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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

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

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.

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.

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.

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.

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.

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.

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.

Background Necrotizing enterocolitis (NEC) is the most frequent life-threatening gastrointestinal disease experienced by premature infants in neonatal intensive care units. The challenge for neonatologists is to detect early clinical manifestations of NEC. One strategy would be to identify specific markers that could be used as early diagnostic tools to identify preterm infants most at risk of developing NEC or in the event of a diagnostic dilemma of suspected disease. As a first step in this direction, we sought to determine the specific gene expression profile of NEC. Methods Deep sequencing (RNA-Seq) was used to establish the gene expression profiles in ileal samples obtained from preterm infants diagnosed with NEC and non-NEC conditions. Data were analyzed with Ingenuity Pathway Analysis and ToppCluster softwares. Results Data analysis indicated that the most significant functional pathways over-represented in NEC neonates were associated with immune functions, such as altered T and B cell signaling, B cell development, and the role of pattern recognition receptors for bacteria and viruses. Among the genes that were strongly modulated in neonates with NEC, we observed a significant degree of similarity when compared with those reported in Crohn’s disease, a chronic inflammatory bowel disease. Conclusions Gene expression profile analysis revealed a predominantly altered immune response in the intestine of NEC neonates. Moreover, comparative analysis between NEC and Crohn’s disease gene expression repertoires revealed a surprisingly high degree of similarity between these two conditions suggesting a new avenue for identifying NEC biomarkers. Electronic supplementary material The online version of this article (doi:10.1186/s12920-016-0166-9) contains supplementary material, which is available to authorized users.