Short-chain fatty acid butyrate, a breast milk metabolite, enhances immature intestinal barrier function genes in response to inflammation in vitro and in vivo.

Infants born under 1,500 g have an increased incidence of necrotizing enterocolitis in the ileum and the colon, which is a life-threatening intestinal necrosis. This is in part due to excessive inflammation in the immature intestine to colonizing bacteria because of an immature innate immune response. Breastmilk complex carbohydrates create metabolites of colonizing bacteria in the form of short-chain fatty acids (SCFAs). We studied the effect of breastmilk metabolites, SCFAs, on immature intestine with regard to anti-inflammatory effects. This showed that acetate, propionate, and butyrate were all anti-inflammatory to an IL-1ß inflammatory stimulus. In this study, to further define the mechanism of anti-inflammation, we created transcription profiles of RNA from immature human enterocytes after exposure to butyrate with and without an IL-1ß inflammatory stimulus. We demonstrated that butyrate stimulates an increase in tight-junction and mucus genes and if we inhibit these genes, the anti-inflammatory effect is partially lost. SCFAs, products of microbial metabolism of complex carbohydrates of breastmilk oligosaccharides, have been found with this study to induce an anti-IL-1ß response that is associated with an upregulation of tight junctions and mucus genes in epithelial cells (H4 cells). These studies suggest that breastmilk in conjunction with probiotics can reduce excessive inflammation with metabolites that are anti-inflammatory and stimulate an increase in the mucosal barrier.NEW & NOTEWORTHY This study extends previous observations to define the anti-inflammatory properties of butyrate, a short-chain fatty acid produced by the metabolism of breastmilk oligosaccharides by colonizing bacteria. Using transcription profiling of immature enterocyte genes, after exposure to butyrate and an IL-1ß stimulus, we showed that tight-junction genes and mucus genes were increased, which contributed to the anti-inflammatory effect.

Indole-3-lactic acid, a metabolite of tryptophan, secreted by Bifidobacterium longum subspecies infantis is anti-inflammatory in the immature intestine.

BACKGROUND: Necrotizing enterocolitis (NEC), a necrotic inflammation of the intestine, represents a major health problem in the very premature infant. Although prevention is difficult, the combination of ingestion of maternal-expressed breastmilk in conjunction with a probiotic provides the best protection. In this study, we establish a mechanism for breastmilk/probiotic protection. METHODS: Ultra-high-performance liquid chromatography-tandem mass spectrometry of Bifidobacterium longum subsp. infantis (B. infantis) secretions was used to identify an anti-inflammatory molecule. Indole-3-lactic acid (ILA) was then tested in an established human immature small intestinal cell line, necrotizing colitis enterocytes, and other immature human enteroids for anti-inflammatory effects and to establish developmental function. ILA was also examined in immature and mature enterocytes. RESULTS: We have identified ILA, a metabolite of breastmilk tryptophan, as the anti-inflammatory molecule. This molecule is developmentally functional in immature but not mature intestinal enterocytes; ILA reduces the interleukin-8 (IL-8) response after IL-1ß stimulus. It interacts with the transcription factor aryl hydrocarbon receptor (AHR) and prevents transcription of the inflammatory cytokine IL-8. CONCLUSIONS: This molecule produced by B. infantis (ATCC No. 15697) interaction with ingested breastmilk functions in a complementary manner and could become useful in the treatment of all at-risk premature infants for NEC if safety and clinical studies are performed.

The developmentally regulated fetal enterocyte gene, ZP4, mediates anti-inflammation by the symbiotic bacterial surface factor polysaccharide A on Bacteroides fragilis.

Initial colonizing bacteria play a critical role in completing the development of the immune system in the gastrointestinal tract of infants. Yet, the interaction of colonizing bacterial organisms with the developing human intestine favors inflammation over immune homeostasis. This characteristic of bacterial-intestinal interaction partially contributes to the pathogenesis of necrotizing enterocolitis (NEC), a devastating premature infant intestinal inflammatory disease. However, paradoxically some unique pioneer bacteria (initial colonizing species) have been shown to have a beneficial effect on the homeostasis of the immature intestine and the prevention of inflammation. We have reported that one such pioneer bacterium, Bacteroides fragilis (B. fragilis), and its surface component polysaccharide A (PSA) inhibit IL-1ß-induced inflammation in a human primary fetal small intestinal cell line (H4 cells). In this study, using transcription profiling of H4 cellular RNA after pretreatment with or without PSA before an inflammatory stimulation of IL-1ß, we have begun to further determine the cellular mechanism for anti-inflammation. We show that a developmentally regulated gene, zona pellucida protein 4 (ZP4), is uniquely elevated after IL-1ß stimulation and reduced with PSA exposure. ZP4 was known as a sperm receptor-mediating species-specific binding protein in the initial life of mammals. However, its intestinal epithelial function is unclear. We found that ZP4 is a developmentally regulated gene involved with immune function and regulated by both Toll-like receptor 2 and 4. Knockdown of ZP4-affected PSA inhibited IL-8 mRNA expression in response to IL-1ß. This represents an initial study of ZP4 innate immune function in immature enterocytes. This study may lead to new opportunity for efficient treatment of NEC.NEW & NOTEWORTHY This study extends previous observations to define the cellular mechanisms of polysaccharide A-induced anti-inflammation in immature enterocytes using transcription profiling of enterocyte genes after preexposure to polysaccharide A before an inflammatory stimulus with IL-1ß.

The importance of appropriate initial bacterial colonization of the intestine in newborn, child, and adult health.

The fetus does not reside in a sterile intrauterine environment and is exposed to commensal bacteria from the maternal gut/blood stream that cross the placenta and enter the amniotic fluid. This intestinal exposure to colonizing bacteria continues at birth and during the first year of life and has a profound influence on lifelong health. Why is this important? Intestinal crosstalk with colonizing bacteria in the developing intestine affects the infant's adaptation to extrauterine life (immune homeostasis) and provides protection against disease expression (allergy, autoimmune disease, obesity, etc.) later in life. Colonizing intestinal bacteria are critical to the normal development of host defense. Disrupted colonization (dysbiosis) due to maternal dysbiosis, cesarean section delivery, use of perinatal antibiotics, or premature delivery may adversely affect the gut development of host defense and predispose to inflammation rather than to homeostasis, leading to increased susceptibility to disease later in life. Babies born by cesarean section have a higher incidence of allergy, type 1 diabetes, and obesity. Infants given repeated antibiotic regimens during the first year of life are more likely to have asthma as adolescents. This research breakthrough helps to explain the shift in disease paradigms from infections to immune-mediated in children from developed countries. This review will develop this research breakthrough.

Hydrocortisone-induced anti-inflammatory effects in immature human enterocytes depend on the timing of exposure.

The immature human gut has a propensity to exaggerated inflammatory responses that are thought to play a role in the pathogenesis of necrotizing enterocolitis (NEC). Prenatal exposure to corticosteroids has been reported to reduce the risk of NEC, while postnatal dexamethasone treatment is associated with adverse neurodevelopmental outcomes in preterm infants. The aim of this study was to investigate the direct role of hydrocortisone in gene expression patterns and inflammatory responses in immature human enterocytes. Time-dependent hydrocortisone effects in nontransformed primary human fetal intestinal epithelial cell line H4 were investigated by cDNA microarray. Fetal intestinal organ culture and cell culture experiments were conducted. Inflammatory responses were induced by stimulation with IL-1ß and TNF-α with and without hydrocortisone. IL-8 and IL-6 expression and secretion were measured as functional readout. Here we report time-dependent hydrocortisone-induced changes in gene expression patterns detected by cDNA microarray. Hydrocortisone significantly attenuated IL-1ß-induced inflammatory responses in the immature human gut when administered at the time of the proinflammatory insult: IL-1ß-induced IL-8 and IL-6 secretion in the fetal ileum as well as H4 cells were significantly reduced. Hydrocortisone also inhibited IL-8 secretion in response to TNF-α. In contrast, TNF-α-induced IL-8 secretion was not reduced in cells treated with hydrocortisone for 48 h before stimulation. Our observations provide a physiological basis for understanding the differential clinical effects of corticosteroids in the immature human gut depending on the timing of treatment.

Anti-inflammatory effects of Bifidobacterium longum subsp infantis secretions on fetal human enterocytes are mediated by TLR-4 receptors.

The therapeutic and preventive application of probiotics for necrotizing enterocolitis (NEC) has been supported by more and more experimental and clinical evidence in which Toll-like receptor 4 (TLR-4) exerts a significant role. In immune cells, probiotics not only regulate the expression of TLR-4 but also use the TLR-4 to modulate the immune response. Probiotics may also use the TLR-4 in immature enterocytes for anti-inflammation. Here we demonstrate that probiotic conditioned media (PCM) from Bifidobacterium longum supp infantis but not isolated organisms attenuates interleukin-6 (IL-6) induction in response to IL-1ß by using TLR-4 in a human fetal small intestinal epithelial cell line (H4 cells), human fetal small intestinal xenografts, mouse fetal small intestinal organ culture tissues, and primary NEC enterocytes. Furthermore, we show that PCM, using TLR-4, downregulates the mRNA expression of interleukin-1 receptor-associated kinase 2 (IRAK-2), a common adapter protein shared by IL-1ß and TLR-4 signaling. PCM also reduces the phosphorylation of the activator-protein 1 (AP-1) transcription factors c-Jun and c-Fos in response to IL-1ß stimulation in a TLR-4-dependent manner. This study suggests that PCM may use TLR-4 through IRAK-2 and via AP-1 to prevent IL-1ß-induced IL-6 induction in immature enterocytes. Based on these observations, the combined use of probiotics and anti-TLR-4 therapy to prevent NEC may not be a good strategy.

The very low birth weight infant microbiome and childhood health.

This review describes current understandings about the nature of the very low birth weight infant (VLBW) gut microbiome. VLBW infants often experience disruptive pregnancies and births, and prenatal factors can influence the maturity of the gut and immune system, and disturb microbial balance and succession. Many VLBWs experience rapid vaginal or Caesarean births. After birth these infants often have delays in enteral feeding, and many receive little or no mother's own milk. Furthermore the stressors of neonatal life in the hospital environment, common use of antibiotics, invasive procedures and maternal separation can contribute to dysbiosis. These infants experience gastrointestinal dysfunction, sepsis, transfusions, necrotizing enterocolitis, oxygen toxicity, and other pathophysiological conditions that affect the normal microbiota. The skin is susceptible to dysbiosis, due to its fragility and contact with NICU organisms. Dysbiosis in early life may resolve but little is known about the timing of the development of the signature gut microbiome in VLBWs. Dysbiosis has been associated with a number of physical and behavioral problems, including autism spectrum disorders, allergy and asthma, gastrointestinal disease, obesity, depression, and anxiety. Dysbiosis may be prevented or ameliorated in part by prenatal care, breast milk feeding, skin to skin contact, use of antibiotics only when necessary, and vigilance during infancy and early childhood.

Activation of epidermal growth factor receptor mediates mucin production stimulated by p40, a Lactobacillus rhamnosus GG-derived protein.

The mucus layer coating the gastrointestinal tract serves as the first line of intestinal defense against infection and injury. Probiotics promote mucin production by goblet cells in the intestine. p40, a Lactobacillus rhamnosus GG-derived soluble protein, has been shown to transactivate the EGF receptor (EGFR) in intestinal epithelial cells, which is required for inhibition of apoptosis and preservation of barrier function in the colon, thereby ameliorating intestinal injury and colitis. Because activation of EGFR has been shown to up-regulate mucin production in goblet cells, the purpose of this study was to investigate the effects and mechanisms of p40 regulation of mucin production. p40 activated EGFR and its downstream target, Akt, in a concentration-dependent manner in LS174T cells. p40 stimulated Muc2 gene expression and mucin production in LS174T cells, which were abolished by inhibition of EGFR kinase activity, down-regulation of EGFR expression by EGFR siRNA transfection, or suppression of Akt activation. Treatment with p40 increased mucin production in the colonic epithelium, thus thickening the mucus layer in the colon of wild type, but not of Egfr(wa5) mice, which have a dominant negative mutation in the EGFR kinase domain. Furthermore, inhibition of mucin-type O-linked glycosylation suppressed the effect of p40 on increasing mucin production and protecting intestinal epithelial cells from TNF-induced apoptosis in colon organ culture. Thus, these results suggest that p40-stimulated activation of EGFR mediates up-regulation of mucin production, which may contribute to the mechanisms by which p40 protects the intestinal epithelium from injury.

Breast milk, microbiota, and intestinal immune homeostasis.

Newborns adjust to the extrauterine environment by developing intestinal immune homeostasis. Appropriate initial bacterial colonization is necessary for adequate intestinal immune development. An environmental determinant of adequate colonization is breast milk. Although the full-term infant is developmentally capable of mounting an immune response, the effector immune component requires bacterial stimulation. Breast milk stimulates the proliferation of a well-balanced and diverse microbiota, which initially influences a switch from an intrauterine TH2 predominant to a TH1/TH2 balanced response and with activation of T-regulatory cells by breast milk-stimulated specific organisms (Bifidobacteria, Lactobacillus, and Bacteroides). As an example of its effect, oligosaccharides in breast milk are fermented by colonic bacteria producing an acid milieu for bacterial proliferation. In addition, short-chain fatty acids in breast milk activate receptors on T-reg cells and bacterial genes, which preferentially mediate intestinal tight junction expression and anti-inflammation. Other components of breast milk (defensins, lactoferrin, etc.) inhibit pathogens and further contribute to microbiota composition. The breast milk influence on initial intestinal microbiota also prevents expression of immune-mediated diseases (asthma, inflammatory bowel disease, type 1 diabetes) later in life through a balanced initial immune response, underscoring the necessity of breastfeeding as the first source of nutrition.

Toll-like receptor-4 in human and mouse colonic epithelium is developmentally regulated: a possible role in necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is an immature intestinal condition resulting in devastating intestinal inflammation due to unknown mechanisms. Evidence has suggested that intestinal maturation attenuates the severity of NEC and Toll-like receptor 4 (TLR4) has been suggested to play a critical role in its pathogenesis. We investigated whether maturational effects of TLR4 expression in immature colon might contribute to the development of NEC. METHODS: TLR4 colonocyte expression was detected by immunofluorescence confocal microscopy. Interleukin-6 (IL-6) levels were assayed by an enzyme-linked immunosorbent assay (ELISA). RESULTS: TLR4 expression was high in fetal colonic epithelium in human and mouse, with earlier gestation having a higher surface/cytoplasm distribution. TLR4 remained high in mouse postnatal day 1 but the surface/cytoplasm distribution was reduced. TLR4 decreased in amount and then was expressed in crypts in the mature human and mouse colon. Hydrocortisone (HC) reduced the surface/cytoplasm distribution of TLR4 in human fetal colon. Elevated IL-6 levels in immature colon after lipopolysaccharide were attenuated by HC in human and mouse. CONCLUSION: Expression, localization, and signaling of TLR4 in colonic epithelium may be developmentally regulated. HC may accelerate the TLR developmental pathway change to an adult type, which may account for its impact on TLR4 signaling.

Long-chain polyunsaturated fatty acids attenuate the IL-1ß-induced proinflammatory response in human fetal intestinal epithelial cells.

BACKGROUND: Evidence suggests that excessive inflammation of the immature intestine may predispose premature infants to necrotizing enterocolitis (NEC). We investigated the anti-inflammatory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) in human fetal and adult intestinal epithelial cells (IEC) in primary culture. METHODS: Human fetal IEC in culture were derived from a healthy fetal small intestine (H4) or resected small intestine of a neonate with NEC (NEC-IEC). Intestinal cell lines Caco2 and NCM460 in culture were used as models for mature IEC. IEC in culture were pretreated with 100 µmol/l palmitic acid (PAL), DHA, EPA, ARA, or ARA+DHA for 48 h and then stimulated with proinflammatory IL-1ß. RESULTS: DHA significantly attenuated IL-1ß induced proinflammatory IL-8 and IL-6 protein and mRNA in fetal H4, NEC-IEC, and mature Caco2, NCM460 IEC, compared to control and PAL treatment. DHA downregulated IL-1R1 (IL-1ß receptor) and NFk ß1 mRNA expression in fetal and adult IEC. ARA had potent anti-inflammatory effects with lower IL-8 and IL-6 (protein and mRNA) in fetal H4 but not in NEC-IEC or adult IEC. CONCLUSION: The present study provides evidence that DHA and ARA may have important anti-inflammatory functions for prevention of NEC in premature infants.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

BACKGROUND: Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. METHODS: Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. RESULTS: Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. CONCLUSION: The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

From Birth to "Immunohealth," Allergies and Enterocolitis.

Microbial signals stimulate development and maintenance of the neonatal immune system. The process begins in utero, with limited exposure to microbes in the intrauterine environment, as well as maternal immune signals priming the developing immune system. After birth and initial colonization, the immune system must be able to activate against pathogens, but also achieve oral tolerance of food and resident gut microbes. Through microbial signals and appropriate nutrition, the immune system is able to achieve homeostasis. Major challenges to successful colonization and immune system regulation include abnormal microbial inoculi (cesarean section, hygiene) and antibiotics. When normal colonization is interrupted, dysbiosis occurs. This imbalance of microbes and subsequently of the immune system can result in allergic diseases, asthma, or necrotizing enterocolitis. Probiotics and probiotic-derived therapies represent an exciting avenue to replete the population of commensal microbes and to prevent the immune-mediated sequelae of dysbiosis.

Recommendations for Probiotic Use--2015 Update: Proceedings and Consensus Opinion.

This paper describes the consensus opinion of the participants in the 4th Triennial Yale/Harvard Workshop on Probiotic Recommendations. The recommendations update those of the first 3 meetings that were published in 2006, 2008, and 2011. Recommendations for the use of probiotics in necrotizing enterocolitis, childhood diarrhea, inflammatory bowel disease, irritable bowel syndrome and Clostridium difficile diarrhea are reviewed. In addition, we have added recommendations for liver disease for the first time. As in previous publications, the recommendations are given as A, B, or C ratings.

Why is initial bacterial colonization of the intestine important to infants' and children's health?

Microbial colonization of the infant occurs during a critical time window for immune and gastrointestinal development. Infant colonization sets the stage for the adult microbiome. This review is a broad survey of the factors affecting infant colonization and the downstream effects on gastrointestinal health and disease. Major topics affecting colonization include initial inoculation dependent on birth mode, the impact of breast-feeding, and inside-out modulation of the developing microbiome by the immune system. Major outcomes of colonization include the timing-dependent education of the neonatal immune system, which is interconnected with barrier function and metabolism. These all engage in further continuing cross-talk with the microbiome, genetics, and nutrition. This review also briefly examines mechanisms of disease resulting from disrupted colonization as well as nutritional and microbial therapies.

Conditioned medium from Bifidobacteria infantis protects against Cronobacter sakazakii-induced intestinal inflammation in newborn mice.

Necrotizing enterocolitis (NEC) is associated with a high morbidity and mortality in very low birth weight infants. Several hypotheses regarding the pathogenesis of NEC have been proposed but to date no effective treatment is available. Previous studies suggest that probiotic supplementation is protective. We recently reported that probiotic (Bifidobacterium infantis) conditioned medium (PCM) has an anti-inflammatory effect in cultured fetal human intestinal cells (H4) and fetal intestine explants. In this study, we tested in vivo whether PCM protects neonatal mice from developing intestinal inflammation induced by exposure to Cronobacter sakazakii (C. sakazakii), an opportunistic pathogen associated with NEC. We found that infected neonatal mice had a significantly lower body weight than control groups. Infection led to ileal tissue damage including villous rupture, disruption of epithelial cell alignment, intestinal inflammation, apoptotic cell loss, and decreased mucus production. Pretreatment with PCM prevented infection caused decrease in body weight, attenuated enterocyte apoptotic cell death, mitigated reduced mucin production, and maintained ileal structure. Infected ileum expressed reduced levels of IκBα, which could be restored upon pretreatment with PCM. We also observed a nuclear translocation of NF-κB p65 in H4 cells exposed to C. sakazakii, which was prevented in PCM-pretreated cells. Finally, treatment of neonatal mice with PCM prior to infection sustained the capacity of ileal epithelial proliferation. This study suggests that an active component(s) released into the culture medium by B. infantis may prevent ileal damage by a pathogen linked to NEC.

Helminth infection impairs autophagy-mediated killing of bacterial enteropathogens by macrophages.

Autophagy is an important mechanism used by macrophages to kill intracellular pathogens. The results reported in this study demonstrate that autophagy is also involved in the macrophage killing of the extracellular enteropathogen Citrobacter rodentium after phagocytosis. The process was significantly impaired in macrophages isolated from mice chronically infected with the helminth parasite Heligmosomoides polygyrus. The H. polygyrus-mediated inhibition of autophagy was Th2 dependent because it was not observed in macrophages isolated from helminth-infected STAT6-deficient mice. Moreover, autophagy of Citrobacter was inhibited by treating macrophages with IL-4 and IL-13. The effect of H. polygyrus on autophagy was associated with decreased expression and processing of L chain protein 3 (LC3), a key component of the autophagic machinery. The helminth-induced inhibition of LC3 expression and processing was STAT6 dependent and could be recapitulated by treatment of macrophages with IL-4 and IL-13. Knockdown of LC3 significantly inhibited autophagic killing of Citrobacter, attesting to the functional importance of the H. polygyrus-mediated downregulation of this process. These observations reveal a new aspect of the immunosuppressive effects of helminth infection and provide mechanistic insights into our earlier finding that H. polygyrus significantly worsens the in vivo course of Citrobacter infection.

Probiotics prevent necrotizing enterocolitis by modulating enterocyte genes that regulate innate immune-mediated inflammation.

Necrotizing enterocolitis (NEC), an extensive intestinal inflammatory disease of premature infants, is caused, in part, by an excessive inflammatory response to initial bacterial colonization due to the immature expression of innate immune response genes. In a randomized placebo-controlled clinical trial, supplementation of very low birth weight infants with probiotics significantly reduced the incidence of NEC. The primary goal of this study was to determine whether secreted products of these two clinically effective probiotic strains, Bifidobacterium infantis and Lactobacillus acidophilus, prevented NEC by accelerating the maturation of intestinal innate immune response genes and whether both strains are required for this effect. After exposure to probiotic conditioned media (PCM), immature human enterocytes, immature human intestinal xenografts, and primary enterocyte cultures of NEC tissue (NEC-IEC) were assayed for an IL-8 and IL-6 response to inflammatory stimuli. The latter two models were also assayed for innate immune response gene expression. In the immature xenograft, PCM exposure significantly attenuated LPS and IL-1ß-induced IL-8 and IL-6 expression, decreased TLR2 mRNA and TLR4 mRNA, and increased mRNA levels of specific negative regulators of inflammation, SIGIRR and Tollip. In NEC-IEC, PCM decreased TLR2-dependent IL-8 and IL-6 induction and increased SIGIRR and Tollip expression. The attenuated inflammatory response with PCM was reversed with Tollip siRNA-mediated knockdown. The anti-inflammatory secreted factor is a 5- to 10-kDa molecule resistant to DNase, RNase, protease, heat stress, and acid exposure. B. infantis-conditioned media showed superior anti-inflammatory properties to that of L. acidophilus in immature human enterocytes, suggesting a strain specificity to this effect. We conclude that PCM promotes maturation of innate immune response gene expression, potentially explaining the protective effects of probiotics in clinical NEC.

Method development for fecal lipidomics profiling.

Robust methodologies for the analysis of fecal material will facilitate the understanding of gut (patho)physiology and its role in health and disease and will help improve care for individual patients, especially high-risk populations, such as premature infants. Because lipidomics offers a biologically and analytically attractive approach, we developed a simple, sensitive, and quantitatively precise method for profiling intact lipids in fecal material. The method utilizes two separate, complementary extraction chemistries, dichloromethane (DCM) and a methyl tert-butyl ether/hexafluoroisopropanol (MTBE) mixture, alone or with high pressure cycling. Extracts were assessed by liquid chromatography-high-resolution mass spectrometry-based profiling with all ion higher energy collisional dissociation fragmentation in both positive and negative ionization modes. This approach provides both class-specific and lipid-specific fragments, enhancing lipid characterization. Solvents preferentially extracted lipids based on hydrophobicity. More polar species preferred MTBE; more hydrophobic compounds preferred DCM. Pressure cycling differentially increased the yield of some lipids. The platform enabled analysis of >500 intact lipophilic species with over 300 lipids spanning 6 LIPID MAPS categories identified in the fecal matter from premature infants. No previous report exists that provides these data; thus, this study represents a new paradigm for assessing nutritional health, inflammation, and infectious disease in vulnerable populations.