effects in early life. Methods Neonatal mice were treated with a combination of amoxicillin, vancomycin, and metronidazole from postnatal day 10 to 20. Intestinal permeability and whole-intestine gene and protein expression were analyzed. IECs were sorted by a fluorescence-activated cell sorter and their genome-wide gene expression was analyzed. Mouse fetal intestinal organoids were treated with the same AB combination and their gene and protein expression and metabolic capacity were determined. Results We found that in vivo treatment of neonatal mice led to decreased intestinal permeability and a reduced number of specialized vacuolated cells, characteristic of the neonatal period and necessary for absorption of milk macromolecules. In addition, the expression of genes typically present in the neonatal intestinal epithelium was lower, whereas the adult gene expression signature was higher. Moreover, we found altered epithelial defense and transepithelial-sensing capacity. In vitro treatment of intestinal fetal organoids with AB showed that part of the consequences observed in vivo is a result of the direct action of the ABs on IECs. Lastly, ABs reduced the metabolic capacity of intestinal fetal organoids. Conclusions Our results show that early life AB treatment induces direct and indirect effects on IECs, influencing their maturation and functioning., Graphical abstract

Birth by Caesarean (C)-section impacts early gut microbiota colonization and is associated with an increased risk of developing immune and metabolic disorders. Moreover, alterations of the microbiome have been shown to affect neurodevelopmental trajectories. However, the long-term effects of C-section on neurobehavioral processes remain unknown. Here, we demonstrated that birth by C-section results in marked but transient changes in microbiome composition in the mouse, in particular, the abundance of Bifidobacterium spp. was depleted in early life. Mice born by C-section had enduring social, cognitive, and anxiety deficits in early life and adulthood. Interestingly, we found that these specific behavioral alterations induced by the mode of birth were also partially corrected by co-housing with vaginally born mice. Finally, we showed that supplementation from birth with a Bifidobacterium breve strain, or with a dietary prebiotic mixture that stimulates the growth of bifidobacteria, reverses selective behavioral alterations in C-section mice. Taken together, our data link the gut microbiota to behavioral alterations in C-section-born mice and suggest the possibility of developing adjunctive microbiota-targeted therapies that may help to avert long-term negative consequences on behavior associated with C-section birth mode.

Knowledge about how molecular properties of proteins affect their digestion kinetics is crucial to understand protein postprandial plasma amino acid (AA) responses. Previously it was found that a native whey protein isolate (NWPI) and heat denatured whey protein isolate (DWPI) elicit comparable postprandial plasma AA peak concentrations in neonatal piglets, while a protein base ingredient for infant formula (PBI, a β-casein-native whey protein mixture) caused a 39% higher peak AA concentration than NWPI. We hypothesized that both whey protein denaturation by heat as well as changing protein composition by including β-casein, increases the rate of intact protein loss, and that changing the protein composition (by including β-casein), but not whey protein denaturation, yields a faster absorbable product release. Therefore NWPI (91% native), DWPI (91% denatured) and PBI hydrolysis was investigated in a semi-dynamic in vitro digestion model (SIM). NWPI and DWPI hydrolysis were also compared in a dynamic digestion model with dialysis (TIM-1) to exclude potential product inhibition effects that may occur in a closed vessel digestion model as SIM. In both models, the degree of hydrolysis (DH), loss of intact protein, and release of absorbable products (SIM

The postprandial plasma essential amino acid (AA) peak concentrations of infant formula (IF) are higher than those of human milk (HM) in infants. In addition, several HM proteins have been recovered intact in infant stool and appeared digestion resistant in vitro. We, therefore, hypothesized that gastrointestinal protein hydrolysis of IF is faster than HM and leads to accelerated absorbable digestion product release. HM and IF protein hydrolysis kinetics were compared in a two-step semi-dynamic in vitro infant digestion model, and the time course of degree of protein hydrolysis (DH), loss of intact protein, and release of free AA and peptides was evaluated. Gastric DH increase was similar for IF and HM, but the rate of intestinal DH increase was 1.6 times higher for IF than HM. Intact protein loss in IF was higher than HM from 120 min gastric phase until 60 min intestinal phase. Intestinal phase total digestion product (free AA + peptides

In the first 2–3 years of life, the gut microbiota of infants quickly becomes diverse and rich. Disruptions in the evolving gut microbiota during this critical developmental period can impact brain development. Communication between the microbiota, gut and brain is driven by hormonal and neural regulation, as well as immune and metabolic pathways, however, our understanding of how the parallel developments that may underlie this communication are limited. In this paper, we review the known associations between the gut microbiota and brain development and brain function in early life, speculate on the potential mechanisms involved in this complex relationship and describe how nutritional intervention can further modulate the microbiota and, ultimately, brain development and function.

Antibiotic treatment is common practice in the neonatal ward for the prevention and treatment of sepsis, which is one of the leading causes of mortality and morbidity in preterm infants. Although the effect of antibiotic treatment on microbiota development is well recognised, little attention has been paid to treatment duration. We studied the effect of short and long intravenous antibiotic administration on intestinal microbiota development in preterm infants. Faecal samples from 15 preterm infants (35 ± 1 weeks gestation and 2871 ± 260 g birth weight) exposed to no, short (≤ 3 days) or long (≥ 5 days) treatment with amoxicillin/ceftazidime were collected during the first six postnatal weeks. Microbiota composition was determined through 16S rRNA gene sequencing and by quantitative polymerase chain reaction (qPCR). Short and long antibiotic treat ment significantly lowered the abundance of Bifidobacterium right after treatment (p = 0.027) till postnatal week three (p = 0.028). Long treatment caused Bifidobacterium abundance to remain decreased till postnatal week six (p = 0.009). Antibiotic treatment was effective against members of the Enterobacteriaceae family, but allowed Enterococcus to thrive and remain dominant for up to two weeks after antibiotic treatment discontinuation. Community richness and diversity were not affected by antibiotic treatment, but were positively associated with postnatal age (p

Human milk is the optimal diet for infant development, but infant milk formula (IMF) must be available as an alternative. To develop high-quality IMF, bovine milk processing is required to ensure microbial safety and to obtain a protein composition that mimics human milk. However, processing can impact the quality of milk proteins, which can influence gastro-intestinal (GI) tolerance by changing digestion, transit time and/or absorption. The aim of this study was to evaluate the impact of structural changes of proteins due to thermal processing on gastro-intestinal tolerance in the immature GI tract. Preterm and near-term piglets received enteral nutrition based on whey protein concentrate (WPC) either mildly pasteurized (MP-WPC) or extensively heated (EH-WPC). Clinical symptoms, transit time and gastric residuals were evaluated. In addition, protein coagulation and protein composition of coagulates formed during in vitro digestion were analyzed in more detail. Characterization of MP-WPC and EH-WPC revealed that mild pasteurization maintained protein nativity and reduced aggregation of &beta, lactoglobulin and &alpha, lactalbumin, relative to EH-WPC. Mild pasteurization reduced the formation of coagulates during digestion, resulting in reduced gastric residual volume and increased intestinal tract content. In addition, preterm piglets receiving MP-WPC showed reduced mucosal bacterial adherence in the proximal small intestine. Finally, in vitro digestion studies revealed less protein coagulation and lower levels of &beta, lactalbumin in the coagulates of MP-WPC compared with EH-WPC. In conclusion, minimal heat treatment of WPC compared with extensive heating promoted GI tolerance in immature piglets, implying that minimal heated WPC could improve the GI tolerance of milk formulas in infants.

Background. The human digestive tract is structurally mature at birth, yet maturation of gut functions such as digestion and mucosal barrier continues for the next 1&ndash, 2 years. Human milk and infant milk formulas (IMF) seem to impact maturation of these gut functions differently, which is at least partially related to high temperature processing of IMF causing loss of bioactive proteins and formation of advanced glycation end products (AGEs). Both loss of protein bioactivity and formation of AGEs depend on heating temperature and time. The aim of this study was to investigate the impact of mildly pasteurized whey protein concentrate (MP-WPC) compared to extensively heated WPC (EH-WPC) on gut maturation in a piglet model hypersensitive to enteral nutrition. Methods. WPC was obtained by cold filtration and mildly pasteurized (73 &deg, C, 30 s) or extensively heat treated (73 &deg, C, 30 s + 80 &deg, C, 6 min). Preterm (~90% gestation) and near-term piglets (~96% gestation) received enteral nutrition based on MP-WPC or EH-WPC for five days. Macroscopic and histologic lesions in the gastro-intestinal tract were evaluated and intestinal responses were further assessed by RT-qPCR, immunohistochemistry and enzyme activity analysis. Results. A diet based on MP-WPC limited epithelial intestinal damage and improved colonic integrity compared to EH-WPC. MP-WPC dampened colonic IL1-&beta, IL-8 and TNF-&alpha, expression and lowered T-cell influx in both preterm and near-term piglets. Anti-microbial defense as measured by neutrophil influx in the colon was only observed in near-term piglets, correlated with histological damage and was reduced by MP-WPC. Moreover, MP-WPC stimulated iALP activity in the colonic epithelium and increased differentiation into enteroendocrine cells compared to EH-WPC. Conclusions. Compared to extensively heated WPC, a formula based on mildly pasteurized WPC limits gut inflammation and stimulates gut maturation in preterm and near-term piglets and might therefore also be beneficial for preterm and (near) term infants.

Objective: Development of the gastrointestinal tract and immune system can be modulated by the gut microbiota. Establishment of the intestinal microbiota, in its turn, is affected by host and environmental factors. As such, development of the gut microbiota is greatly impacted in preterm infants, who have an immature gut and are exposed to factors like hospitalization, caesarean section, antibiotics, and respiratory support. Design: We analyzed fecal microbiota composition and activity of ten preterm infants (gestational age 25–30 weeks; birthweight 630–1750 g) during the first six postnatal weeks through metaproteomics (LC-MS/MS) and 16S-rRNA gene sequencing. Results: A gestational-age-dependent microbial signature is observed, enabling microbiota-based differentiation between extremely preterm (25–27 weeks gestation) and very preterm (30 weeks gestation) infants. In very preterm infants, the intestinal microbiota developed toward a Bifidobacterium-dominated community and was associated with high abundance of proteins involved in carbohydrate and energy metabolism. Extremely preterm infants remained predominantly colonized by facultative anaerobes and were associated with proteins involved in membrane transport and translation. Delayed colonization by obligate anaerobes could be associated with antibiotic treatment and respiratory support. Conclusion: We speculate that gestational age and its associated intensity of care (e.g. antibiotics and respiratory support) affects intestinal microbiota composition and activity in preterm infants. As the gut microbiota plays a major role in development of the neonate, gestational age and its associated factors could set the stage for early and later life health complications via interference with microbiota development.

Background Multiple studies have indicated that formula-fed infants show a different growth trajectory compared with breastfed infants. The observed growth rates are suggested to be linked to higher postprandial levels of branched chain amino acids (BCAAs) and insulin related to differences in protein quality. Objective We evaluated the effects of milk protein denaturation and milk protein composition on postprandial plasma and hormone concentrations. Methods Neonatal piglets were bolus-fed randomly, in an incomplete crossover design, 2 of 3 milk protein solutions: native whey protein isolate (NWPI), denatured whey protein isolate (DWPI), or protein base ingredient, comprising whey and casein (PBI). Postprandial plasma amino acids (AAs), insulin, glucagon-like peptide 1, glucose, and paracetamol concentrations were assayed. Plasma responses were fitted with a model of first-order absorption with linear elimination. Results DWPI (91% denatured protein) compared with NWPI (91% native protein) showed lower essential amino acids (EAAs) (∼10%) and BCAA (13-19%) concentrations in the first 30-60 min. However, total amino acid (TAA) concentration per time-point and area under the curve (AUC), as well as EAA and BCAA AUC were not different. PBI induced a ∼30% lower postprandial insulin spike than NWPI, yet plasma TAA concentration at several time-points and AUC was higher in PBI than in NWPI. The TAA rate constant for absorption (k a) was twofold higher in PBI than in NWPI. Plasma BCAA levels from 60 to 180 min and AUC were higher in PBI than in NWPI. Plasma EAA concentrations and AUCs in PBI and NWPI were not different. Conclusions Denaturation of WPI had a minimal effect on postprandial plasma AA concentration. The differences between PBI and NWPI were partly explained by the difference in AA composition, but more likely differences in protein digestion and absorption kinetics. We conclude that modifying protein composition, but not denaturation, of milk protein solutions impacts the postprandial amino acid availability in neonatal piglets.

Background: Preterm infants are born with an immature gut, brain, and immune system, predisposing them to short- and long-term complications. Objective: We hypothesized that a milk diet supplemented with pre- and probiotics (i.e. synbiotics) and glutamine would improve gut, brain, and immune maturation in preterm neonates, using preterm pigs as a model. Methods: Preterm pigs (Landrace x Yorkshire x Duroc, n = 40, delivered by c-section at 90% of gestation) were reared individually until day 23 after birth under highly standardized conditions. Piglets in the intervention group (PPG, n = 20) were fed increasing volumes of bovine milk supplemented with prebiotics (short-chain galacto- and long chain fructo-oligosaccharides 9:1, 4-12 g/L), probiotics (Bifidobacterium breve M16-V, 3 × 109 CFU/d) and l-glutamine [0.15-0.30 g/(kg · d)], and compared with pigs fed bovine milk with added placebo compounds as control (CON, n = 20). Clinical, gastrointestinal, immunological, cognitive, and neurological endpoints were measured. Results: The PPG pigs showed more diarrhea but weight gain, body composition, and gut parameters were similar between the groups. Cognitive performance, assessed in a T-maze, was significantly higher in PPG pigs (P

During the suckling‐to‐weaning transition, the intestinal epithelium matures, allowing digestion of solid food. Transplantation experiments with rodent fetal epithelium into subcutaneous tissue of adult animals suggest that this transition is intrinsically programmed and occurs in the absence of dietary or hormonal signals. Here, we show that organoids derived from mouse primary fetal intestinal epithelial cells express markers of late fetal and neonatal development. In a stable culture medium, these fetal epithelium‐derived organoids lose all markers of neonatal epithelium and start expressing hallmarks of adult epithelium in a time frame that mirrors epithelial maturation in vivo . In vitro postnatal development of the fetal‐derived organoids accelerates by dexamethasone, a drug used to accelerate intestinal maturation in vivo . Together, our data show that organoids derived from fetal epithelium undergo suckling‐to‐weaning transition, that the speed of maturation can be modulated, and that fetal organoids can be used to model the molecular mechanisms of postnatal epithelial maturation.

Background: Our aims were (1) to correlate changes in the microbiota to intestinal gene expression before and during the development of colitis in Muc2(-/-) mice and (2) to investigate whether the heterozygote Muc2(+/-) mouse would reveal host markers of gut barrier stress. Methods: Colon histology, transcriptomics, and microbiota profiling of faecal samples was performed on wild type, Muc2(+/-), and Muc2(-/-) mice at 2, 4, and 8 weeks of age. Results: Muc2(-/-) mice develop colitis in proximal colon after weaning, resulting in inflammatory and adaptive immune responses, and expression of genes associated with human inflammatory bowel disease. Muc2(+/-) mice do not develop colitis, but produce a thinner mucus layer. The transcriptome of Muc2(+/-) mice revealed differential expression of genes participating in mucosal stress responses and exacerbation of a transient inflammatory state around the time of weaning. Young wild type and Muc2(+/-) mice have a more constrained group of bacteria as compared with the Muc2(-/-) mice, but at 8 weeks the microbiota composition is more similar in all mice. At all ages, microbiota composition discriminated the groups of mice according to their genotype. Specific bacterial clusters correlated with altered gene expression responses to stress and bacteria, before colitis development, including colitogenic members of the genus Bacteroides. Conclusions: The abundance of Bacteroides pathobionts increased before histological signs of pathology suggesting they may play a role in triggering the development of colitis. The Muc2(+/-) mouse produces a thinner mucus layer and can be used to study mucus barrier stress in the absence of colitis.

Microbial colonization of the gastrointestinal tract is an essential process that modulates host physiology and immunity. Recently, researchers have begun to understand how and when these microorganisms colonize the gut and the early-life factors that impact their natural ecological establishment. The vertical transmission of maternal microbes to the offspring is a critical factor for host immune and metabolic development. Increasing evidence also points to a role in the wiring of the gut-brain axis. This process may be altered by various factors such as mode of delivery, gestational age at birth, the use of antibiotics in early life, infant feeding, and hygiene practices. In fac(t), these early exposures that impact the intestinal microbiota have been associated with the development of diseases such as obesity, type 1 diabetes, asthma, allergies, and even neurodevelopmental disorders. The present review summarizes the impact of cesarean birth on the gut microbiome and the health status of the developing infant and discusses possible preventative and restorative strategies to compensate for early-life microbial perturbations.

During human embryonic and fetal development of the gastrointestinal tract, the gene encoding the MUC5AC mucin has a spatio-temporal pattern of expression restricted to the stomach. In order to better understand the molecular mechanisms responsible for this restricted pattern of expression, we have studied Muc5ac expression in the developing stomach of the mouse and correlated it to that of transcription factors known to be involved in cell differentiation programs during development. Our results indicate that GATA-6 and HNF-4α expression increased concomitantly with the induction of Muc5ac expression in embryonic stomach. We then studied Muc5ac transcriptional regulation by these transcription factors and showed that they all transactivate Muc5ac promoter. We also identified several active GATA-4/-5/-6 and HNF-1/-4 cis-elements using gel shift assays, chromatin immunoprecipitation and site-directed mutagenesis. Among all Muc5ac regulators, only GATA-6 and HNF-4a expression was concomitant to that of Muc5ac in the developing stomach. This is thus in favor of an important role for these two transcription factors as regulators of expression of the Muc5ac mucin during stomach development and in epithelial cancer cells.

Threonine is an essential amino acid necessary for synthesis of intestinal (glyco)proteins such as mucin MUC2 to maintain adequate gut barrier function. In premature infants, reduced barrier function may contribute to the development of necrotizing enterocolitis (NEC). Human milk protects against NEC compared with infant formula. Therefore, we hypothesized that formula feeding decreases the MUC2 synthesis rate concomitant with a decrease in intestinal first-pass threonine utilization, predisposing the preterm neonate to NEC. Preterm pigs were delivered by caesarian section and received enteral feeding with formula (FORM; n = 13) or bovine colostrum (COL; n = 6) for 2 d following 48 h of total parenteral nutrition. Pigs received a dual stable isotope tracer infusion of threonine to determine intestinal threonine kinetics. NEC developed in 38% of the FORM pigs, whereas none of the COL pigs were affected (P = 0.13). Intestinal fractional first-pass threonine utilization was lower in FORM pigs (49 6 2%) than in COL pigs (60 6 4%) (P = 0.02). In FORM pigs compared with COL pigs, protein synthesis (369 6 31 mg·kg 21 ·d 21 vs. 615 6 54 mg·kg 21 ·d 21 ; P = 0.003) and MUC2 synthesis (121 6 17%/d vs. 184 6 15%/d; P = 0.02) were lower in the distal small intestine (SI). Our results suggest that formula feeding compared with colostrum feeding in preterm piglets reduces mucosal growth with a concomitant decrease in first-pass splanchnic threonine utilization, protein synthesis, and MUC2 synthesis in the distal SI. Hence, decreased intestinal threonine metabolism and subsequently impaired gut barrier function may predispose the formula-fed infant to developing NEC. J. Nutr. 141: 1306‐1311, 2011.

Paneth cell dysfunction has been suggested in necrotizing enterocolitis (NEC). The aim of this study was to i) study Paneth cell presence, protein expression, and developmental changes in preterm infants with NEC and ii) determine Paneth cell products and antimicrobial capacity in ileostomy outflow fluid. Intestinal tissue from NEC patients (n = 55), preterm control infants (n = 22), and term controls (n = 7) was obtained during surgical resection and at stoma closure after recovery. Paneth cell abundance and protein expression were analyzed by immunohistochernistry. RNA levels of Paneth cell proteins were determined by real-time quantitative RTPCR. In ileostomy outflow fluid, Paneth cell products were quantified, and antimicrobial activity was measured in vitro. In acute NEC, Paneth cell abundance in small intestinal tissue was not significantly different from preterm controls. After recovery from NEC, Paneth cell hyperplasia was observed in the small intestine concomitant with elevated human alpha-defensin 5 mRNA levels. In the colon, metaplastic Paneth cells were observed. Ileostomy fluid contained Paneth cell proteins and inhibited bacterial growth. In conjunction, these data suggest an important role of Paneth cells and their products in various phases of NEC. (Pediatr Res 69: 217-223, 2011)

INTRODUCTION: The colonic mucus layer plays an important role in the protection of the intestinal epithelium and mainly consists of mucin glycoproteins (primarily MUC2 in the colon) trefoil factor 3 (TFF3) and secretory IgA. Butyrate is a major end product of fermentation of dietary fibres and is associated with beneficial effects on colonic health. Earlier in-vitro and animal studies showed that butyrate modulates MUC2 and TFF3 expression and mucin secretion, although data from human studies are not yet available. Methods: Sixteen healthy volunteers and 35 ulcerative colitis (UC) patients in clinical remission self-administered a 60'ml rectal enema containing 100'mmol/l butyrate or placebo once daily for 2 and 3 weeks, respectively. After each treatment, biopsies were taken from the distal sigmoid for quantitative RT-PCR and immunohistochemical analysis of MUC2 and TFF3. In addition, mucosal sections were stained with high iron diamine-alcian blue to distinguish between sialomucins and sulphomucins. To analyse total mucin secretion and secretory IgA concentrations, 24'h faeces were collected during the day before the endoscopic examination. Results: The butyrate intervention did not significantly modulate the expression of MUC2 (fold change: 1.04 and 1.05 in healthy volunteers and ulcerative colitis patients, respectively) or TFF3 (fold change: 0.91 and 0.94 in healthy volunteers and UC patients, respectively). Furthermore, the percentage of sialomucins, mucus secretion and secretory IgA concentrations were not affected by the butyrate intervention in both the groups. Conclusion: Butyrate exposure in healthy volunteers and UC patients in remission did not affect the measured parameters of the colonic mucus layer. Copyright © 2010 Lippincott Williams & Wilkins.

Schaart MW, de Bruijn ACJM, Schierbeek H, Tibboel D, Renes IB, van Goudoever JB. Small intestinal MUC2 synthesis in human preterm infants. Am J Physiol Gastrointest Liver Physiol 296: G1085-G1090, 2009. First published February 26, 2009; doi:10.1152/ajpgi.90444.2008.-Mucin2 (MUC2) is the structural component of the intestinal protective mucus layer, which contains high amounts of threonine in its peptide backbone. MUC2 synthesis rate might be a potential parameter for intestinal barrier function. In this study, we aimed to determine whether systemic threonine was used for small intestinal MUC2 synthesis and to calculate the MUC2 fractional synthetic rate (FSR) in human preterm infants. Seven preterm infants with an enterostomy following bowel resection for necrotizing enterocolitis received intravenous infusion of [U-(13)C] threonine to determine incorporation of systemic threonine into secreted MUC2 in intestinal outflow fluid. Small intestinal MUC2 was isolated using cesium chloride gradient ultracentrifugation and gravity gel filtration chromatography. MUC2-containing fractions were identified by SDS-PAGE/periodic acid-Schiff staining and Western blot analysis and were subsequently pooled. Isotopic enrichment of threonine, measured in MUC2 using gas chromatography isotopic ratio mass spectrometry, was used to calculate the FSR of MUC2. Systemically derived threonine was indeed incorporated into small intestinal MUC2. Median FSR of small intestinal MUC2 was 67.2 (44.3-103.9)% per day. Systemic threonine is rapidly incorporated into MUC2 in the small intestine of preterm infants, and thereby MUC2 has a very high synthesis rate.

Objectives: Information on epithelial functions of the residual small or colonic bowel after resection for necrotising enterocolitis (NEC) in human infants is scarce. Our aim is to evaluate epithelial functions in the intestinal resection margins of tissue obtained at bowel resection for acute NEC and consecutive stoma closure. Materials and Methods: Epithelial morphology, proliferation, and protein expression were (immuno) histochemically studied. Results: Acute NEC was associated with severe and mild epithelial damage varying from epithelial loss to fairly unaffected epithelium. Epithelial proliferation was increased both at acute NEC and at stoma closure. In acute NEC, lactase, glucose transporter-2 and -5 expression was downregulated in severely affected epithelium, whereas sucraseisomaltase and intestinal fatty acid binding protein expression was maintained. Goblet cells continued to express mucin 2 and trefoil factor 3, however, their numbers were decreased. Moreover, in acute NEC, Paneth cells were weakly lysozyme positive and were reduced in number. At stoma closure, expression of the above cell type-specific markers had completely been re-established. Conclusions: Residual bowel after resection for acute NEC shows a disturbed epithelial proliferation/differentiation balance. Acute NEC was associated with downregulation of distinct enterocyte-specific proteins. Because of goblet cell and Paneth cell loss in acute NEC, mucosal barrier, and defense functions may be impaired. JPGN 49:31-41, 2009.

The intestine serves numerous purposes. In addition to digestion and absorption, the intestine functions as an organ that provides specific and nonspecific protection against pathological bacteria and noxious agents. At birth, and certainly when birth occurs prematurely, these functions are not yet fully developed. This article addresses the specific needs of the neonatal gut to perform these functions adequately and describes efforts to modulate intestinal barrier function by preterm formula supplemented with insulin-like growth factor 1.

International audience; The human gene MUC4 encodes a large transmembrane mucin that is developmentally regulated and expressed along the undifferentiated pseudostratified epithelium, as early as 6.5 weeks during fetal development. Immunohistochemical analysis of Muc4 expression in developing mouse lung and gastroin-testinal tract showed a different spatio-temporal pattern of expression before and after cytodifferentiation. The molecular mechanisms governing MUC4 expression during development are, however, unknown. Hepatocyte nuclear factors (HNF), forkhead box A (FOXA), GATA, and caudal-related homeobox transcription factors (TFs) are known to control cell differentiation of gut endoderm derived-tissues during embryonic development. They also control the expression of cell-and tissue-specific genes and may thus control MUC4 expression. To test this hypothesis, we studied and deciphered the molecular mechanisms responsible for MUC4 transcriptional regulation by these TFs. Experiments using small interfering RNA, cell co-transfection, and site-directed mutagenesis indicated that MUC4 is regulated at the transcriptional level by CDX-1 and-2, HNF-1␣ and-1␤, FOXA1/A2, HNF-4␣ and-4␥, and GATA-4,-5, and-6 factors in a cell-specific manner. Binding of TFs was assessed by chromatin immunoprecipitation, and gel-shift assays. Altogether, these results demonstrate that MUC4 is a target gene of endodermal TFs and thus point out an important role for these TFs in regulating MUC4 expression during epithelial differentiation during development, cancer, and repair.

Background: The intestine plays a key role in the absorption of dietary proteins, which determines growth of human neonates. Bowel resection in the neonatal period brings loss of absorptive and protective surface and may consequently lead to malabsorption of dietary nutrients. However, there are no data on net dietary protein absorption of the small intestine in the period after intestinal surgery in human neonates. We therefore evaluated dietary feeding tolerance and quantified net dietary protein absorption capacity of the small intestine in human neonates in whom a temporary jejunostomy or ileostomy was created. Methods: Seventeen patients were included in the study. We collected small intestinal outflow fluid at the level of the enterostomy weekly for 24-48 hours during weeks 3 through 6 postoperatively. Protein levels in the intestinal outflow fluid were determined by bicinchoninic acid (BCA) assay. Results: In 14 patients, an enteral intake of >100 mL/kg/d was reached at a median of 17 days (range, 8-32 days) postoperatively. Three patients did not reach this level within the study period. Overall, the net dietary protein absorption capacity was 70%-90% of the total enteral protein intake. Conclusions: This study demonstrates that the dietary protein absorption capacity of the small intestine is intact in most human neonates after intestinal surgery in a very critical period of their lives. Furthermore, our results do not support the use of hydrolyzed or elemental formula in newborns with an enterostomy to improve amino acid uptake.

Preterm birth interrupts normal fetal growth with consequences for postnatal growth and organ development. In preterm infants, many physiological deficits adapt and disappear with advancing postnatal age, but some may persist into childhood. We hypothesized that preterm birth would induce impaired organ growth and function during the first postnatal week in pigs, while motor abilities and behavioral characteristics would show more persistent developmental delay. Cesarean-delivered preterm ( n = 112, 90% gestation) or term ( n = 56, 100% gestation) piglets were reared under identical conditions and euthanized for blood and organ collection on postnatal days 0, 5, or 26. Body weight gain remained lower in preterm vs. term pigs up to day 26 (25.5 ± 1.5 vs. 31.0 ± 0.5 g·kg−1·day−1, P < 0.01) when relative weights were higher for brain and kidneys and reduced for liver and spleen. Neonatal preterm pigs had reduced values for blood pH, Po2, glucose, lactate, hematocrit, and cortisol, but at day 26, most values were normalized, although plasma serotonin and IGF 1 levels remained reduced. Preterm pigs showed delayed neonatal arousal and impaired physical activity, coordination, exploration, and learning, relative to term pigs (all P < 0.05). Supplementation of parenteral nutrition during the first 5 days with an enteral milk diet did not affect later outcomes. In preterm pigs, many physiological characteristics of immaturity disappeared by 4 wk, while some neurodevelopmental deficits remained. The preterm pig is a relevant animal model to study early dietary and pharmacological interventions that support postnatal maturation and neurodevelopment in preterm infants.

Background:Muc2-deficient mice show no signs of ileal pathology but the mechanisms remained unknown.Methods:Wild-type (WT), Muc2(+/-), and Muc2(-/-) mice were killed at 2, 4, and 8 weeks of age. Total RNA from ileum was used for full genome transcriptome analysis and qPCR. Microbiota composition was determined using a mouse intestinal chip (MITChip). Morphological and immunohistological studies were performed on segments of ileum.Results:The ileum was colonized by more diverse microbiota in young (week 4) WT than in Muc2(-/-) mice, and composition was influenced by genotype. Weaning was associated with major changes in the transcriptome of all mice, and the highest number of differentially expressed genes compared with adults, reflecting temporal changes in microbiota. Although the spatial compartmentalization of bacteria was compromised in Muc2(-/-) mice, gene set enrichment analysis revealed a downregulation of Toll-like receptor, immune, and chemokine signaling pathways compared to WT mice. The predicted effects of enhanced IL-22 signaling were identified in the Muc2(-/-) transcriptome as the upregulation of epithelial cell proliferation altered expression of mitosis and cell-cycle control pathways. This is consistent with increased villus length and number of Ki67(+) epithelial cells in Muc2(-/-) mice. Additionally, expression of the network of IL-22 regulated defense genes, including Fut2, Reg3, Reg3, Relmb, and the Defensin Defb46 were increased in Muc2(-/-) mice.Conclusions:These findings highlight a role for the IL-22-STAT3 pathway in maintaining ileal homeostasis when the mucus barrier is compromised and its potential as a target for novel therapeutic strategies in inflammatory bowel disease.

Objectives: Bowel segments distal to a congenital intestinal obstruction have been suggested to be immature. In other words, luminal components such as amniotic fluid (before birth) and/or enteral nutrition (after birth) may be required to activate intestinal epithelial protein expression, thereby influencing epithelial differentiation. We investigated cell-type–specific protein expression proximal and distal to jejunal and ileal atresias in human newborns. Patients and Methods: We immunohistochemically studied intestinal tissue specimens of 16 newborns who had undergone surgery for jejunal or ileal atresia. Sections were taken from both the proximal and distal sides of the atresias. Results: For all patients, the enterocyte-specific markers lactase, sucrase-isomaltase, sodium glucose cotransporter 1, glucose transporters 2 and 5, intestinal fatty acid–binding protein and alkaline phosphatase were expressed at a mean 3 ± 1 days after birth, both proximal and distal to jejunal and ileal atresias. Expression of goblet cell–specific markers mucin 2 and trefoil factor 3 and that of the Paneth cell marker lysozyme was maintained at either side of the atretic segment. Conclusions: With respect to the markers used, the human small intestinal epithelium is already differentiated shortly after birth. The absence of intestinal continuity in case of a jejunal or ileal atresia does not affect epithelial protein expression. This would seem to indicate that the developing small intestinal epithelium matures independently of luminal components.

Background & Aims: Expression of mucin MUC2, the structural component of the colonic mucus layer, is lowered in inflammatory bowel disease. Our aim was to obtain insight in the role of Muc2 in epithelial protection. Methods: Muc2 knockout (Muc2−/−) and Muc2 heterozygous (Muc2+/−) mice were characterized and challenged by a colitis-inducing agent, dextran sulfate sodium (DSS). We monitored clinical symptoms, intestinal morphology, and differences in intestine-specific protein and messenger RNA levels. Results: The Muc2−/− mice showed clinical signs of colitis (as of 5 weeks), aggravating as the mice aged. Microscopic analysis of the colon of Muc2−/− mice showed mucosal thickening, increased proliferation, and superficial erosions. Colonic goblet cells in the Muc2−/− mice were negative for Muc2, but trefoil factor 3 was still detectable. In Muc2−/− mice, transient de novo expression of Muc6 messenger RNA was observed in the distal colon. On day 2 of DSS treatment, the histologic damage was more severe in Muc2+/− versus wild-type (Muc2+/+) mice, but the disease activity index was not yet different. By day 7, the disease activity index and histologic score were significantly elevated in Muc2+/− versus Muc2+/+ mice. The disease activity index of the Muc2−/− mice was higher (versus both Muc2+/+ and Muc2+/− mice) throughout DSS treatment. The histologic damage in the DSS-treated Muc2−/− mice was different compared with Muc2+/+ and Muc2+/− mice, with many crypt abscesses instead of mucosal ulcerations. Conclusions: This study shows that Muc2 deficiency leads to inflammation of the colon and contributes to the onset and perpetuation of experimental colitis.

Chemotherapy-induced intestinal damage is a very important dose-limiting side effect for which there is no definitive prophylaxis or treatment. This is in part due to the lack of understanding of its pathophysiology and impact on intestinal differentiation. The objective of this study was to investigate the gene expression of the small intestinal transcription factors HNF-1 alpha, Cdx2, GATA-4 in an experimental model of methotrexate (MTX)-induced intestinal damage, and to correlate these alterations with histological damage, epithelial proliferation and differentiation. HNF-1 alpha, Cdx2 and GATA-4 are critical transcription factors in epithelial differentiation, and in combination they act as promoting factors of the sucrase-isomaltase (SI) gene, an enterocyte-specific differentiation marker which is distinctly downregulated after MTX treatment. Mice received two doses of MTX i.v. on two consecutive days and were sacrificed 1, 3 and 7 or 9 days after final injection. Segments of the jejunum were taken for morphological, immunohistochemical and quantitative analyses. Intestinal damage was most severe at day 3 and was associated with decreased expression of the transcriptional factors HNF-1 alpha, Cdx2 and GATA-4, which correlated well with decreased expression of SI, and seemed inversely correlated with enhanced proliferation of epithelial crypt cells. During severe damage, the epithelium was preferentially concerned with proliferation rather than differentiation, most likely in order to restore the small intestinal barrier function rather than maintaining its absorptive function. Since HNF-1 alpha, Cdx2 and GATA-4 are critical for intestine-specific gene expression and therefore crucial in epithelial differentiation, these results may explain, at least in part, why intestinal differentiation is compromised during MTX treatment.

The crypt and villus epithelium associated with Peyer's patches (PPs) is largely spared from methotrexate (MTX)-induced damage, compared with the non-patch (NP) epithelium. To assess the mechanism(s) preventing damage to the PP epithelium after MTX treatment, epithelial proliferation, apoptosis, and cell functions were studied in a rat-MTX model. Small intestinal segments containing PPs were excised after MTX treatment. Epithelial proliferation and apoptosis were assessed by detection of incorporated BrdU and cleaved caspase-3, respectively. Epithelial functions were determined by the expression of cell type-specific gene products at mRNA and protein level. Before and after MTX treatment, the number of BrdU-positive cells was higher in PP crypts than in NP crypts. BrdU incorporation was diminished in NP crypts, while in PP crypts incorporation was hardly affected. In PP and NP crypts, similar and increased levels of cleaved caspase-3-positive cells were observed after MTX. The enterocyte markers, sucrase-isomaltase, sodium-glucose co-transporter 1, glucose transporters 2 and 5, and intestinal and liver fatty acid binding protein, were down-regulated after MTX in NP epithelium but not in PP epithelium. In contrast, expression of the goblet cell markers, Muc2 and trefoil factor 3, and the Paneth cell marker, lysozyme, was maintained after MTX in both PP and NP epithelium. In conclusion, as MTX-induced apoptosis was similar in PP and NP crypts, the protection of the PP epithelium seems to be based on differences in the regulation of epithelial proliferation. Enterocyte function in the PP epithelium was unaffected by MTX treatment. Goblet and Paneth cell function was maintained in both NP and PP epithelium.

Background and aims. To gain insight in intestinal epithelial proliferation, cell death, and gene expression during experimental colitis rats were treated with dextran sulfate sodium (DSS) for 7 days. Materials and methods. Proximal and distal colonic segments were excised on days 2, 5, 7, and 28. Epithelial proliferation, cell death, enterocyte gene expression (carbonic anhydrase I (CA I) and goblet cell gene expression (mucin, MUC2; trefoil factor 3, TFF3) were studied immunohistochemically and biochemically. Results. Proliferative activity was decreased in the proximal and distal colon at the onset of disease (day 2). However, during active disease (days 5–7) epithelial proliferation was increased in the entire proximal colon and in the proximity of ulcerations in the distal colon. During DSS treatment the number of apoptotic cells in the epithelium of both colonic segments was increased. In the entire colon surface enterocytes became flattened and CA I negative during active disease (day 5–7). Additionally, CA I levels in the distal colon significantly decreased during this phase. In contrast, during the regenerative phase (day 28) CA I levels were restored in the distal colon and up-regulated in the proximal colon. During all disease phases increased numbers of goblet cells were observed in the surface epithelium of the entire colon. In the distal colon TFF3 expression extended to the bottom of the crypts during active disease. Finally, MUC2 and TFF3 expression was increased in the proximal colon during disease. Conclusion. DSS affected the epithelium by inhibiting proliferation and inducing apoptosis. DSS-induced inhibition of CA I expression indicates down-regulation of specific enterocyte functions. Accumulation of goblet cells in the surface epithelium and up-regulation of MUC2 and TFF3 expression in the proximal colon underline the importance of goblet cells in epithelial protection and repair, respectively.

SUMMARY The rapidly dividing small intestinal epithelium is very sensitive to the cytostatic drug methotrexate. We investigated the regulation of epithelial gene expression in rat jejunum during methotrexate-induced damage and regeneration. Ten differentiation markers were localized on tissue sections and quantified at mRNA and protein levels relative to control levels. We analyzed correlations in temporal expression patterns between markers. mRNA expression of enterocyte and goblet cell markers decreased significantly during damage for a specific period. Of these, sucrase-isomaltase ( � 62%) and CPS ( � 82%) were correlated. Correlations were also found between lactase ( � 76%) and SGLT1 ( � 77%) and between I-FABP ( � 52%) and L-FABP ( � 45%). Decreases in GLUT5 ( � 53%), MUC2 ( � 43%), and TFF3 ( � 54%) mRNAs occurred independently of any of the other markers. In contrast, lysozyme mRNA present in Paneth cells increased ( � 76%). At the protein level, qualitative and quantitative changes were in agreement with mRNA expression, except for Muc2 ( � 115%) and TFF3 ( � 81%), which increased significantly during damage, following independent patterns. During regeneration, expression of each marker returned to control levels. The enhanced expression of cytoprotective molecules (Muc2, TFF3, lysozyme) during damage represents maintenance of goblet cell and Paneth cell functions, most likely to protect the epithelium. Decreased expression of enterocyte-specific markers represents decreased enterocyte function, of which fatty acid transporters were least affected.

Background An infant formula that contained milk fermented by the bacteria Bifidobacterium breve and Streptococcus thermophilus (Lactofidus) was reported to alleviate functional digestive symptoms in infants. It was hypothesized that improved protein digestibility of the fermented infant formula could contribute to this effect. Objective The aim of this study was to evaluate the protein digestibility of a specific fermented (FF), a standard (SF), and an extensively hydrolyzed protein (HF) formula. Methods Four-week-old piglets (n = 7) were fitted with a T-cannula at the terminal ileum and received each formula in a Latin square design. FF, SF, and HF contained 11.7%, 9.3%, and 11.9% (w/w) crude protein; 1.5%, 5.4%, and 5.6% (w/w) fiber; and had a casein/whey ratio of 60:40, 50:50, and 0:100 per kilogram of powder, respectively. Ileal digesta were collected and analyzed for amino acids and proteolytic activity. Results FF had a significantly higher apparent ileal crude protein digestibility (92.1% ± 1.0%) than SF and HF (84.4% ± 1.0% and 83.9% ± 0.9%, respectively). FF also had a significantly higher dry matter digestibility than SF and HF. The ileal crude protein flow of FF was significantly lower than that of SF and HF. The ileal flow of FF total proteolytic activity was significantly lower than that of SF but not significantly different from that of HF (412 ± 163 kU/8 h vs. 1530 ± 163 and 703 ± 156 kU/8 h, respectively). Conclusions The FF in piglets had a significantly higher apparent ileal crude protein digestibility than the SF and HF and displayed lower ileal proteolytic activity than the SF. Both effects may contribute to the alleviation of functional gastrointestinal symptoms reported in infants fed fermented infant milk formula.

Tumor recurrence and outgrowth of metastases limit the therapeutical effect of radiotherapy. We have tested whether these problems can be overcome by supplementing radiotherapy with locoregional interleukin-2 (IL-2) treatment. The SL2 lymphoma and the M8013 mammary carcinoma were used. Mice bearing a 10-day-old s.c. tumor were locally irradiated and were treated daily with IL-2 peritumorally for 5 or 10 days. Low-dose IL-2 therapy improved local response (LR) and increased disease-free survival (DFS) in both tumor models following either single-dose irradiation or fractionated irradiation. For example, 93% of SL2-bearing mice treated with single-dose irradiation and 10 days of IL-2 experienced long-term DFS, compared with 17% for irradiation alone (p < 0.0001). Additionally, treatment of one tumor with irradiation +IL-2 led to anti-tumor effects in a second, untreated tumor in 80% of SL2-bearing mice. LR was increased to 100% and DFS to 70% when the second, non-irradiated tumor was also treated with peritumoral IL-2. We conclude that supplementing local radiotherapy with low doses of IL-2 results in increased local tumor control and regression of distant, non-irradiated tumors. This type of radioimmunotherapy is a promising new approach for the clinic. Int. J. Cancer 72:1003–1007, 1997. © 1997 Wiley-Liss, Inc.

textabstractBackground: Palmitic-acid esterified to the sn-1,3 positions of the glycerol backbone (alpha, alpha'-palmitate), the predominant palmitate conformation in regular infant formula fat, is poorly absorbed and might cause abdominal discomfort. In contrast, palmitic-acid esterified to the sn-2 position (beta-palmitate), the main palmitate conformation in human milk fat, is well absorbed. The aim of the present study was to examine the influence of high alpha, alpha'-palmitate fat (HAPF) diet and high beta-palmitate fat (HBPF) diet on colitis development in Muc2 deficient (Muc2-/-) mice, a well-described animal model for spontaneous enterocolitis due to the lack of a protective mucus layer. Methods: Muc2-/- mice received AIN-93G reference diet, HAPF diet or HBPF diet for 5 weeks after weaning. Clinical symptoms, intestinal morphology and inflammation in the distal colon were analyzed. Results: Both HBPF diet and AIN-93G diet limited the extent of intestinal erosions and morphological damage in Muc2-/- mice compared with HAPF diet. In addition, the immunosuppressive regulatory T (Treg) cell response as demonstrated by the up-regulation of Foxp3, Tgfb1 and Ebi3 gene expression levels was enhanced by HBPF diet compared with AIN-93G and HAPF diets. HBPF diet also increased the gene expression of Pparg and enzymatic antioxidants (Sod1, Sod3 and Gpx1), genes all reported to be involved in promoting an immunosuppressive Treg cell response and to protect against colitis. Conclusions: This study shows for the first time that HBPF diet limits the intestinal mucosal damage and controls the inflammatory response in Muc2-/- mice by inducing an immunosuppressive Treg cell response.

textabstractBackground:Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients.Methods:Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot.Results:Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression.Conclusions:XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury.

We examined whether changes in the gut microbiota induced by clinically relevant interventions would impact the bioavailability of dietary amino acids in neonates. We tested the hypothesis that modulation of the gut microbiota in neonatal pigs receiving no treatment (control), intravenously administered antibiotics, or probiotics affects whole body nitrogen and amino acid turnover. We quantified whole body urea kinetics, threonine fluxes, and threonine disposal into protein, oxidation, and tissue protein synthesis with stable isotope techniques. Compared with controls, antibiotics reduced the number and diversity of bacterial species in the distal small intestine (SI) and colon. Antibiotics decreased plasma urea concentrations via decreased urea synthesis. Antibiotics elevated threonine plasma concentrations and turnover, as well as whole body protein synthesis and proteolysis. Antibiotics decreased protein synthesis rate in the proximal SI and liver but did not affect the distal SI, colon, or muscle. Probiotics induced a bifidogenic microbiota and decreased plasma urea concentrations but did not affect whole body threonine or protein metabolism. Probiotics decreased protein synthesis in the proximal SI but not in other tissues. In conclusion, modulation of the gut microbiota by antibiotics and probiotics reduced hepatic ureagenesis and intestinal protein synthesis, but neither altered whole body net threonine balance. These findings suggest that changes in amino acid and nitrogen metabolism resulting from antibiotic- or probiotic-induced shifts in the microbiota are localized to the gut and liver and have limited impact on whole body growth and anabolism in neonatal piglets.

Objective Colonic ischaemia is frequently observed in clinical practice. This study provides a novel insight into the pathophysiology of colon ischaemia/reperfusion (IR) using a newly developed human and rat experimental model. Design In 10 patients a small part of colon that had to be removed for surgical reasons was isolated and exposed to 60 min of ischaemia (60I) with/without different periods of reperfusion (30R and 60R). Tissue not exposed to IR served as control. In rats, colon was exposed to 60I, 60I/30R, 60I/120R or 60I/240R (n=7 per group). The tissue was snap-frozen or fixed in glutaraldehyde, formalin or methacarn fixative. Mucins were stained with Periodic Acid Schiff/Alcian Blue (PAS/AB) and MUC2/Dolichos biflorus agglutinin (DBA). Bacteria were studied using electron microscopy (EM) and fluorescent in situ hybridisation (FISH). Neutrophils were studied using myeloperoxidase staining. qPCR was performed for MUC2, interleukin (IL)-6, IL-1β and tumour necrosis factor α. Results In rats, PAS/AB and MUC2/DBA staining revealed mucus layer detachment at ischaemia which was accompanied by bacterial penetration (in EM and FISH). Human and rat studies showed that, simultaneously, goblet cell secretory activity increased. This was associated with expulsion of bacteria from the crypts and restoration of the mucus layer at 240 min of reperfusion. Inflammation was limited to minor influx of neutrophils and increased expression of proinflammatory cytokines during reperfusion. Conclusions Colonic ischaemia leads to disruption of the mucus layer facilitating bacterial penetration. This is rapidly counteracted by increased secretory activity of goblet cells, leading to expulsion of bacteria from the crypts as well as restoration of the mucus barrier.

International audience; Background: Mucin Muc2 is the structural component of the intestinal mucus layer. Absence of Muc2 leads to loss of this layer allowing direct bacterial-epithelial interactions. We hypothesized that absence of the mucus layer leads to increased expression of innate defense peptides. Specifically, we aimed to study the consequence of Muc2 deficiency (Muc2−/−) on the expression of regenerating islet-derived protein 3 beta (Reg3β), regenerating islet-derived protein 3 gamma (Reg3γ), and angiogenin-4 (Ang4) in the intestine shortly before and after weaning.Methods: Intestinal tissues of Muc2−/− and wild-type (WT) mice were collected at postnatal day 14 (P14, i.e. pre-weaning) and P28 (i.e. post-weaning). Reg3β, Reg3γ, and Ang4 expression was studied by quantitative real-time PCR, Western-blot, in situ hybridization, and immunohistochemistry.Results: Reg3β and Reg3γ were expressed by diverging epithelial cell types; namely enterocytes, Paneth cells, and goblet cells. Additionally, Ang4 expression was confined to Paneth cells and goblet cells. Expression of Reg3β, Reg3γ, and Ang4 differed between WT and Muc2−/− mice before and after weaning. Interestingly, absence of Muc2 strongly increased Reg3β and Reg3γ expression in the small intestine and colon. Finally, morphological signs of colitis were only observed in the distal colon of Muc2−/− mice at P28, where and when expression levels of Reg3β, Reg3γ, and Ang4 were the lowest.Conclusions: Expression of Reg3 proteins and Ang4 by goblet cells point to an important role for goblet cells in innate defense. Absence of Muc2 results in up-regulation of Reg3β and Reg3γ expression, suggesting altered bacterial-epithelial signaling and an innate defense response in Muc2−/− mice. The inverse correlation between colitis development and Reg3β, Reg3γ, and Ang4 expression levels might point toward a role for these innate defense peptides in regulating intestinal inflammation.

The mucin Muc2 is the structural component of the colonic mucus layer. Adult Muc2 knockout (Muc2−/−) mice suffer from severe colitis. We hypothesized that Muc2 deficiency induces inflammation before weaning of mother's milk [ postnatal day (P) 14] with aggravation of colitis after weaning (P28). Muc2−/−and wild-type mice were killed at embryonic day 18.5 and P1.5, P7.5, P14, P21, and P28. Colonic morphology, influx of T cells, and goblet cell-specific protein expression was investigated by (immuno)histochemistry. Cytokine and Toll-like receptor (TLR) profiles in the colon were analyzed by quantitative RT-PCR. Muc2−/−mice showed an increased and persistent influx of Cd3ε-positive T cells in the colonic mucosa as of P1.5. This was accompanied by mucosal damage at P28 in the distal colon but not in the proximal colon. At P14, the proinflammatory immune response [i.e., increased interleukin (IL)-12 p35, IL-12 p40, and tumor necrosis factor-α, expression] in the distal colon of Muc2−/−mice presented with an immune suppressive response [i.e., increased Foxp3, transforming growth factor (TGF)-β1, IL-10, and Ebi3 expression]. In contrast, at P28, a proinflammatory response remained in the distal colon, whereas the immune suppressive response (i.e., Foxp3 and TGF-β1 expression) declined. The proximal colon of Muc2−/−mice did not show morphological damage and was dominated by an immune suppressive response at P14 and P28. Interestingly, changes in expression of TLRs and TLR-related molecules were observed in the distal colon at P14 and P28 and in the proximal colon only at P28. Colitis in Muc2−/−mice is limited before weaning by immune suppressive responses and exacerbates in the distal colon after weaning because of the decline in the immune suppressive response.

International audience; MUC5B is one of the major mucin genes expressed in the respiratory tract. Previous studies in our laboratory have demonstrated that MUC5B is expressed in human lung adenocarcinomas and during lung morphogenesis. Moreover, in human lung adenocarcinoma tissues, a converse correlation between MUC5B and thyroid transcription factor-1 (TTF-1) expression, a lung-specific transcription factor, has been established. However, the molecular mechanisms that govern the regulation of MUC5B expression in the lung are largely unknown. In order to better understand the biological role of MUC5B in lung pathophysiology, we report the characterization of the promoter region of the mouse Muc5b mucin gene. The promoter is flanked by a TATA box (TACATAA) identical to that in the human gene. Human and murine promoters share 67.5% similarity over the first 170 nucleotides. By RT-PCR, co-transfection studies and gel-shift assays, we show that Muc5b promoter activity is completely inhibited by TTF-1, whereas factors of the GATA family (GATA-4/GATA-5/GATA-6) are activators. Together, these results demonstrate, for the first time, that Muc5b is a target gene of transcription factors (TTF-1, GATA-6) involved in lung differentiation programs during development and carcinogenesis, and identify TTF-1 as a strong repressor of Muc5b. The characterization of the structural and functional features of the Muc5b mucin gene will provide us with a strong base to develop studies in murine models aimed at the identification of its biological role in lung pathophysiology.

Background: Mucin Muc2 knockout (Muc2−/−) mice spontaneously develop colitis. Methods: To identify genes and biological responses which play a pivotal role during colitis development in Muc2−/− mice, gene expression profiles of colonic tissues from 2- and 4-week-old Muc2−/− and wildtype mice were determined using microarrays. Results: The majority of highly upregulated genes in 2-week-old as well as 4-week-old Muc2−/− mice were primarily involved in immune responses related to antigen processing/presentation, B-cell and T-cell receptor signaling, leukocyte transendothelial migration, and Jak-STAT signaling. Specifically, Muc2−/− mice expressed high levels of immunoglobulins, murine histocompatibility-2, proinflammatory cytokines, chemokines, and antimicrobial proteins. Additionally, in 4-week-old Muc2−/− mice, expression of genes involved in cell structure related pathways was significantly altered. Particularly, the tight junction-associated gene claudin-10 was upregulated, whereas claudin-1 and claudin-5 were downregulated. Furthermore, 4-week-old Muc2−/− mice showed increased expression of genes regulating cell growth in conjunction with increased crypt length and increased epithelial proliferation. Conclusions: Muc2-deficiency leads to an active inflammatory response in 2- and 4-week-old Muc2−/− mice as demonstrated by the altered expression in immune response related genes. In addition, 4-week-old Muc2−/− mice also showed a decrease in epithelial barrier function and an increase in epithelial proliferation as indicated by, respectively, the altered expression in tight junction-related genes and upregulation of genes stimulating cell growth. Remarkably, upregulation of genes stimulating cell growth correlated with increased crypt length and increased epithelial proliferation in 4-week-old Muc2−/− mice. Together, these data demonstrate that there are distinct phases in colitis development in 2–4-week-old Muc2−/− mice. (Inflamm Bowel Dis 2011;)

MUC5B is one of the major mucin genes expressed in the respiratory tract. Previous studies in our laboratory have demonstrated that MUC5B is expressed in human lung adenocarcinomas and during lung morphogenesis. Moreover, in human lung adenocarcinoma tissues, a converse correlation between MUC5B and thyroid transcription factor-1 (TTF-1) expression, a lung-specific transcription factor, has been established. However, the molecular mechanisms that govern the regulation of MUC5B expression in the lung are largely unknown. In order to better understand the biological role of MUC5B in lung pathophysiology, we report the characterization of the promoter region of the mouse Muc5b mucin gene. The promoter is flanked by a TATA box (TACATAA) identical to that in the human gene. Human and murine promoters share 67.5% similarity over the first 170 nucleotides. By RT-PCR, co-transfection studies and gel-shift assays, we show that Muc5b promoter activity is completely inhibited by TTF-1, whereas factors of the GATA family (GATA-4/GATA-5/GATA-6) are activators. Together, these results demonstrate, for the first time, that Muc5b is a target gene of transcription factors (TTF-1, GATA-6) involved in lung differentiation programs during development and carcinogenesis, and identify TTF-1 as a strong repressor of Muc5b. The characterization of the structural and functional features of the Muc5b mucin gene will provide us with a strong base to develop studies in murine models aimed at the identification of its biological role in lung pathophysiology.

Mucin Muc2 knockout (Muc2(-/-)) mice spontaneously develop colitis.To identify genes and biological responses which play a pivotal role during colitis development in Muc2(-/-) mice, gene expression profiles of colonic tissues from 2- and 4-week-old Muc2(-/-) and wildtype mice were determined using microarrays.The majority of highly upregulated genes in 2-week-old as well as 4-week-old Muc2(-/-) mice were primarily involved in immune responses related to antigen processing/presentation, B-cell and T-cell receptor signaling, leukocyte transendothelial migration, and Jak-STAT signaling. Specifically, Muc2(-/-) mice expressed high levels of immunoglobulins, murine histocompatibility-2, proinflammatory cytokines, chemokines, and antimicrobial proteins. Additionally, in 4-week-old Muc2(-/-) mice, expression of genes involved in cell structure related pathways was significantly altered. Particularly, the tight junction-associated gene claudin-10 was upregulated, whereas claudin-1 and claudin-5 were downregulated. Furthermore, 4-week-old Muc2(-/-) mice showed increased expression of genes regulating cell growth in conjunction with increased crypt length and increased epithelial proliferation.Muc2-deficiency leads to an active inflammatory response in 2- and 4-week-old Muc2(-/-) mice as demonstrated by the altered expression in immune response related genes. In addition, 4-week-old Muc2(-/-) mice also showed a decrease in epithelial barrier function and an increase in epithelial proliferation as indicated by, respectively, the altered expression in tight junction-related genes and upregulation of genes stimulating cell growth. Remarkably, upregulation of genes stimulating cell growth correlated with increased crypt length and increased epithelial proliferation in 4-week-old Muc2(-/-) mice. Together, these data demonstrate that there are distinct phases in colitis development in 2-4-week-old Muc2(-/-) mice.