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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.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

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.

Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

In their recent commentary on our manuscript entitled “Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In vitro,” Dufour et al. suggest that the level of cytokine response generated in our paper is due to remaining bacterial debris rather than true differences between individual phages (Dufour et al., 2016).

Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (109 PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1–4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

Introduction In early‐life, the immature mucosal barrier allows contact between the gut microbiota and the developing immune system. Due to their strategic location and their ability to sample luminal antigen, dendritic cells (DC) play a central role in the interaction of microbes and immune cells in the gut. Here, we investigated how two bacteria associated with opposite immune profiles in children, that is, Lactobacillus (L.) reuteri and Staphylococcus (S.) aureus, influenced the differentiation of monocytes in vitro as well how the generated DC impacted T cell responses. Methods We exposed monocyte cultures to cell‐free supernatants (CFS) from these bacteria during their differentiation to DC. Results The presence of L. reuteri‐CFS during DC differentiation resulted in DC with a more mature phenotype, in terms of up‐regulated surface markers (HLA‐DR, CD86, CD83, CCR7) and enhanced cytokine production (IL6, IL10, and IL23), but had a reduced phagocytic capacity compared with non‐treated monocyte‐derived DC (Mo‐DC). However, upon LPS activation, L. reuteri‐CFS‐generated DC displayed a more regulated phenotype than control Mo‐DC with notable reduction of cytokine responses both at mRNA and protein levels. In contrast, S. aureus‐CFS‐generated DC were more similar to control Mo‐DC both without and after LPS stimulation, but they were still able to induce responses in autologous T cells, in the absence of further T cell stimulation. Conclusions We show that bacterial signals during DC differentiation have a profound impact on DC function and possibly also for shaping the T cell pool.