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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.

Background In the first weeks after birth, enteral feeding and bacterial colonization interact to influence gut maturation in preterm infants. Bovine colostrum (BC) has been suggested as a relevant supplementary diet when own mother's milk (MM) is insufficient or absent. In a pilot trial whether the supplement type, BC or human donor milk (DM), affects gut colonization in preterm infants during the first week of life was tested. Methods On day 7 faecal samples were collected from preterm infants (n = 24) fed BC or DM as a supplement to MM. The gut microbiome (GM) was analysed by 16S rRNA amplicon sequencing. Correlations between the relative abundance of specific bacterial taxa and blood chemistry variables, including amino acids, were explored. Results BC-supplemented infants showed a lower relative abundance of the families Lactobacillaceae and Enterococcaceae than DM infants. Planococcaceae was more abundant in infants delivered by caesarean section versus vaginally. The relative abundance of bacterial families, specifically Enterobacteriaceae, correlated negatively with plasma levels of multiple essential and non-essential amino acids (Val, Ile, Lys, His, Arg). Conclusion The nature of nutritional supplements (BC or DM) just after birth may affect GM development and nutrient metabolism in the neonatal period of preterm infants. The exploratory nature of our study calls for confirmation of these results and their possible long-term clinical implications for preterm infants. This article is protected by copyright. All rights reserved.

Background Chorioamnionitis (CA) is a risk factor for preterm birth and is associated with neurodevelopmental delay and cognitive disorders. Prenatal inflammation-induced brain injury may resolve during the immediate postnatal period, which is a time of rapid metabolic and brain remodeling. Cerebrospinal fluid (CSF) collected at birth may be a critical source of predictive biomarkers. Using pigs as a model of preterm infants exposed to CA, we hypothesized that prenatal lipopolysaccharide (LPS) exposure induces proteome changes in the CSF and brain both at birth and later. Methods Fetal piglets (103 days gestation of the full-term 117 days) were administered intra-amniotic (IA) lipopolysaccharide (LPS) 3 days before preterm delivery by caesarian section. CSF and brain tissue were collected on postnatal Days 1 and 5 (P1 and P5). CSF and hippocampal proteins were profiled by LC–MS-based quantitative proteomics. Neuroinflammatory responses in the cerebral cortex, periventricular white matter and hippocampus were evaluated by immunohistochemistry, and gene expression was evaluated by qPCR. Results Pigs exposed to LPS in utero showed changes in CSF protein levels at birth but not at P5. Complement protein C3, hemopexin, vasointestinal peptide, carboxypeptidase N subunit 2, ITIH1 and plasminogen expression was upregulated in the CSF, while the expression of proteins associated with axon growth and synaptic functions (FGFR1, BASP1, HSPD1, UBER2N, and RCN2), adhesion (Talin1), and neuronal survival (Atox1) was downregulated. Microglia, but not astrocytes, were activated by LPS at P5 in the hippocampus but not in other brain regions. At this time, marginal increases in complement protein C3, LBP, Hif1a, Basp1, Minpp1 and FGFR1 transcription indicated hippocampal proinflammatory responses. Conclusion A brief period of prenatal endotoxin exposure induces proteome changes in the CSF and brain at birth, but most changes resolve a few days later. The developing hippocampus has high neuronal plasticity in response to perinatal inflammation. Changes in CSF protein expression at birth may help to predict later structural brain damage in preterm infants exposed to variable types and durations of CA-related inflammation in utero.

Infant formulas offer an alternative to breast milk for both normal birth weight (NBW) and immunocompromised intrauterine growth restricted (IUGR) infants. Although the lipid fraction in formulas is often derived from vegetable oils, it is unclear if this alters immunological outcomes relative to milk fats or whether these effects differ between IUGR and NBW infants. We hypothesized that replacing vegetable oil with bovine milk fat in infant formula would improve immune development in IUGR and NBW neonates. Two-day old piglets were selected (NBW, n = 18, IUGR, n = 18) and each group of animals were fed formula based on either vegetable oil (VEG) or bovine milk fat (MILK). Animals were reared until day 23/24 and systemic immune parameters were evaluated. Milk-fat feeding decreased blood neutrophil counts and improved neutrophil function while transiently reducing leucocytes’ expression of genes related to adaptive and innate immunity as well as energy metabolism, following in vitro stimulation by live Staphylococcus epidermidis (whole blood, 2 h). However, there were only a few interactions between milk-fat type and birthweight status. Thus, piglets fed milk-fat-based formula had improved neutrophil maturation and suppressed pro-inflammatory responses, compared to those fed vegetable-oil-based formula.

Preterm infants are susceptible to bloodstream infection that can lead to sepsis. High parenteral glucose supplement is commonly used to support their growth and energy expenditure, but may exceed endogenous regulation during infection, causing dysregulated immune response and clinical deterioration. Using a preterm piglet model of neonatal sepsis induced by Staphylococcus epidermidis infection, we demonstrate the delicate interplay between immunity and energy metabolism to regulate the host infection response. Circulating glucose levels, glycolysis and inflammatory response to infection are closely connected across the states of tolerance, resistance and immunoparalysis. Further, high parenteral glucose provision during infection induces hyperglycemia, elevated glycolysis and inflammation, leading to lactate acidosis and sepsis, whereas glucose restricted individuals are clinically unaffected with increased gluconeogenesis to maintain moderate hypoglycemia. Finally, pharmacological glycolysis inhibition during normoglycemia enhances bacterial clearance and dampens inflammation but fails to prevent sepsis. Our results uncover how blood glucose controls immune cell metabolism and function, in turn determining the clinical fate of infected preterm neonates. This also questions the current practice of parenteral glucose supply for infected preterm infants.

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.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.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).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.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.

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.

Preterm infants frequently develop necrotizing enterocolitis (NEC), a severe intestinal disorder associated with high mortality. Early detection of NEC is difficult due to poor specificity and sensitivity of clinical signs. We hypothesized that early development of NEC, before clear clinical symptoms appear, might affect expression of blood genes, potentially related to early systemic immune responses. Using preterm pigs as models for preterm infants, a retrospective analysis was performed on 129 infant formula fed pigs that had NEC diagnosis at necropsy on day 5. Clinical data including growth, activity, hematology, gastric residuals, incidence of diarrhea, bloody stool and abdominal distention were retrospectively reviewed. During this early postnatal period, except that bloody stool was observed in 19% of NEC pigs and absent in healthy pigs, no other clinical outcomes showed difference between NEC and healthy pigs. Whole blood transcriptome was compared between NEC pigs (NEC, n=20) and their litter-mate healthy controls (CON, n=19) on day 5, and revealed 344 differentially expressed genes (DEGs). PubMed literature search identified 123 genes that co-occurred with at least one of 9 NEC-related keywords (NEC, colitis, necrotic, hemorrhage, epithelial apoptosis, intestinal inflammation, inflammatory bowel disease, Crohn’s disease, ulcerative colitis). Co-expression network analysis suggested PAK2 as one of the hub genes. Using whole blood and dried blood spots (DBS) from another group of preterm pigs for validation, up-regulation of PAK2 and genes that co-occurred with NEC and other keywords in PubMed literatures (AOAH, ARG2, FKBP5 and STAT3) was confirmed in severe NEC cases. Specifically, ex vivo stimulation of cord blood with S.epidermidis increased ARG2. Our results show that whole blood gene expressions are affected in preterm pigs at an early stage when NEC is suspicious. Expression of target genes may be used to indicate NEC severity and associated bacterial infection. Routinely collected neonatal DBS may be used to develop early biomarkers for identifying infants with severe NEC lesions, thus providing better intervention strategy.

Neonatal sepsis remains a significant cause of morbidity and mortality especially in the preterm infant population. The ability to promptly and accurately diagnose neonatal sepsis based on clinical evaluation and laboratory blood tests remains challenging. Advances in high-throughput molecular technologies have increased investigations into the utility of transcriptomic, proteomic and metabolomic approaches as diagnostic tools for neonatal sepsis. A systems-level understanding of neonatal sepsis, obtained by using omics-based technologies (at the transcriptome, proteome or metabolome level), may lead to new diagnostic tools for neonatal sepsis. In particular, recent omic-based studies have identified distinct transcriptional signatures and metabolic or proteomic biomarkers associated with sepsis. Despite the emerging need for a systems biology approach, future studies have to address the challenges of integrating multi-omic data with laboratory and clinical meta-data in order to translate outcomes into precision medicine for neonatal sepsis. Omics-based analytical approaches may advance diagnostic tools for neonatal sepsis. More research is needed to validate the recent systems biology findings in order to integrate multi-dimensional data (clinical, laboratory and multi-omic) for future translation into precision medicine for neonatal sepsis. This review will discuss the possible applications of omics-based analyses for identification of new biomarkers and diagnostic signatures for neonatal sepsis, focusing on the immune-compromised preterm infant and considerations for clinical translation.