Skeletal consequences of preterm birth in pigs as a model for preterm infants.

Preterm birth affects about 10% of all live births with many resultant health challenges, including metabolic bone disease of prematurity (MBDP) which is characterized by elevated alkaline phosphatase, suppressed phosphate, and deficient skeletal development. Because of the lack of an animal model, very little is known about bone structure, strength, and quality after preterm birth. This study investigated the utility of a pig model to replicate clinical features of preterm birth, including MBDP, and sought to determine if early postnatal administration of insulin-like growth factor (IGF)-1 was an effective treatment. Preterm pigs, born by caesarean section at 90% gestation, were reared in intensive care facilities (respiratory, thermoregulatory and nutritional support) and compared with sow-reared term pigs born vaginally. Preterm pigs were systemically treated with vehicle or IGF-1 (recombinant human IGF-1/BP-3, 2.25 mg/kg/day). Tissues were collected at postnatal days 1, 5, and 19 (the normal weaning period in pigs). Most bone-related outcomes were affected by preterm birth throughout the study period whereas IGF-1 supplementation had almost no effect. By day 19, alkaline phosphatase was elevated, phosphate and calcium were reduced, and the bone resorption marker CTX-1 was elevated in preterm pigs compared to term pigs. Preterm pigs also had decrements in femoral cortical cross-sectional properties, consistent with reduced whole-bone strength. Thus, the preterm pig model replicates many features of preterm bone development in infants, including features of MBDP, and allows for direct interrogation of skeletal tissues, enhancing the field's ability to examine underlying mechanisms.

Premature birth interrupts a critical period of skeletal development as the majority of fetal bone mineral accumulation occurs during the last gestational trimester, leaving preterm infants at increased risk for low bone mineral density and fractures. While there are some data on growth in bone mass in preterm infants, very little is known about bone structural properties, quality, and strength during development after preterm birth. In this study we sought to evaluate the pig as a model for postnatal skeletal development after premature birth. Preterm pigs born after approximately 90% of the full gestation period were compared to full-term control pigs through day 19 of life. Levels of two blood markers used to diagnose osteoporosis of prematurity were replicated in the pig model. Bone properties related to strength were reduced even when accounting for their smaller body size, possibly suggesting elevated fracture risk in preterm infants. Based on the similarities between the preterm pig model and preterm human infants, the pig model may prove to be useful to study factors and interventions affecting postnatal bone development after preterm birth.

Adverse Metabolic Phenotypes in Parenterally Fed Neonatal Pigs Do Not Persist into Adolescence.

BACKGROUND: Nutrition during fetal and neonatal life is an important determinant for the risk of adult-onset diseases, especially type 2 diabetes and obesity. OBJECTIVES: We aimed to determine whether total parenteral nutrition (TPN) compared with enteral formula feeding [enteral nutrition (EN)] in term piglets during the first 2 wk after birth would increase the long-term (5-mo) development of metabolic syndrome phenotypes with adverse glucose homeostasis, fatty liver disease, and obesity. METHODS: Neonatal female pigs were administered TPN (n = 12) or fed enterally with a liquid enteral milk-replacer formula (EN, n = 12) for 14 d. After transitioning TPN pigs to enteral feeding of liquid formula (days 15-26), both groups were adapted to a solid high-fat diet (30% of the total diet) and sucrose (20% of the total diet) diet (days 27-33), which was fed until the end of the study (140 d). Body composition was measured by dual-energy X-ray absorptiometry at 14, 45, and 140 d. Serum biochemistry and glucose-insulin values (after a fasting intravenous glucose tolerance test) were obtained at 140 d. Liver and muscle were analyzed for insulin receptor signaling and triglycerides. RESULTS: Body weight was similar, but percent fat was higher, whereas percent lean and bone mineral density were lower in TPN than in EN pigs (P < 0.01) at 45 d of age but not at 140 d. At 140 d, there were no differences in serum markers of liver injury or lipidemia. Intravenous glucose tolerance test at 140 d showed a lower (P < 0.05) AUC for both glucose and insulin in TPN than in EN pigs, but the ratio of AUCs of insulin and glucose was not different between groups. CONCLUSIONS: Administration of TPN during the neonatal period increased adipose deposition that transiently persisted in early adolescence when challenged with a high-fat diet but was not sustained or manifested as glucose intolerance.

Trial-related blood sampling and red blood cell transfusions in preterm infants.

AIM: To determine if trial-related blood sampling increases the risk of later red blood cell (RBC) transfusion in very preterm infants, we compared the volume of clinical- and trial-related blood samples, in a specific trial and correlated to subsequent RBC transfusion. METHODS: For 193 very preterm infants, participating in the FortiColos trial (NCT03537365), trial-related blood volume drawn was in accordance with ethical considerations established by the European Commission. Medical records were reviewed to assess the number and accumulated volume (mL/kg) of blood samples (both clinical- and trial-related). Data were compared with the need of RBC transfusions during the first 28 days of life. RESULTS: Mean (SD) gestational age and birth weight was 28 ± 1 weeks and 1168 ± 301 g. In total, 11% of total blood volume was drawn for sampling (8.1 ± 5.1 mL/kg) and trial-related sampling accounted for 1.6 ± 0.6 mL/kg. Trial-related blood sampling had no impact on RBC transfusion (p = 0.9). CONCLUSION: Clinical blood sampling in very preterm infants is associated with blood loss and subsequent need for RBC transfusions. In a specific trial requiring blood samples, we found no additional burden of trial-related blood sampling. The study suggests that trial-related sampling is safe if European criteria are followed.

Insulin-like growth factor 1 supplementation supports motor coordination and affects myelination in preterm pigs.

Introduction: Preterm infants have increased risk of impaired neurodevelopment to which reduced systemic levels of insulin-like growth factor 1 (IGF-1) in the weeks after birth may play a role. Hence, we hypothesized that postnatal IGF-1 supplementation would improve brain development in preterm pigs, used as a model for preterm infants. Methods: Preterm pigs delivered by cesarean section received recombinant human IGF-1/IGF binding protein-3 complex (rhIGF-1/rhIGFBP-3, 2.25 mg/kg/day) or vehicle from birth to postnatal day 19. Motor function and cognition were assessed by monitoring of in-cage and open field activities, balance beam test, gait parameters, novel object recognition and operant conditioning tests. Collected brains were subject to magnetic resonance imaging (MRI), immunohistochemistry, gene expression analyses and protein synthesis measurements. Results: The IGF-1 treatment increased cerebellar protein synthesis rates (both in vivo and ex vivo). Performance in the balance beam test was improved by IGF-1 but not in other neurofunctional tests. The treatment decreased total and relative caudate nucleus weights, without any effects to total brain weight or grey/white matter volumes. Supplementation with IGF-1 reduced myelination in caudate nucleus, cerebellum, and white matter regions and decreased hilar synapse formation, without effects to oligodendrocyte maturation or neuron differentiation. Gene expression analyses indicated enhanced maturation of the GABAergic system in the caudate nucleus (decreased NKCC1:KCC2 ratio) with limited effects in cerebellum or hippocampus. Conclusion: Supplemental IGF-1 during the first three weeks after preterm birth may support motor function by enhancing GABAergic maturation in the caudate nucleus, despite reduced myelination. Supplemental IGF-1 may support postnatal brain development in preterm infants, but more studies are required to identify optimal treatment regimens for subgroups of very or extremely preterm infants.

Hypothyroidism impairs development of the gastrointestinal tract in the ovine fetus.

Growth and maturation of the fetal gastrointestinal tract near term prepares the offspring for the onset of enteral nutrition at birth. Structural and functional changes are regulated by the prepartum rise in cortisol in the fetal circulation, although the role of the coincident rise in plasma tri-iodothyronine (T3) is unknown. This study examined the effect of hypothyroidism on the structural development of the gastrointestinal tract and the activity of brush-border digestive enzymes in the ovine fetus near term. In intact fetuses studied between 100 and 144 days of gestation (dGA; term ∼145 days), plasma concentrations of T3, cortisol and gastrin; the mucosal thickness in the abomasum, duodenum, jejunum and ileum; and intestinal villus height and crypt depth increased with gestational age. Removal of the fetal thyroid gland at 105-110 dGA suppressed plasma thyroxine (T4) and T3 concentrations to the limit of assay detection in fetuses studied at 130 and 144 dGA, and decreased plasma cortisol and gastrin near term, compared to age-matched intact fetuses. Hypothyroidism was associated with reductions in the relative weights of the stomach compartments and small intestines, the outer perimeter of the intestines, the thickness of the gastric and intestinal mucosa, villus height and width, and crypt depth. The thickness of the mucosal epithelial cell layer and muscularis propria in the small intestines were not affected by gestational age or treatment. Activities of the brush border enzymes varied with gestational age in a manner that depended on the enzyme and region of the small intestines studied. In the ileum, maltase and dipeptidyl peptidase IV (DPPIV) activities were lower, and aminopeptidase N (ApN) were higher, in the hypothyroid compared to intact fetuses near term. These findings highlight the importance of thyroid hormones in the structural and functional development of the gastrointestinal tract near term, and indicate how hypothyroidism in utero may impair the transition to enteral nutrition and increase the risk of gastrointestinal disorders in the neonate.

Nurses' and Physicians' Rationale Behind Clinical Performance and Interpretation of Routine Prefeed Gastric Aspiration in Preterm Infants: A Cross-sectional Study.

This study aims at understanding the rationale behind performing prefeed gastric aspirations in preterm infants, how nurses and physicians interpret the gastric aspiration and variations between them, and illuminating potential barriers for omitting routine prefeed aspiration. Nurses and physicians from all Danish neonatal intensive care units completed a questionnaire. Of 682 participants, the majority (94%) indicated that they routinely performed prefeed aspiration, primarily to check the feeding tube placement (nurses: 88%, physicians: 46%). Nurses feared necrotizing enterocolitis when observing a large gastric residual (GR) volume (31%) and green-stained GR (63%). Fewer nurses relative to physicians had "no worries" related to large volumes (15% vs 34%) or green-stained GR (14% vs 24%, both P < .01). More nurses than physicians intended to pause enteral feeding when observing green-stained GR (31% vs 16%, P < .01) and more nurses were concerned of completely omitting routine gastric aspirations (90% vs 46%, P < .05). The rationale behind the clinical use of GR volume and color as markers of necrotizing enterocolitis and feeding intolerance differs markedly between nurses and physicians in Denmark. If routine prefeed gastric aspiration should be omitted, special focus on information about early signs of necrotizing enterocolitis and methods to check tube placement is needed.

Subclinical necrotizing enterocolitis-induced systemic immune suppression in neonatal preterm pigs.

Preterm infants are at high risks of sepsis and necrotizing enterocolitis (NEC). Some develop sepsis shortly after suspected or confirmed NEC, implying that NEC may predispose to sepsis but the underlying mechanisms are unknown. Using NEC-sensitive preterm pigs as models, we investigated the immune status in animals following development of subclinical NEC-like lesions with variable severities. Caesarean-delivered preterm pigs were reared until day 5 or day 9. Blood was analyzed for T-cell subsets, neutrophil phagocytosis, transcriptomics, and immune responses to in vitro LPS challenge. Gut tissues were used for histology and cytokine analyses. Pigs with/without macroscopic NEC lesions were scored as healthy, mild, or severe NEC. Overall NEC incidence was similar on day 5 and day 9 (61%-62%) but with lower severity on day 9, implying gradual mucosal repair following the early phase of NEC. Pigs with NEC showed decreased goblet cell density and increased MPO+ and CD3+ cell infiltration in the distal small intestine or colon. Mild or severe NEC lesions had limited effects on circulating parameters on day 5. On day 9, pigs with NEC lesions (especially severe lesions) showed systemic immune suppression, as indicated by elevated Treg frequency, impaired neutrophil phagocytosis, low expression of genes related to innate immunity and Th1 polarization, and diminished LPS-induced immune responses. In conclusion, we shows evidence for NEC-induced systemic immune suppression, even with mild and subclinical NEC lesions. The results help to explain that preterm infants suffering from NEC may show high sensitivity to later secondary infections and sepsis.NEW & NOTEWORTHY Necrotizing enterocolitis (NEC) and sepsis are common diseases in preterm infants. Many develop sepsis following an episode of suspected NEC, suggesting NEC as a predisposing factor for sepsis but mechanisms are unclear. Using preterm pigs as a model, now we show that subclinical NEC lesions, independent of clinical confounding factors, induces systemic immune suppression. The results may help to explain the increased risks of infection and sepsis in preterm infants with previous NEC diagnosis.

Dairy-Derived Emulsifiers in Infant Formula Show Marginal Effects on the Plasma Lipid Profile and Brain Structure in Preterm Piglets Relative to Soy Lecithin.

Breastfed infants have higher intestinal lipid absorption and neurodevelopmental outcomes compared to formula-fed infants, which may relate to a different surface layer structure of fat globules in infant formula. This study investigated if dairy-derived emulsifiers increased lipid absorption and neurodevelopment relative to soy lecithin in newborn preterm piglets. Piglets received a formula diet containing soy lecithin (SL) or whey protein concentrate enriched in extracellular vesicles (WPC-A-EV) or phospholipids (WPC-PL) for 19 days. Both WPC-A-EV and WPC-PL emulsions, but not the intact diets, increased in vitro lipolysis compared to SL. The main differences of plasma lipidomics analysis were increased levels of some sphingolipids, and lipid molecules with odd-chain (17:1, 19:1, 19:3) as well as mono- and polyunsaturated fatty acyl chains (16:1, 20:1, 20:3) in the WPC-A-EV and WPC-PL groups and increased 18:2 fatty acyls in the SL group. Indirect monitoring of intestinal triacylglycerol absorption showed no differences between groups. Diffusor tensor imaging measurements of mean diffusivity in the hippocampus were lower for WPC-A-EV and WPC-PL groups compared to SL indicating improved hippocampal maturation. No differences in hippocampal lipid composition or short-term memory were observed between groups. In conclusion, emulsification of fat globules in infant formula with dairy-derived emulsifiers altered the plasma lipid profile and hippocampal tissue diffusivity but had limited effects on other absorptive and learning abilities relative to SL in preterm piglets.

Gut transit time, using radiological contrast imaging, to predict early signs of necrotizing enterocolitis.

BACKGROUND: Immature gut motility in preterm neonates may be a risk factor for necrotizing enterocolitis (NEC). Using preterm pigs as a model for infants, we hypothesized that intestinal dysmotility precedes NEC development. METHODS: Eighty-five preterm pigs were fed increasing amounts of milk diets to induce NEC lesions, as detected at autopsy on day 5. Gut transit time was determined on day 4 by x-ray imaging after oral intake of contrast solution. RESULTS: No clinical or radiological signs of NEC were detected on day 4, but macroscopic NEC lesions were recorded in 59% of pigs (n = 50) on day 5. Relative to pigs without NEC (noNEC, n = 35), pigs with small intestinal lesions (siNEC, n = 18) showed delayed stomach emptying time (StEmpty) and time for contrast to reach cecum (ToCecum) already on day 4. Pigs with lesions only in colon (coNEC, n = 20) showed more diarrhea, shorter ToCecum time, but longer small intestinal emptying time (SiEmpty). ToCecum time predicted siNEC and coNEC lesions with a receiver-operator characteristic area under the curve of 78-81%. CONCLUSIONS: Region-dependent changes in gut transit time is associated with early NEC development in preterm pigs. How gut dysmotility is related to NEC in preterm infants requires further investigations. IMPACT: Using preterm pigs as a model for preterm infants, we show that gut transit time, using serial x-ray contrast imaging, was changed in individuals with NEC-like lesions before they showed the typical radiological signs of NEC. Thus prolonged transit time across the entire gut was recorded when NEC lesions appeared in the small intestine but not when lesions were detected only in the colon. Until now, recordings of food transit have mainly investigated changes in the upper gut. Using serial x-rays, this study describes food transit across the entire gut and documents a region-dependent effect of NEC lesions on gut transit changes in preterm individuals. The findings provide proof of concept for use of x-ray contrast imaging as a tool to monitor gut transit in preterm pigs as models for infants. Delayed passage across the entire gut may be an early sign of small intestinal NEC, at least in pigs. More studies are needed to confirm relations in infants. In the future, it might be possible to use x-ray contrast imaging in preterm infants to better understand gut motility in relation to early NEC progression and need for medical NEC treatment.

Early Use of Antibiotics Is Associated with a Lower Incidence of Necrotizing Enterocolitis in Preterm, Very Low Birth Weight Infants: The NEOMUNE-NeoNutriNet Cohort Study.

OBJECTIVE: To determine whether commencement of antibiotics within 3 postnatal days in preterm, very low birth weight (VLBW; ≤1500 g) infants is associated with the development of necrotizing enterocolitis (NEC). STUDY DESIGN: Preplanned statistical analyses were done to study the association between early antibiotic treatment and later NEC development, using the NEOMUNE-NeoNutriNet cohort of VLBW infants from 13 neonatal intensive care units (NICUs) in 5 continents (n = 2831). NEC incidence was compared between infants who received early antibiotics and those who did not, with statistical adjustments for NICU, gestational age, birth weight, sex, delivery mode, antenatal steroid use, Apgar score, and type and initiation of enteral nutrition. RESULTS: The incidence of NEC was 9.0% in the group of infants who did not receive early antibiotics (n = 269), compared with 3.9% in those who did receive early antibiotics (n = 2562). The incidence remained lower in the early antibiotic group after stepwise statistical adjustments for NICU (OR, 0.57; 95% CI, 0.35-0.94, P < .05) and other potential confounders (OR, 0.25; 95% CI, 0.12-0.47; P < .0001). CONCLUSIONS: In this large international cohort of preterm VLBW infants, a small proportion of infants did not receive antibiotics just after birth, and these infants had a higher incidence of NEC. It is important to better understand the role of such variables as time, type, and duration of antibiotic treatment on NEC incidence, immune development, gut colonization, and antibiotic resistance in the NICU.

Rapid Gut Adaptation to Preterm Birth Involves Feeding-Related DNA Methylation Reprogramming of Intestinal Genes in Pigs.

Following preterm birth, the immature gut function and immunology must rapidly adapt to cope with bacterial colonization and enteral milk feeding. We hypothesized that intestinal epigenetic changes are involved in the gut response to preterm birth and the first feeding. Using piglets as models for infants, preterm, and term pigs were fed total parenteral nutrition (TPN) or partial enteral feeding for 5 days, followed by exclusive enteral feeding with bovine milk until day 26 (weaning age). Intestinal structure, function, microbiome, DNA methylome, and gene expressions were compared between preterm and term pigs on days 0, 5, and 26 (n = 8 in each group). At birth, the intestine of preterm pigs showed villus atrophy and global hypermethylation, affecting genes related to the Wnt signaling pathway. Hypermethylation-associated lowered expression of lipopolysaccharide-binding protein and genes related to the Toll-like receptor 4 pathway were evident during the first 5 days of life, but most early methylation differences disappeared by day 26. Regardless, sucrase and maltase activities (adult-type brush border enzymes) remained reduced, and the gut microbiota altered (fewer Akkermansia, more Lachnoclostridia and Lactobacilli) until day 26 in preterm pigs. During the 0- to 5-day period, many new preterm-term methylation differences appeared, but mainly when no enteral feed was provided (TPN feeding). These methylation differences affected intestinal genes related to cell metabolism, including increased GCK (glucokinase) expression via promoter hypomethylation. In conclusion, the immature intestine has a remarkable capacity to adapt its gene methylation and expression after preterm birth, and only few preterm-related defects persisted until weaning. Early enteral feeding may be important to stimulate the methylation reprogramming of intestinal genes, allowing rapid intestinal adaptation to preterm birth.

Postnatal Gut Immunity and Microbiota Development Is Minimally Affected by Prenatal Inflammation in Preterm Pigs.

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.

Bioactive proteins in bovine colostrum and effects of heating, drying and irradiation.

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, ß-LgA, ß-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.

Prenatal inflammation suppresses blood Th1 polarization and gene clusters related to cellular energy metabolism in preterm newborns.

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.

Malnutrition Predisposes to Endotoxin-Induced Edema and Impaired Inflammatory Response in Parenterally Fed Piglets.

BACKGROUND: Poor nutrition status is common among hospitalized children and children in low-income countries and may be associated with increased susceptibility to edema and infections. We hypothesized that poor nutrition status, established with a suboptimal composition of parenteral nutrition (PN), predisposes to endotoxemia-induced edema, oxidative stress, and dysregulated immune responses. METHODS: Using a 2 × 2 factorial design, 3-day-old piglets (n = 40) were given either optimal or suboptimal composition of PN for 7 days and then infused with either saline or lipopolysaccharide (LPS) for 9 hours to induce an acute-phase reaction. Abdominal tissue edema and blood markers of immunity, inflammation, and oxidative stress were assessed. RESULTS: Piglets receiving suboptimal nutrition showed signs of malnutrition with restricted growth, signs of inflammation (elevated C-reactive protein [CRP], interleukin-6, and serum amyloid A levels), oxidative stress (lower erythrocyte glutathione/hemoglobin and α-tocopherol/cholesterol ratios), and liver dysfunction (increased liver weight and blood bilirubin levels). Perirenal edema was more excessive in malnourished LPS-infused animals, relative to healthy LPS-infused control animals (P < .01). Malnutrition reduced the inflammatory response to LPS (lower CRP, tumor necrosis factor-α, haptoglobin, and neutrophil to lymphocyte ratio) but did not influence LPS-induced oxidative stress markers. CONCLUSIONS: We conclude that endotoxemia and malnutrition in combination lead to acute-phase hyporesponsiveness and perirenal edema in piglets. This finding may have implications for pediatric patients that suffer from malnutrition, as their response to bacterial infections may differ substantially from patients of normal nutrition status.

Gut and immune effects of bioactive milk factors in preterm pigs exposed to prenatal inflammation.

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-1ß 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.

FortiColos - a multicentre study using bovine colostrum as a fortifier to human milk in very preterm infants: study protocol for a randomised controlled pilot trial.

BACKGROUND: Very preterm infants (< 32 weeks gestation) have a relatively high nutrient requirement for growth and development. The composition of human milk is often inadequate to ensure optimal growth so it is common to fortify human milk for very preterm infants with nutrient fortifiers based on bovine milk. However, there are concerns that bovine milk-based fortifiers may increase the risk of feeding intolerance, necrotizing enterocolitis and late-onset sepsis. We hypothesize that a bovine colostrum-based product is a suitable alternative to bovine milk-based products when used as a fortifier to human milk in very preterm infants. METHODS/DESIGN: In an open-label multicentre randomised controlled pilot trial, 200 very preterm infants (26 + 0 to 30 + 6 weeks gestation at birth) will be randomly allocated to a bovine colostrum-based or a bovine milk-based fortifier added to mother's own milk and/or human donor milk. Outcomes are growth rate, incidence of necrotizing enterocolitis and late-onset sepsis, a series of paraclinical endpoints, and practical feasibility of using the novel fortifier for very preterm infants. DISCUSSION: The optimal enteral diet and feeding regimen for very preterm infants remain debated; this clinical trial will document the feasibility, safety and preliminary efficacy of using bovine colostrum, rich in nutrients and bioactive factors, as a novel fortifier for human milk to very preterm infants. Data on infant growth, metabolism, gut function and immunity will be assessed from clinical data as well as blood and stool samples. TRIAL REGISTRATION: Registered retrospectively 25 May 2018 at ClinicalTrials.gov: NCT03537365 .

Synbiotics Combined with Glutamine Stimulate Brain Development and the Immune System in Preterm Pigs.

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 < 0.01), whereas motor function and exploratory interest were similar between the groups. Using ex vivo diffusion imaging, the orientation dispersion index in brain cortical gray matter was 50% higher (P = 0.04), and fractional anisotropy value was 7% lower (P = 0.05) in PPG pigs compared with CON pigs, consistent with increased dendritic branching in PPG. In associative fibers, radial diffusivity was lower and fractional anisotropy was higher in PPG pigs compared with CON pigs (all P < 0.05), while measures in the internal capsule showed a tendency towards reduced radial diffusivity and mean diffusivity (both P = 0.09). On day 23 pigs in the PPG group showed higher blood leukocyte numbers (+43%), neutrophil counts (+100%), and phagocytic rates (+24%), relative to CON, all P < 0.05. Conclusion: Preterm pigs supplemented with Bifidobacterium breve, galacto- and fructo-oligosaccharides, and l-glutamine showed enhanced neuronal and immunological development. The findings indicate the potential for targeted nutritional interventions after preterm birth, to support development of important systems such as immunity and brain.