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Background: Necrotizing enterocolitis (NEC), a severe gut disorder in preterm infants, is difficult to predict due to poor specificity and sensitivity of clinical signs and biomarkers. Using preterm piglets as a model, we hypothesized that early development of NEC affects blood gene expression, potentially related to early systemic immune responses. Methods: A retrospective analysis of clinical, tissue, and blood data was performed on 129 formula-fed piglets with NEC diagnosis at necropsy on day 5. Subgroups of NEC (n = 20) and control piglets (CON, n = 19) were analyzed for whole-blood transcriptome. Results: Preterm piglets had variable NEC lesions, especially in the colon region, without severe clinical signs (e.g. normal growth, activity, hematology, digestion, few piglets with bloody stools). Transcriptome analysis showed 344 differentially expressed genes (DEGs) between NEC and CON piglets. Validation experiment showed that AOAH, ARG2, FKBP5, PAK2, and STAT3 were among the genes affected by severe lesions on day 5, when analyzed in whole blood and in dried blood spots (DBS). Conclusion: Whole-blood gene expressions may be affected in preterm pigs before clinical signs of NEC get severe. Blood gene expression analysis, potentially using DBS samples, is a novel tool to help identify new early biomarkers of NEC. Impact: Preterm pig model was used to investigate if blood transcriptomics could be used to identify new early blood biomarkers of NEC progression.Whole-blood transcriptome revealed upregulation of target genes in NEC cases when clinical symptoms are subtle, and mainly colon regions were affected.Differential NEC-associated gene expressions could be detected also in dried blood spots, potentially allowing easy collection of small blood volumes in infants.

Aim: To examine gene expression in different clinical phenotypes of allopurinol-induced severe cutaneous adverse reactions (SCARs). Materials & methods: Gene expression profiling was performed using microarray on 11 RNA samples (four controls, three hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms, four Stevens–Johnson syndrome/toxic epidermal necrolysis) followed by quantitative real-time PCR in a total of 11 SCARs patients and 11 controls. Results: The biological pathways which were significantly enriched in differentially expressed genes in Stevens–Johnson syndrome/toxic epidermal necrolysis compared with hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms patients included; cell surface interactions at the vascular wall, immunoregulatory interactions at the immunological synapse and MyD88 signaling pathways. Overexpression of miR146a occurred in allopurinol-tolerant HLA-B*58:01 carriers. Conclusion: Biological pathways are identified which appear to be implicated in determining clinical phenotypes in allopurinol-induced SCARs. Overexpression of miR146a is potentially important for allopurinol tolerance in HLA-B*58:01 carriers.

Bovine colostrum (BC) contains bioactive proteins, such as immunoglobulin G (IgG), lactoferrin (LF) and lactoperoxidase (LP). BC was subjected to low-temperature, long-time pasteurization (LTLT, 63 °C, 30 min) or high-temperature, short-time pasteurization (HTST, 72 °C, 15 s) and spray-drying (SD), with or without γ-irradiation (GI, ∼14 kGy) to remove microbial contamination. Relative to unpasteurized liquid BC, SD plus GI increased protein denaturation by 6 and 11%, respectively, increasing to 19 and 27% after LTLT and to 48% after HTST, with no further effects after GI (all P < 0.05). LTLT, without or with GI, resulted in 15 or 29% denaturation of IgG, compared with non-pasteurized BC, and 34 or 58% for HTST treatment (all P < 0.05, except LTLT without GI). For IgG, only GI, not SD or LTLT, increased denaturation (30–38%, P < 0.05) but HTST increased denaturation to 40%, with further increases after GI (60%, P < 0.05). LTLT and HTST reduced LP levels (56 and 81% respectively) and LTLT reduced LF levels (21%), especially together with GI (47%, P < 0.05). Denaturation of BSA, β-LgA, β-LgB and α-La were similar to IgG. Methionine, a protective amino acid against free oxygen radicals, was oxidised by LTLT + GI (P < 0.05) while LTLT and HTST had no effect. Many anti-inflammatory proteins, including serpin anti-proteinases were highly sensitive to HTST and GI but preserved after LTLT pasteurization. LTLT, followed by SD is an optimal processing technique preserving bioactive proteins when powdered BC is used as a diet supplement for sensitive patients.

Deficient levels of milk osteopontin (OPN) in infant formula may partly account for developmental differences between infants fed formula or maternal milk. We hypothesized that a milk diet supplemented with bovine milk OPN improves gut, immunity and brain development and tested this in a preterm pig model. Preterm pigs delivered by cesarean section (90% gestation) were fed raw bovine milk (CON, n = 19) or the same diet supplemented with a physiologically relevant dose of OPN (46 mg/(kg·d), n = 16). Endpoints related to clinical outcomes, systemic immunity and neurocognitive development were assessed during the study and gut tissues were collected at Day 19. Growth pattern, early motor development and most systemic immune parameters were similar between OPN and CON pigs. The OPN pigs had higher villus-to-crypt ratios than CON pigs and higher monocyte and lymphocyte counts on Day 8. Gut digestive and absorptive functions and cognitive performance (T-maze test) were similar between OPN and CON pigs. In conclusion, dietary supplementation with OPN above basal bovine milk levels induced minor improvements in gut structure and systemic immunity without any effects on cognitive performance. The minimal levels of OPN in infant formula to secure optimal adaptation in the immediate neonatal period remain to be determined.

Colostrum contains bioactive components protecting the newborn intestine against bacteria. It is unclear how to optimize processing conditions with highest product bioactivity. Non-pasteurized (BC00), standard-pasteurized (72 °C-15 s, BC72), gently-pasteurized bovine colostrums without (63 °C-30 min, BC63) and with gamma-irradiation (BC63g) were tested for effects on bacterial growth inhibition (Escherichia coli, Staphylococcus aureus and S. epidermidis), intestinal epithelial cell (IEC) proliferation and cytokine secretion in vitro. Thermal processing decreased endogenous bacteria and IgG levels. All BCs inhibited bacterial growth 1–2 h after inoculation, but only BC00, BC63 and BC63g retained activity after 4–24 h. After 4 h, the activity against S. epidermidis of BC63g was lower than BC00 but still potent when mixed with formula. All BCs stimulated IEC proliferation, with the most pronounced responses for BC00. Only BC00 and BC63 increased IL-8 secretion in lipopolysaccharide-stimulated IECs. Thermal processing reduced bioactivity and combined gentle pasteurization and gamma-irradiation improved BC sterility and bioactivity, relative to standard pasteurization.

Prenatal inflammation is a major risk for preterm birth and neonatal morbidity, but its effects on postnatal immunity and organ functions remain unclear. Using preterm pigs as a model for preterm infants, we investigated whether prenatal intra-amniotic (IA) inflammation modulates postnatal systemic immune status and organ functions. Preterm pigs exposed to IA lipopolysaccharide (LPS) for 3 days were compared with controls at birth and postnatal day 5 after formula feeding. IA LPS induced mild chorioamnionitis but extensive intra-amniotic inflammation. There were minor systemic effects at birth (increased blood neutrophil counts), but a few days later, prenatal LPS induced delayed neonatal arousal, systemic inflammation (increased blood leukocytes, plasma cytokines, and splenic bacterial counts), altered serum biochemistry (lower albumin and cholesterol and higher iron and glucose values), and increased urinary protein and sodium excretion. In the gut and lungs, IA LPS-induced inflammatory responses were observed mainly at birth (increased LPS, CXCL8, and IL-1 beta levels and myeloperoxidase-positive cell density, multiple increases in innate immune gene expressions, and reduced villus heights), but not on postnatal day 5 (except elevated lung CXCL8 and diarrhea symptoms). Finally, IA LPS did not affect postnatal gut brush-border enzymes, hexose absorption, permeability, or sensitivity to necrotizing enterocolitis on day 5. Short-term IA LPS exposure predisposes preterm pigs to postnatal systemic inflammation after acute fetal gut and lung inflammatory responses.

Background: After very preterm birth, male infants show higher mortality than females, with higher incidence of lung immaturity, neurological deficits, infections, and growth failure. In modern pig production, piglets dying in the perinatal period (up to 20%) often show signs of immature organs, but sex-specific effects are not clear. Using preterm pigs as model for immature infants and piglets, we hypothesized that neonatal survival and initial growth and immune development depend on sex.Methods: Using data from a series of previous intervention trials with similar delivery and rearing procedures, we established three cohorts of preterm pigs (90% gestation), reared for 5, 9, or 19 days before sample collection (total n = 1,938 piglets from 109 litters). Partly overlapping endpoints among experiments allowed for multiple comparisons between males and females for data on mortality, body and organ growth, gut, immunity, and brain function.Results: Within the first 2 days, males showed higher mortality than females (18 vs. 8%, P < 0.001), but less severe immune response to gram-positive infection. No effect of sex was observed for thermoregulation or plasma cortisol. Later, infection resistance did not differ between sexes, but growth rate was reduced for body (up to −40%) and kidneys (−6%) in males, with higher leucocyte counts (+15%) and lower CD4 T cell fraction (−5%) on day 9 and lower monocyte counts (−18%, day 19, all P < 0.05). Gut structure, function and necrotizing enterocolitis (NEC) incidence were similar between groups, but intestinal weight (−3%) and brush-border enzyme activities were reduced at day 5 (lactase, DPP IV, −8%) in males. Remaining values for blood biochemistry, hematology, bone density, regional brain weights, and visual memory (tested in a T maze) were similar.Conclusion: Following preterm birth, male pigs show higher mortality and slower growth than females, despite limited differences in organ growth, gut, immune, and brain functions. Neonatal intensive care procedures may be particularly important for compromised newborns of the male sex. Preterm pigs can serve as good models to study the interactions of sex- and maturation-specific survival and physiological adaptation in mammals.

OBJECTIVES: Human milk (HM) is most appropriate for neonates, but it does not provide enough nutrients for preterm infants. HM fortifiers based on highly processed mature bovine milk (BMF) are routinely used for preterm infants despite risks of causing gut dysfunction and systemic infection. Bovine colostrum processed by mild thermal treatment as a fortifier (BCF) may better protect against infection and inflammation. We hypothesized that BCF-fortified HM has enhanced antimicrobial activity against pathogens commonly causing neonatal sepsis, relative to BMF-fortified HM. METHODS: Holder-pasteurized HM samples (10 mothers) were aliquoted into three fractions: unfortified HM, HM fortified with either BMF or BCF. The samples were analysed for pH, lactoferrin concentrations, and antimicrobial activities against S. epidermidis, E.coli and E. faecalis. RESULTS: HM+BCF had a lower pH and higher levels of lactoferrin than HM+BMF, with HM being intermediate. Relative to IF, HM decreased the growth of S. epidermidis and E.coli and E.faecalis, with no difference between preterm and term HM. Addition of BMF abolished the antimicrobial effect of HM against S. epidermidis and E.faecalis, but not E.coli. In contrast, addition of BCF into HM enhanced antimicrobial activity against S. epidermidis and E.coli, relative to unfortified HM. HM+BCF was superior to HM+BMF in inhibiting growth of all tested bacteria. CONCLUSION: BMF fortification decreased while BCF fortification enhanced in vitro antimicrobial activity of HM. This effect may partly be derived from the high levels of antimicrobial factors found in BCF, including lactoferrin. BCF may be a better fortifier than BMF for preterm infants. This article is protected by copyright. All rights reserved.

Promoting sustainable food consumption contributes to the achievement of the United Nation&rsquo, s Sustainable Development Goals. This study investigated the key determinants of consumer intention to purchase organic meat in an emerging market and a unique model was developed by incorporating environmental concern and guilt with the key components of the Theory of Planned Behavior. This model was then validated by obtaining data from a sample of 402 Vietnamese consumers at five food stores in Ho Chi Minh City, using a paper-based survey. The findings reveal that consumers who are concerned about the environment are likely to formulate favorable attitudes and downplay monetary barriers associated with organic food purchase. In addition, while attitudes and guilt about buying conventional meat have a positive effect on organic meat purchase intention, perceived monetary barriers significantly reduce the intention. These findings highlight both the rational and emotional aspects of organic food purchase intention and have important implications for key stakeholders and the encouragement of organic meat consumption.

Preterm infants are at risk of multiple morbidities including necrotizing enterocolitis (NEC). Suspected NEC patients receive intravenous antibiotics (AB) to prevent sepsis, although enteral AB is arguably more effective at reducing NEC but is rarely used due to the risk of AB resistance. Fecal microbiota transplantation (FMT) has shown protective effects against NEC in animal experiments, but the interaction between AB and FMT has not been investigated in neonates. We hypothesized that administration of enteral AB followed by rectal FMT would effectively prevent NEC with negligible changes in AB resistance and systemic immunity. Using preterm piglets, we examined host and gut microbiota responses to AB, FMT, or a sequential combination thereof, with emphasis on NEC development. In a saline-controlled experiment, preterm piglets (n = 67) received oro-gastric neomycin (50 mg/kg/d) and amoxicillin-clavulanate (50/12.5 mg/kg/d) (hereafter AB) for four days after cesarean delivery, and were subsequently given rectal FMT from healthy suckling piglet donors. Whereas AB protected the stomach and small intestine, and FMT primarily protected the colon, the sequential combination treatment surprisingly provided no NEC protection. Furthermore, minor changes in the gut microbiota composition were observed in response to either treatment, although AB treatment decreased species diversity and increased AB resistance among coliform bacteria and Enterococci, which were both partly reversed by FMT. Besides, enteral AB treatment suppressed cellular and functional systemic immune development, which was not prevented by subsequent FMT. We discovered an antagonistic relationship between enteral AB and FMT in terms of NEC development. The outcome may depend on choice of AB compounds, FMT composition, doses, treatment duration, and administration routes, but these results challenge the applicability of enteral AB and FMT in preterm infants.

Aims: To determine genetic susceptibility markers for carbamazepine (CBZ) and allopurinol-induced severe cutaneous adverse reactions (SCARs) in Vietnamese. Methods: A case–control study was performed involving 122 patients with CBZ or allopurinol-induced SCARs and 120 drug tolerant controls. Results: HLA-B*58:01 was strongly associated with allopurinol-induced SCARs and strongly correlated with SNP rs9263726. HLA-B*15:02 was associated with CBZ-induced Stevens–Johnson syndrome/toxic epidermal necrolysis but not with drug-induced hypersensitivity syndrome/drug rash with eosinophilia and systemic symptoms. No association was found between HLA-A*31:01 and CBZ-induced SCARs. HLA-B*58:01 and rs3909184 allele A with renal insufficiency were shown to increase the risk of allopurinol-induced SCARs. Conclusion: HLA-B*58:01 and HLA-B*15:02 confer susceptibility to allopurinol-induced SCARs and CBZ-induced SJS/TEN in Vietnamese. SNP rs9263726 can be used as a surrogate marker in identifying HLA-B*58:01.

Chorioamnionitis (CA) predisposes to preterm birth and affects the fetal mucosal surfaces (i.e., gut, lungs, and skin) via intra-amniotic (IA) inflammation, thereby accentuating the proinflammatory status in newborn preterm infants. It is not known if CA may affect more distant organs, such as the kidneys, before and after preterm birth. Using preterm pigs as a model for preterm infants, we investigated the impact of CA on fetal and neonatal renal status and underlying mechanisms. Fetal pigs received an IA dose of lipopolysaccharide (LPS), were delivered preterm by cesarean section 3 days later (90% gestation), and compared with controls (CON) at birth and at postnatal day 5. Plasma proteome and inflammatory targets in kidney tissues were evaluated. IA LPS-exposed pigs showed inflammation of fetal membranes, higher fetal plasma creatinine, and neonatal urinary microalbumin levels, indicating renal dysfunction. At birth, plasma proteomics revealed LPS effects on proteins associated with renal inflammation (up-regulated LRG1, down-regulated ICA, and ACE). Kidney tissues of LPS pigs at birth also showed increased levels of kidney injury markers (LRG1, KIM1, NGLA, HIF1A, and CASP3), elevated molecular traits related to innate immune activation (infiltrated MPO+ cells, complement molecules, oxidative stress, TLR2, TLR4, S100A9, LTF, and LYZ), and Th1 responses (CD3+ cells, ratios of IFNG/IL4, and TBET/GATA3). Unlike in plasma, innate and adaptive immune responses in kidney tissues of LPS pigs persisted to postnatal day 5. We conclude that prenatal endotoxin exposure induces fetal and postnatal renal inflammation in preterm pigs with both innate and adaptive immune activation, partly explaining the potential increased risks of kidney injury in preterm infants born with CA.

Background: Infants born preterm or small for gestational age (SGA, due to fetal growth restriction) both show an increased risk of neonatal infection. However, it remains unclear how the co-occurrence of preterm birth and SGA may affect neonatal immunity and infection risk. We hypothesized that fetal growth restricted (FGR) preterm newborns possess impaired immune competence and increased susceptibility to systemic infection and sepsis, relative to corresponding normal birth weight (NBW) newborns. Methods: Using preterm pigs as a model for preterm infants, gene expression in lipopolysaccharide (LPS) stimulated cord blood was compared between NBW and FGR (lowest 25% birth weight percentile) preterm pigs. Next, clinical responses to a systemic Staphylococcus epidermidis (SE) challenge were investigated in newborn FGR and NBW preterm pigs. Finally, occurrence of spontaneous infections were investigated in 9 d-old FGR and NBW preterm pigs, with or without neonatal antibiotics treatment. Results: At birth, preterm FGR piglets showed diminished ex vivo cord blood responses to LPS for genes related to both innate and adaptive immunity, and also more severe septic responses following SE infection (e.g., higher blood lactate, decreased blood pH, neutrophil and platelet counts, relative to NBW pigs). After 9 d, FGR pigs had higher incidence and severity of spontaneous infections (e.g., higher bacterial densities in the bone marrow), increased regulatory T cell numbers, reduced neutrophil phagocytosis capacity, and impaired ex vivo blood gene responses to LPS, especially when receiving neonatal antibiotics. Conclusion: FGR at preterm birth is associated with poor immune competence, impaired infection resistance, and greater sepsis susceptibility in the immediate postnatal period. Our results may explain the increased morbidity and mortality of SGA preterm infants and highlight the need for clinical vigilance for this highly sensitive subgroup of preterm neonates.

Objectives: Maternal milk is often absent or in limited supply just after preterm birth. Many preterm infants are therefore fed infant formula as their first enteral feed despite an increased risk of feeding intolerance, necrotizing enterocolitis (NEC), and infection. Using preterm pigs as a model for preterm infants, we hypothesized that bovine colostrum given before or after formula feeding would alleviate formula-induced detrimental effects during the first days after preterm birth. Methods: A total of 74 preterm pigs received gradually increasing volumes of formula (F) or bovine colostrum (C) until day 5, when they were euthanized or transitioned to either C or F for another 4 days, resulting in six groups: C or F until day 5 (C5, F5, n = 11 each), C or F until day 9 (CC, FF n = 12–13 each), C followed by F (CF, n = 14), and F followed by C (FC, n = 13). Results: Systemically, colostrum feeding stimulated circulating neutrophil recruitment on day 5 (C5 vs. F5, P < 0.05). Relative to initial formula feeding, initial colostrum feeding promoted the development of systemic immune protection as indicated by a decreased T-helper cell population and an increased regulatory T-cell population (CC + CF vs. FC + FF, P < 0.01). In the gut, colostrum feeding improved intestinal parameters such as villus heights, enzymes, hexose absorption, colonic goblet cell density, and decreased the incidence of severe NEC (27 vs. 64%), diarrhea (16 vs. 49%), and gut permeability on day 5, coupled with lowered expression of LBP, MYD88, IL8, HIF1A, and CASP3 (C5 vs. F5, all P < 0.05). On day 9, the incidence of severe NEC was similarly low across groups (15–21%), but diarrhea resistance and intestinal parameters were further improved by colostrum feeding, relative to exclusive formula feeding (CC, CF, or FC vs. FF, respectively, all P < 0.05). The expression of MYD88 and CASP3 remained downregulated by exclusive colostrum feeding (CC vs. FF, P < 0.01) and colostrum before or after formula feeding down regulated HIF1A and CASP3 expression marginally. Conclusion: Colostrum feeding ameliorated detrimental effects of formula feeding on systemic immunity and gut health in preterm newborns, especially when given immediately after birth.

Background: Most hospitalized preterm infants receive antibiotics in the first days of life to prevent or treat infections. Short-term, early antibiotic treatment may also prevent the microbiota-dependent gut inflammatory disorder, necrotizing enterocolitis (NEC). It remains a challenge to predict NEC, and a few early blood diagnostic markers exist. Using preterm pigs as model for infants, blood parameters and plasma proteins affected by early progression of NEC were profiled in preterm pigs subjected to oral, systemic, or no antibiotics after preterm birth. Methods: Preterm newborn pigs were treated with saline (CON) or antibiotics (ampicillin, gentamicin, and metronidazole) given enterally (ENT) or parenterally (PAR), and fed formula for 4 days to induce variable microbiome-dependent sensitivities to NEC. The gut was collected for macroscopic scoring of NEC lesions and blood for hematology, blood biochemistry, and LC/MS-based plasma proteomics. Statistical modeling was applied to detect plasma proteins affected by NEC and/or antibiotics. Results: Analyzed across different antibiotic regimens, NEC progression was associated with altered blood parameters and abundance of 89 plasma proteins that were functionally involved in extracellular membrane destruction, lipid metabolism, coagulopathy, and acute phase response. Large NEC-related changes were observed in abundance of RBP4, FGA, AHSG, C5, PTPRG, and A-1-antichymotrypsin 2, indicating potential serving as early markers of NEC. Conversely, antibiotic treatment, independent of NEC, affected only 4 proteins with main differences found between ENT and CON pigs. Conclusion: Early postnatal development of NEC lesions is associated with marked plasma protein changes that may be used for early NEC diagnosis.

Measurement of intestinal permeability (IP) is often used in the examination of inflammatory gastrointestinal disorders. IP can be assessed by measurement of urinary recovery of ingested non-metabolizable lactulose (L) and mannitol (M). The present study aimed to examine how measurements of IP can be integrated in a NMR-based metabolomics approach for a simultaneous quantification of L/M ratio and biomarker exploration. For this purpose, plasma and urine samples were collected from five-day-old preterm piglets (n = 20) with gastrointestinal disorders (subjected to intra-amniotic lipopolysaccharide (LPS, 1 mg/fetus)) after they had been administrated a 5% lactulose and 5% mannitol solution (15 mL/kg). The collected plasma and urine samples were analyzed by 1H NMR-based metabolomics. Urine L/M ratio measured by 1H NMR spectroscopy showed high correlation with the standard measurement of the urinary recoveries by enzymatic assays (r = 0.93, p < 0.05). Partial least squares (PLS) regressions and correlation analyses between L/M ratio and NMR metabolomics data revealed that L/M ratio was positively correlated with plasma lactate, acetate and succinate levels and negatively correlated with urinary hippuric acid and glycine. In conclusion, the present study demonstrated that NMR metabolomics enables simultaneous IP testing and discovery of biomarkers associated with an impaired intestinal permeability.

Human milk is rich in nutritional factors, such as alpha-lactalbumin (&alpha, Lac), and important for neonatal development, but nutrient supplementation may be required for optimal growth. Using a pig model, we hypothesized that &alpha, Lac-enriched whey protein concentrate (WPC) supplementation improves neonatal development. Cesarean-delivered preterm pigs were fed either dilute bovine milk (REF) or REF milk supplemented with WPC with normal (STANDARD-ALPHA) or high (HIGH-ALPHA) &alpha, Lac. Clinical, gut, immune and cognitive endpoints (open field, T-maze) were assessed and tissues collected at Day 19. The growth of STANDARD-ALPHA and HIGH-ALPHA were higher than REF (31 vs. 19 g/kg/d). Most organ weights, gut, immunity and brain variables were similar between WPC groups. HIGH-ALPHA had a higher bone mineral content, colon microbial diversity and an abundance of specific bacteria and microbial metabolites, and tended to show a faster food transit time (p = 0.07). Relative to REF, WPC pigs showed higher relative organ weights, blood amino acids, blood neutrophil function, and microbial metabolites, but lower brush-border enzyme activities and plasma cortisol. Cognition outcomes did not differ among the groups. In conclusion, WPC supplementation of milk improved some growth, gut and immunity parameters in preterm pigs. However, increasing the &alpha, Lac content beyond human milk levels had limited effects on the immature gut and developing brain.

Chorioamnionitis (CA), resulting from intra-amniotic inflammation, is a frequent cause of preterm birth and exposes the immature intestine to bacterial toxins and/or inflammatory mediators before birth via fetal swallowing. This may affect intestinal immune development, interacting with the effects of enteral feeding and gut microbiota colonization just after birth. Using preterm pigs as model for preterm infants, we hypothesized that prenatal exposure to gram-negative endotoxin influences postnatal bacterial colonization and gut immune development. Pig fetuses were given intra-amniotic lipopolysaccharide (LPS) 3 days before preterm delivery by cesarean section and were compared with littermate controls (CON) at birth and after 5 days of formula feeding and spontaneous bacterial colonization. Amniotic fluid was collected for analysis of leukocyte counts and cytokines, and the distal small intestine was analyzed for endotoxin level, morphology, and immune cell counts. Intestinal gene expression and microbiota were analyzed by transcriptomics and metagenomics, respectively. At birth, LPS-exposed pigs showed higher intestinal endotoxin, neutrophil/macrophage density, and shorter villi. About 1.0% of intestinal genes were affected at birth, and DMBT1, a regulator of mucosal immune defense, was identified as the hub gene in the co-expression network. Genes related to innate immune response (TLR2, LBP, CD14, C3, SFTPD), neutrophil chemotaxis (C5AR1, CSF3R, CCL5), and antigen processing (MHC II genes and CD4) were also affected, and expression levels correlated with intestinal neutrophil/macrophage density and amniotic fluid cytokine levels. On day 5, LPS and CON pigs showed similar sensitivity to necrotizing enterocolitis, endotoxin levels, morphology, immune cell counts, gene expressions, and microbiota composition (except for difference in some low-abundant species). Our results show that CA markedly affects intestinal genes at preterm birth, including genes related to immune cell infiltration. However, a few days later, following the physiological adaptations to preterm birth, CA had limited effects on intestinal structure, function, gene expression, bacterial colonization, and necrotizing enterocolitis sensitivity. We conclude that short-term, prenatal intra-amniotic inflammation is unlikely to exert marked effects on intestinal immune development in preterm neonates beyond the immediate neonatal period.

Epigenetic regulation may play an important role in mediating microbe–host interactions and adaptation of intestinal gene expression to bacterial colonization just after birth. This is particularly important after preterm birth because the immature intestine is hypersensitive to invading bacteria. We compared the intestinal DNA methylome and microbiome between conventional (CON) and antibiotics-treated (AB) preterm pigs, used as a model for preterm infants. Oral AB treatment reduced bacterial density (∼100-fold), diversity and fermentation, improved the resistance to necrotizing enterocolitis (NEC) and changed the genome-wide DNA methylation in the distal small intestine. Integration of epigenome data with previously obtained proteome data showed that intestinal immune–metabolic pathways were affected by the AB-induced delay in bacterial colonization. DNA methylation and expression of intestinal genes, related to innate immune response, phagocytosis, endothelial homeostasis and tissue metabolism (e.g. CPN1, C3, LBP, HIF1A, MicroRNA-126, PTPRE), differed between AB and CON pigs even before any evidence of NEC lesions. Our findings document that the newborn immature intestine is influenced by bacterial colonization via DNA methylation changes. Microbiota-dependent epigenetic programming of genes related to gut immunity, vascular integrity and metabolism may be critical for short- and long-term intestinal health in preterm neonates.

Formula feeding is associated with compromised intestinal health in preterm neonates compared with maternal milk, but the mechanisms behind this are unclear. We hypothesized that the use of maltodextrin and whey protein concentrates (WPCs) with reduced bioactivity owing to thermal processing are important factors.Ninety-two cesarean-delivered preterm pigs were fed increasing doses of formulas for 5 days (24-120 mL · kg · day). In experiment 1, 4 groups of pigs (n = 15-16) were fed lactose- or maltodextrin-dominant formulas (lactose/maltodextrin ratios 3:1 or 1:3, respectively), containing WPC with either high or low levels of IgG (WPC1 or WPC2, respectively). In experiment 2, 2 groups of pigs (n = 15-16) were fed lactose-dominant formulas with either a bioactive WPC (BioWPC, produced by reduced thermal-processing) or a conventional WPC (ConWPC).In experiment 1, pigs fed formula with WPC1 had higher villi, hexose absorption, and lactase activity in small intestine, relative to WPC2, but predominantly with the lactose-dominant formula (all P 0.05). In experiment 2, the BioWPC product had higher bioactivity, as indicated by higher IgG, lactoferrin, and TGF-β2 levels, and better enterocyte proliferation in vitro. Pigs fed the BioWPC formula showed better feeding tolerance and higher intestinal villi and lactase activity (all P 0.05). The BioWPC formula-fed pigs also had greater physical activity (P 0.05 on day 4) and tended to show improved hexose absorption and decreased gut permeability (both P ≤ 0.09).Infant formulas containing lactose as the main carbohydrate, and WPC with reduced thermal processing, may support gut maturation and health in sensitive, preterm neonates.

Preterm infants are highly susceptible to late-onset sepsis (LOS) and necrotizing enterocolitis (NEC), but disease pathogenesis and specific diagnostic markers are lacking. Circulating cell-free DNA (cfDNA) and immune cell-derived proteins are involved in multiple immune diseases in adults but have not been investigated in preterm neonates. We explored the relation of circulating neutrophil-associated proteins and cfDNA to LOS and/or NEC. Using a clinically relevant preterm pig model of spontaneous LOS and NEC development, we investigated neutrophil-associated proteins and cfDNA in plasma, together with cytokines in gut tissues. The changes in cfDNA levels were further studied in preterm pigs and neonatal mice with induced sepsis, and in preterm infants with or without LOS and/or NEC. Fifteen of 114 preterm pigs spontaneously developed both LOS and NEC, and they showed increased intestinal levels of IL-6 and IL-1β and plasma levels of cfDNA, neutrophil-associated proteins, and proteins involved in platelet-neutrophil interaction during systemic inflammation. The abundance of neutrophil-associated proteins highly correlated with cfDNA levels. Further, Staphylococcus epidermidis challenge of neonatal mice and preterm pigs increased plasma cfDNA levels and bacterial accumulation in the spleen. In infants, plasma cfDNA levels were elevated at LOS diagnosis and 1–6 d before NEC. In conclusion, elevated levels of plasma cfDNA and neutrophil proteins are associated with LOS and NEC diagnosis.

Human milk oligosaccharides (HMOs) may mediate prebiotic and anti-inflammatory effects in newborns. This is particularly important for preterm infants who are highly susceptible to intestinal dysfunction and necrotizing enterocolitis (NEC). We hypothesized that HMO supplementation of infant formula (IF) improves intestinal function, bacterial colonization and NEC resistance immediately after preterm birth, as tested in a preterm pig model. Mixtures of HMOs were investigated in intestinal epithelial cells and in preterm pigs (n=112) fed IF supplemented without (CON) or with a mixture of four HMOs (4-HMO) or25 HMOs (25-HMO, 5-10 g/L given for 5 or 11 days). The 25-HMO blend decreased cell proliferation and both HMO blends decreased lipopolysaccharide-induced interleukin-8 secretion in IPEC-J2 cells, relative to control (P.05). All HMOs were found in urine and feces of HMO-treated pigs, and short-chain fatty acids in the colon were higher in HMO vs. CON pigs (P.05). After 5 days, NEC lesions were similar between HMO and CON pigs and 25-HMO increased colon weights (P.01). After 11 days, the 4-HMO diet did not affect NEC (56 vs. 79%, P=.2) but increased dehydration and diarrhea (P.05) and expression of immune-related genes (IL10, IL12, TGFβ, TLR4; P.05). Bacterial adherence and diversity was unchanged after HMO supplementation.Complex HMO-blends affect intestinal epithelial cells in vitro and gut gene expression and fermentation in preterm pigs. However, the HMOs had limited effects on NEC and diarrhea when supplemented to IF. Longer-term exposure to HMOs may be required to improve the immature intestinal function in formula-fed preterm neonates.

Chorioamnionitis (CA, fetal membrane inflammation) predisposes to preterm birth and is associated with increased neonatal infection risk, but the separate effects of prematurity, CA, and postnatal adaptations on this risk are unclear. Using pigs as models for infants, we examined the systemic immune-metabolic status in cesarean-delivered preterm pigs, with and without CA induced by intra-amniotic (IA) LPS exposure. At birth, cord blood of preterm pigs showed neutropenia and low expressions of innate and adaptive immune genes, relative to term pigs. IA LPS induced CA and fetal systemic innate immune activation via complement and neutrophil-related pathways. These were mainly modulated via cellular regulations rather than granulopoiesis, as validated by the in vitro LPS stimulation of cord blood. After birth, IA LPS-exposed preterm pigs did not follow normal immune-metabolic ontogenies found in fetuses or newborns without prenatal insults, but showed consistently high levels of Treg, impaired Th1 polarization, and reduced expressions of multiple genes related to cellular oxidative phosphorylation and ribosomal activities. In conclusion, our results provide cellular and molecular evidence for CA-induced distinct neonatal immune-metabolic status with increased disease tolerance strategy, suggesting mechanisms for the clinical observation of elevated sepsis risks in immune-compromised preterm infants born with CA.

Background: Preterm infants are born with an immature immune system, limited passive immunity, and are at risk of developing bacteremia and sepsis in the postnatal period. We hypothesized that enteral feeding, with or without added immunoglobulins, improves the clinical response to systemic infection by coagulase negative staphylococci. Methods: Using preterm cesarean delivered pigs as models for preterm infants, we infused live Staphylococcus epidermidis (SE, 5 × 109 colony forming units per kg) systemically 0–3 days after birth across five different experiments. SE infection responses were assessed following different gestational age at birth (preterm vs. term), enteral milk diets (bovine colostrum, infant formula with or without added porcine plasma) and with/without systemic immunoglobulins. Pigs infected with SE were assessed 12–48 h for clinical variables, blood bacteriology, chemistry, hematology, and gut dysfunction (intestinal permeability, necrotizing enterocolitis lesions). Results: Adverse clinical responses and increased mortality were observed in preterm vs. term pigs, when infected with SE just after birth. Feeding bovine colostrum just after birth improved blood SE clearance and clinical status (improved physical activity and intestinal structure, fewer bone marrow bacteria), relative to pigs fed infant formula. A few days later, clinical responses to SE bacteremia (hematology, neutrophil phagocytic capacity, T cell subsets) were less severe, and less affected by different milk diets, with or without added immunoglobulins. Conclusion: Prematurity increases the sensitivity of newborn pigs to SE bacteremia, potentially causing sepsis. Sensitivity to systemic SE infection decreases rapidly in the days after preterm birth. Both age and diet (parenteral nutrition, colostrum, milk, formula) may influence gut inflammation, bacterial translocation and systemic immune development in the days after birth in preterm newborns.

Background: Many preterm infants are born with growth restriction (GR) following maternal or fetal complications before birth. Such infants may continue to grow slowly after birth, regardless of birth weight (BW), due to morbidities related to their immature organs. Severe GR increases the susceptibility to infections, but it is not clear if this is a consequence of impaired systemic immunity or other factors, such as prolonged hospital stay or poor mucosal barrier function. Using preterm pigs as models for preterm infants, we hypothesized that moderate GR, exerting limited clinical effects, does not influence systemic immune development. Methods: Preterm pigs were delivered by cesarean section and fed bovine milk diets until 19 d. Piglets with fetal growth restriction (F-GR, the lowest 25% of BW, n = 27, excluding those with BW

Prenatal inflammation may predispose to preterm birth and postnatal inflammatory disorders such as necrotizing enterocolitis (NEC). Bioactive milk ingredients may help to support gut maturation in such neonates, but mother’s milk is often insufficient after preterm birth. We hypothesized that supplementation with bioactive ingredients from bovine milk [osteopontin (OPN), caseinoglycomacropeptide (CGMP), colostrum (COL)] supports gut, immunity, and NEC resistance in neonates born preterm after gram-negative infection before birth. Using preterm pigs as a model for preterm infants, fetal pigs were given intraamniotic injections of lipopolysaccharide (LPS; 1 mg/fetus) and delivered 3 days later (90% gestation). For 5 days, groups of LPS-exposed pigs were fed formula (FOR), bovine colostrum (COL), or formula enriched with OPN or CGMP. LPS induced intraamniotic inflammation and postnatal systemic inflammation but limited effects on postnatal gut parameters and NEC. Relative to FOR, COL feeding to LPS-exposed pigs showed less diarrhea, NEC severity, reduced gut IL-1β and IL-8 levels, greater gut goblet cell density and digestive enzyme activities, and blood helper T-cell fraction. CGMP improved neonatal arousal and gut lactase activities and reduced LPS-induced IL-8 secretion in intestinal epithelial cells (IECs) in vitro. Finally, OPN tended to reduce diarrhea and stimulated IEC proliferation in vitro. No effects on villus morphology, circulating cytokines, or colonic microbiota were observed among groups. In conclusion, bioactive milk ingredients exerted only modest effects on gut and systemic immune parameters in preterm pigs exposed to prenatal inflammation. Short-term, prenatal exposure to inflammation may render the gut less sensitive to immune-modulatory milk effects. NEW & NOTEWORTHY Prenatal inflammation is a risk factor for preterm birth and postnatal complications including infections. However, from clinical studies, it is difficult to separate the effects of only prenatal inflammation from preterm birth. Using cesarean-delivered preterm pigs with prenatal inflammation, we documented some beneficial gut effects of bioactive milk diets relative to formula, but prenatal inflammation appeared to decrease the sensitivity of enteral feeding. Special treatments and diets may be required for this neonatal population.

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

Background: Neonatal infection and sepsis are common for preterm infants due to their immature immune system. Early diagnosis is important for effective treatment, but few early markers of systemic and neuro-inflammatory responses in neonates are known. We hypothesised that systemic infection with Staphylococcus epidermidis (SE), a Gram-positive bacteria, induces acute changes to proteins in the plasma and cerebrospinal fluid (CSF), potentially affecting the immature brain of preterm neonates.Methods: Using preterm pigs as a model for preterm infants, plasma and CSF samples were collected up to 24 h after SE infection and investigated by untargeted mass spectrometry (MS)-based proteomics. Multiple differentially expressed proteins were further studied in vitro.Results: The clinical signs of sepsis and neuroinflammation in SE-infected piglets were associated with changes of multiple CSF and plasma proteins. Eight plasma proteins, including APOA4, haptoglobin, MBL1, vWF, LBP, and sCD14, were affected 6 h after infection. Acute phase reactants, including complement components, showed a time-dependent activation pattern after infection. Feeding bovine colostrum reduced the sepsis-related changes in clinical indices and plasma proteins. Neuroinflammation-related neuropeptide Y (NPY), IL-18, and MMP-14 showed distinct changes in the CSF and several brain regions (the prefrontal cortex, PVWM, and hippocampus) 24 h after infection. These changes were verified in TLR2 agonist-challenged primary microglia cells, where exogenous NPY suppressed the inflammatory response.Conclusion: Systemic infection with SE induces inflammation with rapid proteome changes in the plasma and CSF in preterm newborn pigs. The observed early markers of sepsis and neuroinflammation in preterm pigs may serve as novel biomarkers for sepsis in preterm infants.

Preterm infants have immature organ functions that predispose them to gut and immune disorders. Developmental delays at preterm birth may affect various organs differently at term-corrected age. We hypothesized that gut and immune maturation in moderately preterm neonates depends more on birth and postnatal factors than on advancing postconceptional age (PCA). Using preterm pigs as models, we investigated how gut and immune parameters develop until term-corrected age and how these differ from those in term counterparts. Preterm ( n = 43, 106 days of gestation) and term pigs ( n = 41, 116 days of gestation) were delivered by caesarean section and euthanized at birth ( day 1) or postnatal day 11 (term-corrected age for preterm pigs) using identical rearing conditions. Relative to term pigs, preterm pigs had lower blood oxygenation, glucose, and cortisol levels, lower gut lactase activity, villus height, and goblet cell density, and lower blood neutrophil, helper T, and cytotoxic T cell numbers at birth. Despite slower growth in preterm pigs, most intestinal and immune parameters increased markedly after birth in both groups. However, some parameters remained negatively affected by preterm birth until postnatal day 11 (goblet cells, gut permeability, and cytotoxic T cells). The colon microbiota showed limited differences between preterm and term pigs at this time. At the same PCA, preterm 11-day-old pigs had higher blood leukocyte numbers and gut enzyme activities but lower villus height and blood cytotoxic T cell numbers relative to newborn term pigs. Birth and postnatal factors, not advancing PCA, are key determinants of gut and immune maturation in moderately preterm neonates. NEW & NOTEWORTHY Postnatally, preterm infants are often considered to reach a physiological maturation similar to that in term infants when they reach term-corrected postconceptional age (PCA). Using preterm pigs as models, we show that PCA may be a poor measure of gut and immune maturation because environmental triggers (regardless of PCA at birth) are critical. Possibly, PCA is only relevant to evaluate physiological maturation of organs that develop relatively independent of the external environment (e.g., the brain).

Whey protein concentrate (WPC) is often subjected to heat treatment during industrial processing, resulting in protein denaturation and loss of protein bioactivity. We hypothesized that WPC samples subjected to different degrees of thermal processing are associated with different levels of bioactive proteins and effects on proliferation and immune response in intestinal epithelial cells (IEC). The results showed that low-heat-treated WPC had elevated levels of lactoferrin and transforming growth factor-β2 compared with that of standard WPC. The level of aggregates depended on the source of whey, with the lowest level being found in WPC derived from acid whey. Following acid activation, WPC from acid whey enhanced IEC proliferation compared with WPC from sweet whey or nonactivated WPC. Low-heat-treated WPC from acid whey induced greater secretion of IL-8 in IEC than either standard WPC from acid whey or low-heat-treated WPC from sweet whey. Following acid activation (to activate growth factors), low-heat-treated WPC from sweet whey induced higher IL-8 levels in IEC compared with standard WPC from sweet whey. In conclusion, higher levels of bioactive proteins in low-heat-treated WPC, especially from acid whey, may enhance proliferation and cytokine responses of IEC. These considerations could be important to maintain optimal bioactivity of infant formulas, including their maturational and immunological effects on the developing intestine.

Immature immunity may predispose preterm neonates to infections and necrotizing enterocolitis (NEC). Intravenous antibiotics are frequently given to prevent and treat sepsis, while oral antibiotics are seldom used. We hypothesized that oral antibiotics promote maturation of systemic immunity and delay gut bacterial colonization and thereby protect preterm neonates against both NEC and bacteremia in the immediate postnatal period. Preterm pigs were given formula and administered saline (CON) or broad-spectrum antibiotics orally (ORA) or systemically (SYS) for 5 d after birth. Temporal changes in blood parameters and bacterial composition in the intestine, blood and immune organs were analyzed. Newborn preterm pigs had few blood neutrophils and a high frequency of progenitor cells. Neutrophils gradually matured after preterm birth with increasing CD14 and decreasing CD172a expressions. Preterm neutrophil and monocyte TLR2 expression and TLR2-mediated blood cytokine responses were low relative to adults. ORA pigs showed enhanced blood neutrophil maturation with reduced cell size and CD172a expression. Only ORA pigs, but not SYS pigs, were protected from a high density of gut Gram-positive bacteria, high gut permeability, Gram-positive bacteremia and NEC. Neonatal oral antibiotics may benefit mucosal and systemic immunity via delayed gut colonization and enhanced blood neutrophil maturation just after preterm birth.

Oligosaccharides support gut development and bacterial colonization in term infants, but it is unknown if they benefit preterm infants. Using preterm pigs, we investigated effects of bovine milk supplements enriched with oligosaccharides to improve gut development and colonization. Caesarean-delivered preterm pigs (n = 57) were reared for 19 days. The pigs were fed bovine milk supplemented with an oligosaccharide-enriched whey containing sialyllactose, or a heterogeneous oligosaccharide ingredient. To evaluate the influence of artificial rearing, near-term, vaginally born pigs raised by their sow (n = 12) were compared with artificially reared, caesarean-delivered near-term pigs (n = 14). In preterm pigs, the clinical outcome, gut function, gut microbiota, and systemic immunity were similar among dietary treatments. Natural rearing increased growth rates, gut functions, colon short chain fatty acid concentrations and bacterial diversity, relative to artificial rearing. In conclusion, supplements with bovine milk oligosaccharides were well tolerated, but did not improve gut maturation or clinical outcomes in artificially reared preterm piglets. Immaturity at birth, coupled with artificial rearing, may render the neonate unresponsive to the gut-protective effects of milk oligosaccharides. Whether bovine milk oligosaccharides may affect other endpoints (e.g., brain functions) in conditions of immaturity remains to be investigated.

BACKGROUND: Nutrient fortification of human milk is often required to secure adequate growth and organ development for very preterm infants. There is concern that formula-based fortifiers (FFs) induce intestinal dysfunction, feeding intolerance, and necrotizing enterocolitis (NEC). Bovine colostrum (BC) may be an alternative nutrient fortifier, considering its high content of protein and milk bioactive factors.OBJECTIVE: We investigated whether BC was superior to an FF product based on processed bovine milk and vegetable oil to fortify donor human milk (DHM) for preterm pigs, used as a model for infants.METHODS: Sixty preterm pigs from 4 sows (Danish Landrace × Large White × Duroc, birth weight 944 ± 29 g) received decreasing volumes of parenteral nutrition (96-72 mL ⋅ kg-1 ⋅ d-1) and increasing volumes of enteral nutrition (24-132 mL ⋅ kg-1 ⋅ d-1) for 8 d. Pigs were fed donor porcine milk (DPM) and DHM with or without FF or BC fortification (+4.6 g protein ⋅ kg-1 ⋅ d-1).RESULTS: DPM-fed pigs showed higher growth (10-fold), protein synthesis (+15-30%), villus heights, lactase and peptidase activities (+30%), and reduced intestinal cytokines (-50%) relative to DHM pigs (all P < 0.05). Fortification increased protein synthesis (+20-30%), but with higher weight gain and lower urea and cortisol concentrations for DHM+BC compared with DHM+FF pigs (2- to 3-fold differences, all P ≤ 0.06). DHM+FF pigs showed more diarrhea and reduced lactase and peptidase activities, hexose uptake, and villus heights relative to DHM+BC or DHM pigs (30-90% differences, P < 0.05). Fortification did not affect NEC incidence but DHM+BC pigs had lower colonic interleukin (IL)-6 and IL-8 concentrations relative to the remaining pigs (-30%, P = 0.06). DHM+FF pigs had higher stomach bacterial load than did DHM, and higher bacterial density along intestinal villi than did DHM and DHM+BC pigs (2- to 3-fold, P < 0.05).CONCLUSIONS: The FF product investigated in this study reduced growth, intestinal function, and protein utilization in DHM-fed preterm pigs, relative to BC as fortifier. The relevance of BC as an alternative nutrient fortifier for preterm infants should be tested.

BACKGROUND Fortification of donor human milk (DHM) is required for optimal growth of very preterm infants, but there are concerns of more gut dysfunction and necrotizing enterocolitis (NEC) when using formula-based fortifiers (FFs), especially soon after birth. Intact bovine colostrum (BC) is rich in nutrients and bioactive factors, and protects against NEC in preterm pigs. We hypothesized that fortification of DHM with BC is superior to FFs to prevent gut dysfunction and infections when provided shortly after preterm birth. METHODS Two FF products, Enfamil (ENF; intact protein, vegetable oil) and PreNAN+Nutrilon (NAN; extensively hydrolyzed protein, maltodextrin), were compared with BC as fortifier to DHM fed to preterm pigs for 5 days. RESULTS Relative to the DHM+BC group, DHM+FF groups had higher diarrhea score and lower hexose uptake and lactase activity, and specifically the DHM+NAN group showed higher gut permeability, NEC score, more mucosa-adherent bacteria with altered gut microbiota structure (ie, lower diversity, increased Enterococcus, decreased Staphylococcus abundance). Both DHM+FF groups showed higher expression of intestinal cytokine and inflammation-related genes, more gut-derived bacteria in the bone marrow, lower density of mucin-containing goblet cells, and slightly higher colon lactate, stomach pH and acetate, and blood neutrophil-to-lymphocyte levels than the DHM+BC group. CONCLUSIONS Used as a fortifier to DHM, BC is superior to FFs to support gut function, nutrient absorption, and bacterial defense mechanisms in preterm pigs. It is important to optimize the composition of nutrient fortifiers for preterm infants fed human milk.

Background: Holder pasteurization (HP) destroys multiple bioactive factors in donor human milk (DM), and UV-C irradiation (UVC) is potentially a gentler method for pasteurizing DM for preterm infants. Objective: We investigated whether UVC-treated DM improves gut maturation and resistance toward bacterial infections relative to HP-treated DM. Methods: Bacteria, selected bioactive components, and markers of antioxidant capacity were measured in unpasteurized donor milk (UP), HP-treated milk, and UVC-treated milk (all from the same DM pool). Fifty-seven cesarean-delivered preterm pigs (91% gestation; ratio of males to females, 30:27) received decreasing volumes of parental nutrition (average 69 mL ·kg-1 ·d-1) and increasing volumes of the 3 DM diets (n = 19 each, average 89 mL · kg-1 · d-1) for 8-9 d. Body growth, gut structure and function, and systemic bacterial infection were evaluated. Results: A high bacterial load in the UP (6 × 105 colony forming units/mL) was eliminated similarly by HP and UVC treatments. Relative to HP-treated milk, both UVC-treated milk and UP showed greater activities of lipase and alkaline phosphatase and concentrations of lactoferrin, secretory immunoglobulin A, xanthine dehydrogenase, and some antioxidantmarkers (all P < 0.05). The pigs fed UVC-treated milk and pigs fed UP showed higher relative weight gain than pigs fed HP-treated milk (5.4% and 3.5%), and fewer pigs fed UVC-treated milk had positive bacterial cultures in the bone marrow (28%) than pigs fed HP-treated milk (68%) (P < 0.05). Intestinal healthwas also improved in pigs fed UVC-treated milk compared with those fed HP-treated milk as indicated by a higher plasma citrulline concentration (36%) and villus height (38%) (P < 0.05) and a tendency for higher aminopeptidase N (48%) and claudin-4 (26%) concentrations in the distal intestine (P < 0.08). The gut microbiota composition was similar among groups except for greater proportions of Enterococcus in pigs fed UVC-treated milk than in pigs fed UP and those fed HP-treated milk in both cecum contents (20% and 10%) and distal intestinal mucosa (24% and 20%) (all P < 0.05). Conclusions: UVC is better than HP treatment in preserving bioactive factors in DM. UVC-treated milk may induce better weight gain, intestinal health, and resistance against bacterial infections as shown in preterm pigs as a model for DM-fed preterm infants.

The first genome-scale metabolic network of Cordyceps militaris (iWV1170) was constructed representing its whole metabolisms, which consisted of 894 metabolites and 1,267 metabolic reactions across five compartments, including the plasma membrane, cytoplasm, mitochondria, peroxisome and extracellular space. The iWV1170 could be exploited to explain its phenotypes of growth ability, cordycepin and other metabolites production on various substrates. A high number of genes encoding extracellular enzymes for degradation of complex carbohydrates, lipids and proteins were existed in C. militaris genome. By comparative genome-scale analysis, the adenine metabolic pathway towards putative cordycepin biosynthesis was reconstructed, indicating their evolutionary relationships across eleven species of entomopathogenic fungi. The overall metabolic routes involved in the putative cordycepin biosynthesis were also identified in C. militaris, including central carbon metabolism, amino acid metabolism (glycine, l -glutamine and l -aspartate) and nucleotide metabolism (adenosine and adenine). Interestingly, a lack of the sequence coding for ribonucleotide reductase inhibitor was observed in C. militaris that might contribute to its over-production of cordycepin.

Preterm neonates are highly sensitive to systemic infections in early life but little is known about systemic immune development following preterm birth. We hypothesized that preterm neonates have immature systemic immunity with distinct developmental trajectory for the first several weeks of life, relative to those born at near-term or term. Using pigs as a model, we characterized blood leukocyte subsets, antimicrobial activities and TLR-mediated cytokine production during the first weeks after preterm birth. Relative to near-term and term pigs, newborn preterm pigs had low blood leukocyte counts, poor neutrophil phagocytic rate, and limited cytokine responses to TLR1/2/5/7/9 and NOD1/2 agonists. The preterm systemic responses remained immature during the first postnatal week, but thereafter showed increased blood leukocyte numbers, NK cell proportion, neutrophil phagocytic rate and TLR2-mediated IL-6 and TNF-α production. These immune parameters remained different between preterm and near-term pigs at 2–3 weeks, even when adjusted for post-conceptional age. Our data suggest that systemic immunity follows a distinct developmental trajectory following preterm birth that may be influenced by postnatal age, complications of prematurity and environmental factors. Consequently, the immediate postnatal period may represent a window of opportunity to improve innate immunity in preterm neonates by medical, antimicrobial or dietary interventions.

The Brittle 2 (Bt2) gene encodes a small subunit of ADP-glucose pyrophosphorylase (AGPase) enzyme, one of the enzymes that play an important role in endosperm starch biosynthesis. We carried out a study to transfer the Bt2 gene into several inbred maize lines and regenerate transgenic maize plants possessing the transgene, which can be used to further study the role of the Bt2 gene in starch biosynthesis. Agrobacterium tumefaciens strains C58 and EHA105 harboring the transformation vector pCambia2300/Ubi-Bt2-Nos were used to transfer the Bt2 gene into five inbred maize lines, H14, H26, H240, H95, and N18. Transformed calli were selected on selection medium containing 100 mg/L of kanamycin. The stable integration of the Bt2 gene into transgenic plants was confirmed by Southern hybridizations. We recorded the different transformation frequencies in the experiments with A. Tumefaciens strains C58 and EHA105. Transgenic plants were regenerated from transformed calli of all five studied maize lines. Transformation frequencies varied from 1.09% to 2.33%, depending on maize genotypes. The results obtained show that our transformation system is effective for numerous maize genotypes. H95 and H240 are good lines for gene transfer in maize.

Bovine lactoferrin (bLF) may modulate neonatal intestinal inflammation. Previous studies in intestinal epithelial cells (IECs) indicated that moderate bLF doses enhance proliferation whereas high doses trigger inflammation. To further elucidate cellular mechanisms, we profiled the porcine IEC proteome after stimulation with bLF at 0, 0.1, 1 and 10 g/L by LC-MS-based proteomics. Key pathways were analyzed in the intestine of formula-fed preterm pigs with and without supplementation of 10 g/L bLF. Levels of 123 IEC proteins were altered by bLF. Low bLF doses (0.1–1 g/L) up-regulated 11 proteins associated with glycolysis, energy metabolism and protein synthesis, indicating support of cell survival. In contrast, a high bLF dose (10 g/L) up-regulated three apoptosis-inducing proteins, down-regulated five anti-apoptotic and proliferation-inducing proteins and 15 proteins related to energy and amino acid metabolism, and altered three proteins enhancing the hypoxia inducible factor-1 (HIF-1) pathway. In the preterm pig intestine, bLF at 10 g/L decreased villus height/crypt depth ratio and up-regulated the Bax/Bcl-2 ratio and HIF-1α, indicating elevated intestinal apoptosis and inflammation. In conclusion, bLF dose-dependently affects IECs via metabolic, apoptotic and inflammatory pathways. It is important to select an appropriate dose when feeding neonates with bLF to avoid detrimental effects exerted by excessive doses. Biological significance The present work elucidates dose-dependent effects of bLF on the proteomic changes of IECs in vitro supplemented with data from a preterm pig study confirming detrimental effects of enteral feeding with the highest dose of bLF (10 g/L). The study contributes to further understanding on mechanisms that bLF, as an important milk protein, can regulate the homeostasis of the immature intestine. Results from this study urge neonatologists to carefully consider the dose of bLF to supplement into infant formula used for preterm neonates.

Preterm infants are susceptible to infection and necrotizing enterocolitis (NEC) and are often treated with antibiotics. Simultaneous administration of enteral and parenteral antibiotics during the first days after preterm birth prevents formula-induced NEC lesions in pigs, but it is unknown which administration route is most effective. We hypothesized that only enteral antibiotics suppress gut bacterial colonization and NEC progression in formula-fed preterm pigs. Caesarean-delivered preterm pigs (90–92% of gestation) were fed increasing amounts of infant formula from birth to day 5 and given saline (CON) or antibiotics (ampicillin, gentamicin, and metronidazole) via the enteral (ENT) or parenteral (PAR) route ( n = 16–17). NEC lesions, intestinal morphology, function, microbiology, and inflammatory mediators were evaluated. NEC lesions were completely prevented in ENT pigs, whereas there were high incidences of mild NEC lesions (59–63%) in CON and PAR pigs ( P < 0.001). ENT pigs had elevated intestinal weight, villus height/crypt depth ratio, and goblet cell density and reduced gut permeability, mucosal adherence of bacteria, IL-8 levels, colonic lactic acid levels, and density of Gram-positive bacteria, relative to CON pigs ( P < 0.05). Values in PAR pigs were intermediate with few affected parameters (reduced lactic acid levels and density and adherence of Gram-positive bacteria, relative to CON pigs, P < 0.05). There was no evidence of increased antimicrobial resistance following the treatments. We conclude that enteral, but not parenteral, administration of antibiotics reduces gut bacterial colonization, inflammation, and NEC lesions in newborn, formula-fed preterm pigs. Delayed colonization may support intestinal structure, function, and immunity in the immediate postnatal period of formula-fed preterm neonates.