Bifidobacteria-mediated immune system imprinting early in life.

Immune-microbe interactions early in life influence the risk of allergies, asthma, and other inflammatory diseases. Breastfeeding guides healthier immune-microbe relationships by providing nutrients to specialized microbes that in turn benefit the host's immune system. Such bacteria have co-evolved with humans but are now increasingly rare in modern societies. Here we show that a lack of bifidobacteria, and in particular depletion of genes required for human milk oligosaccharide (HMO) utilization from the metagenome, is associated with systemic inflammation and immune dysregulation early in life. In breastfed infants given Bifidobacterium infantis EVC001, which expresses all HMO-utilization genes, intestinal T helper 2 (Th2) and Th17 cytokines were silenced and interferon ß (IFNß) was induced. Fecal water from EVC001-supplemented infants contains abundant indolelactate and B. infantis-derived indole-3-lactic acid (ILA) upregulated immunoregulatory galectin-1 in Th2 and Th17 cells during polarization, providing a functional link between beneficial microbes and immunoregulation during the first months of life.

Infant feeding practices and parental perceptions during the 2022 United States infant formula shortage crisis.

BACKGROUND: In May of 2022, parents living in the United States experienced a dramatic infant formula shortage caused by supply chain issues and the recall of several infant formula products over contamination concerns. METHODS: An anonymous, electronic, cross-sectional survey was designed to understand infant feeding practices, parental experience and perceived support during the crisis. RESULTS: Ninety-nine parents that lived in the U.S. and fulfilled study criteria completed the survey. 66% of respondents were female, and 75% of respondents were recipients of the Special Supplemental Nutrition Program for Women Infant Children (WIC). Parental mean age was 30.0 years, and the mean infant age was 26.8 weeks. The number of individuals that used at least one unsafe infant feeding practice increased from 8% before the infant formula shortage to 48.5% during the shortage (p < 0.001). 79% of parents fed their infants U.S. infant formula brands and 39% of parents fed their infants imported infant formula brands before the shortage which were significantly reduced during the shortage to 27% (p < 0.005) and 11% (p < 0.005), respectively. The percentage of parents that reported infant feeding practices before and during the infant formula shortage significantly increased from 2 to 28% for banked donor milk use (p < 0.005); 5-26% for use of human milk from informal sharing (p < 0.005); and 2-29% for use of watered-down infant formula (p < 0.005). The resources that parents reported as most helpful in navigating the crisis differed by parental sex and WIC recipient status and included other parents, friends, and family; lactation consultants; healthcare providers; and WIC. CONCLUSIONS: Our study found that feeding practices in response to the infant formula shortage may pose health risks to infants including nutrition and food insecurity. These data suggest the need for policy changes within regulatory and the healthcare system to provide families with clinical prenatal and postnatal lactation support, access to pasteurized banked donor milk, and access to more commercially available products.

Human Milk Oligosaccharide Compositions Illustrate Global Variations in Early Nutrition.

BACKGROUND: Human milk oligosaccharides (HMOs) are an abundant class of compounds found in human milk and have been linked to the development of the infant, and specifically the brain, immune system, and gut microbiome. OBJECTIVES: Advanced analytical methods were used to obtain relative quantitation of many structures in approximately 2000 samples from over 1000 mothers in urban, semirural, and rural sites across geographically diverse countries. METHODS: LC-MS-based analytical methods were used to profile the compounds with broad structural coverage and quantitative information. The profiles revealed their structural heterogeneity and their potential biological roles. Comparisons of HMO compositions were made between mothers of different age groups, lactation periods, infant sexes, and residing geographical locations. RESULTS: A common behavior found among all sites was a decrease in HMO abundances during lactation until approximately postnatal month 6, where they remained relatively constant. The greatest variations in structural abundances were associated with the presence of α(1,2)-fucosylated species. Genomic analyses of the mothers were not performed; instead, milk was phenotyped according to the abundances of α(1,2)-fucosylated structures. Mothers from the South American sites tended to have higher proportions of phenotypic secretors [mothers with relatively high concentrations of α(1,2)-fucosylated structures] in their populations compared to the rest of the globe, with Bolivia at ∼100% secretors, Peru at ∼97%, Brazil at ∼90%, and Argentina at ∼85%. Conversely, the cohort sampled in Africa manifested the lowest proportion of secretors (South Africa ∼ 63%, the Gambia ∼ 64%, and Malawi ∼ 75%). Furthermore, we compared total abundances of HMOs in secretors compared with nonsecretors and found that nonsecretors have lower abundances of HMOs compared to secretors, regardless of geographical location. We also observed compositional differences of the 50+ most abundant HMOs between milk types and geographical locations. CONCLUSIONS: This study represents the largest structural HMO study to date and reveals the general behavior of HMOs during lactation among different populations.

Early probiotic supplementation with B. infantis in breastfed infants leads to persistent colonization at 1 year.

BACKGROUND: Recent studies have reported a dysfunctional gut microbiome in breastfed infants. Probiotics have been used in an attempt to restore the gut microbiome; however, colonization has been transient, inconsistent among individuals, or has not positively impacted the host's gut. METHODS: This is a 2-year follow-up study to a randomized controlled trial wherein 7-day-old infants received 1.8 × 1010 colony-forming unit Bifidobacterium longum subsp. infantis (B. infantis) EVC001 (EVC) daily for 21 days or breast milk alone (unsupplemented (UNS)). In the follow-up study, mothers (n = 48) collected infant stool at 4, 6, 8, 10, and 12 months postnatal and completed the health-diet questionnaires. RESULTS: Fecal B. infantis was 2.5-3.5 log units higher at 6-12 months in the EVC group compared with the UNS group (P < 0.01) and this relationship strengthened with the exclusion of infants who consumed infant formula and antibiotics. Infants in the EVC group had significantly higher Bifidobacteriaceae and lower Bacteroidaceae and Lachnospiraceae (P < 0.05). There were no differences in any health conditions between the two groups. CONCLUSIONS: Probiotic supplementation with B. infantis within the first month postnatal, in combination with breast milk, resulted in stable colonization that persisted until at least 1 year postnatal. IMPACT: A dysfunctional gut microbiome in breastfed infants is common in resource-rich nations and associated with an increased risk of immune diseases. Probiotics only transiently exist in the gut without persistent colonization or altering the gut microbiome. This is the first study to show that early probiotic supplementation with B. infantis with breast milk results in stable colonization of B. infantis and improvements to the gut microbiome 1 year postnatal. This study addresses a key gap in the literature whereby probiotics can restore the gut microbiome if biologically selected microorganisms are matched with their specific food in an open ecological niche.

Immunoglobulin A N-glycosylation Presents Important Body Fluid-specific Variations in Lactating Mothers.

Secretory Immunoglobulin A (SIgA) is central to mucosal immunity: represents one of the main immunological mechanisms of defense against the potential attack of pathogens. During lactation SIgA is produced by plasmablasts in the mammary gland and is present in breast milk, playing a vital role in the passive immunity of the newborn. Interestingly, the different components of SIgA are highly N-glycosylated, and these N-Glycans have an essential role in health maintenance. In this work, we performed a glycomic study to compare N-glycosylation of SIgA purified from mature breast milk and saliva, and plasma IgA from the same lactating participants. Our results revealed a greater diversity than previously reported, with 89 glycan compositions that may correspond to over 250 structures. Among these glycans, 54 glycan compositions were characterized as body-fluid specific. Most of these unique N-Glycan compositions identified in SIgA from mature milk and IgA from plasma were fucosylated and both fucosylated and sialylated species, whereas in salivary SIgA the unique structures were mainly undecorated complex N-Glycans. In addition, we evaluated the effect of delivery mode on (S)IgA glycosylation. Lactating participants who had given birth by vaginal delivery presented an increased proportion of high mannose and fucosylated glycans in salivary SIgA, and selected high mannose, fucosylated, sialylated, and both fucosylated and sialylated glycans in plasma IgA, indicating that the hormonal changes during vaginal delivery could affect plasma and saliva IgA. These results reveal the structural details that provide a new dimension to the roles of (S)IgA N-Glycans in different tissues, and especially in maternal and new-born protection and infant development. The design of optimal recombinant IgA molecules specifically targeted to protect mucosal surfaces will need to include this dimension of structural detail.

Human Milk Proteins and Their Glycosylation Exhibit Quantitative Dynamic Variations during Lactation.

BACKGROUND: Proteins in human milk are essential and known to support the growth, development, protection, and health of the newborn. These proteins are highly modified by glycans that are currently being recognized as vital to protein structure, stability, function, and health of the intestinal mucosa. Although milk proteins have been studied, the quantitative changes in milk proteins and their respective site-specific glycosylation are unknown. OBJECTIVE: This study expanded the analytical tools for milk proteins and their site-specific glycosylation and applied these tools to a large cohort to determine changes in individual protein concentrations and their site-specific N-glycosylation across lactation. DESIGN: A tandem mass spectrometry method was applied to 231 breast-milk samples from 33 mothers in Davis, California, obtained during 7 different periods of lactation. Dynamic changes in the absolute abundances of milk proteins, as well as variation in site-specific N-glycosylation of individual proteins, were quantified. RESULTS: α-Lactalbumin, ß-casein, k-casein, and α-antitrypsin were significantly increased from colostrum to transitional milk (4.37 ± 1.33 g/L to 6.41 ± 0.72 g/L, 2.25 ± 0.86 g/L to 2.59 ± 0.78 g/L, 1.33 ± 0.44 g/L to 1.60 ± 0.39 g/L, and 0.09 ± 0.10 g/L to 0.11 ± 0.04 g/L, respectively; P < 0.002). α-Lactalbumin (37%), ß-casein (9%), and lysozyme (159%) were higher in mature milk than in colostrum. Glycans exhibited different behavior. Fucosylated glycans of lactoferrin and high-mannose, undecorated, fucosylated, sialylated, and combined fucosylated + sialylated glycans of secretory immunoglobulin A increased during lactation even when the concentrations of the parent proteins decreased. CONCLUSIONS: Proteins in healthy mothers vary dynamically through lactation to support the development of infants. Individual milk proteins carried unique glycan modifications that varied systematically in structure even with site specificity. The role of glycosylation in human milk proteins will be important in understanding the functional components of human milk. This trial was registered at clinicaltrials.gov as NCT01817127.

Colonization by B. infantis EVC001 modulates enteric inflammation in exclusively breastfed infants.

BACKGROUND: Infant gut dysbiosis, often associated with low abundance of bifidobacteria, is linked to impaired immune development and inflammation-a risk factor for increased incidence of several childhood diseases. We investigated the impact of B. infantis EVC001 colonization on enteric inflammation in a subset of exclusively breastfed term infants from a larger clinical study. METHODS: Stool samples (n = 120) were collected from infants randomly selected to receive either 1.8 × 1010 CFU B. infantis EVC001 daily for 21 days (EVC001) or breast milk alone (controls), starting at day 7 postnatal. The fecal microbiome was analyzed using 16S ribosomal RNA, proinflammatory cytokines using multiplexed immunoassay, and fecal calprotectin using ELISA at three time points: days 6 (Baseline), 40, and 60 postnatal. RESULTS: Fecal calprotectin concentration negatively correlated with Bifidobacterium abundance (P < 0.0001; ρ = -0.72), and proinflammatory cytokines correlated with Clostridiaceae and Enterobacteriaceae, yet negatively correlated with Bifidobacteriaceae abundance. Proinflammatory cytokines were significantly lower in EVC001-fed infants on days 40 and 60 postnatally compared to baseline and compared to control infants. CONCLUSION: Our findings indicate that gut dysbiosis (absence of B. infantis) is associated with increased intestinal inflammation. Early addition of EVC001 to diet represents a novel strategy to prevent enteric inflammation during a critical developmental phase.

Ontogeny of alkaline phosphatase activity in infant intestines and breast milk.

BACKGROUND: Necrotizing enterocolitis (NEC) is a devastating disease of intestinal inflammation that primarily affects premature infants. A potential risk factor for necrotizing enterocolitis is exposure of the premature neonatal intestine to environmental bacteria and their proinflammatory products such as lipopolysaccharide. The metalloenzyme alkaline phosphatase (ALP) has been shown to reduce lipopolysaccharide-mediated inflammation. Additionally, premature rat pups have reduced alkaline phosphatase activity and expression as compared to full term pups. To explore the possibility that the human premature neonatal intestine has a paucity of alkaline phosphatase activity, we measured endogenously produced intestinal alkaline phosphatase activity in meconium as a function of gestational age. To test whether breast milk could serve as a source of exogenous alkaline phosphatase to the neonatal intestine through ingestion, we measured alkaline phosphatase activity in breast milk across a range of time points post-birth. METHODS: Alkaline phosphatase activity was quantified in 122 meconium samples from infants of gestational ages ranging from 24 to 40 weeks and in 289 breast milk samples collected from 78 individual mothers between days 2-49 post-birth. RESULTS: We observed a strong positive correlation between the meconium alkaline phosphatase activity and gestational age, with preterm infants having lower meconium alkaline phosphatase activities than early term or term infants. Breast milk alkaline phosphatase activity was highest in the first week post-birth, with peak alkaline phosphatase activity at day 2 post-birth, followed by relatively low alkaline phosphatase activity in weeks 2-7. CONCLUSIONS: Our results are consistent with the two major risk factors for necrotizing enterocolitis development, preterm birth and lack of breast milk feeding, both contributing to a paucity of alkaline phosphatase activity and impaired capacity to detoxify proinflammatory bacterial products such as lipopolysaccharide.

Identification of Oligosaccharides in Feces of Breast-fed Infants and Their Correlation with the Gut Microbial Community.

Glycans in breast milk are abundant and found as either free oligosaccharides or conjugated to proteins and lipids. Free human milk oligosaccharides (HMOs) function as prebiotics by stimulating the growth of beneficial bacteria while preventing the binding of harmful bacteria to intestinal epithelial cells. Bacteria have adapted to the glycan-rich environment of the gut by developing enzymes that catabolize glycans. The decrease in HMOs and the increase in glycan digestion products give indications of the active enzymes in the microbial population. In this study, we quantitated the disappearance of intact HMOs and characterized the glycan digestion products in the gut that are produced by the action of microbial enzymes on HMOs and glycoconjugates from breast milk. Oligosaccharides from fecal samples of exclusively breast-fed infants were extracted and profiled using nanoLC-MS. Intact HMOs were found in the fecal samples, additionally, other oligosaccharides were found corresponding to degraded HMOs and non-HMO based compounds. The latter compounds were fragments of N-glycans released through the cleavage of the linkage to the asparagine residue and through cleavage of the chitobiose core of the N-glycan. Marker gene sequencing of the fecal samples revealed bifidobacteria as the dominant inhabitants of the infant gastrointestinal tracts. A glycosidase from Bifidobacterium longum subsp. longum was then expressed to digest HMOs in vitro, which showed that the digested oligosaccharides in feces corresponded to the action of glycosidases on HMOs. Similar expression of endoglycosidases also showed that N-glycans were released by bacterial enzymes. Although bifidobacteria may dominate the gut, it is possible that specific minority species are also responsible for the major products observed in feces. Nonetheless, the enzymatic activity correlated well with the known glycosidases in the respective bacteria, suggesting a direct relationship between microbial abundances and catabolic activity.

Plasma Lipoprotein Particle Subclasses in Preterm Infants.

OBJECTIVE: A pilot study to determine lipoprotein classes and subclasses in premature infants and examine associations with nutritional intake, gestational age (GA), and morbidity. STUDY DESIGN: Plasma lipoprotein particle concentrations were analyzed in a cohort of 15 premature infants in the first 5 days of life and again at 2 weeks. Breast milk samples were analyzed for fatty acid content. Associations between lipoprotein particle subclasses and GA, breast milk intake, milk fatty acid intake, and chronic lung disease (CLD) were determined. RESULTS: At 2 weeks of age, more premature infants had higher concentrations of total very low-density lipoprotein and lower concentrations of total high-density lipoprotein (HDL) and large HDL particles (similar to profiles seen in adults and children with infectious disease, cardiometabolic disease, and diabetes). Lower total HDL, large HDL, and medium HDL and a higher small HDL:total HDL ratio at 2 weeks were each associated with CLD with GA a likely confounder. Intake of human milk C18 and C20 fatty acids was inversely correlated with plasma total LDL concentration at 2 weeks of age. CONCLUSION: Dyslipidemia was common in extremely premature infants and was associated with CLD and with lower intake of specific long chain fatty acids.

Validating Quantitative Untargeted Lipidomics Across Nine Liquid Chromatography-High-Resolution Mass Spectrometry Platforms.

Liquid chromatography-mass spectrometry (LC-MS) methods are most often used for untargeted metabolomics and lipidomics. However, methods have not been standardized as accepted "best practice" documents, and reports lack harmonization with respect to quantitative data that enable interstudy comparisons. Researchers use a wide variety of high-resolution mass spectrometers under different operating conditions, and it is unclear if results would yield different biological conclusions depending on the instrument performance. To this end, we used 126 identical human plasma samples and 29 quality control samples from a nutritional intervention study. We investigated lipidomic data acquisitions across nine different MS instruments (1 single TOF, 1 Q/orbital ion trap, and 7 QTOF instruments). Sample preparations, chromatography conditions, and data processing methods were kept identical. Single-point internal standard calibrations were used to estimate absolute concentrations for 307 unique lipids identified by accurate mass, MS/MS spectral match, and retention times. Quantitative results were highly comparable between the LC-MS platforms tested. Using partial least-squares discriminant analysis (PLS-DA) to compare results between platforms, a 92% overlap for the most discriminating lipids based on variable importance in projection (VIP) scores was achieved for all lipids that were detected by at least two instrument platforms. Importantly, even the relative positions of individual samples on the PLS-DA projections were identical. The key for success in harmonizing results was to avoid ion saturation by carefully evaluating linear dynamic ranges using serial dilutions and adjusting the resuspension volume and/or injection volume before running actual study samples.

Absolute Quantitation of Human Milk Oligosaccharides Reveals Phenotypic Variations during Lactation.

BACKGROUND: The quantitation of human milk oligosaccharides (HMOs) is challenging because of the structural complexity and lack of standards. OBJECTIVE: The objective of our study was to rapidly measure the absolute concentrations of HMOs in milk using LC-mass spectrometry (MS) and to determine the phenotypic secretor status of the mothers. METHODS: This quantitative method for measuring HMO concentration was developed by using ultraperformance LC multiple reaction monitoring MS. It was validated and applied to milk samples from Malawi (88 individuals; 88 samples from postnatal month 6) and the United States (Davis, California; 45 individuals, mean age: 32 y; 103 samples collected on postnatal days 10, 26, 71, or 120, repeated measures included). The concentrations of α(1,2)-fucosylated HMOs were used to determine the mothers' phenotypic secretor status with high sensitivity and specificity. We used Friedman's test and Wilcoxon's signed rank test to evaluate the change in HMO concentration during the course of lactation, and Student's t test was used to compare secretors and nonsecretors. RESULTS: A decrease (P < 0.05) in HMO concentration was observed during the course of lactation for the US mothers, corresponding to 19.3 ± 2.9 g/L for milk collected on postnatal day 10, decreasing to 8.53 ± 1.18 g/L on day 120 (repeated measures; n = 14). On postnatal day 180, the total concentration of HMOs in Malawi milk samples from secretors (6.46 ± 1.74 mg/mL) was higher (P < 0.05) than that in samples from nonsecretors (5.25 ± 2.55 mg/mL ). The same trend was observed for fucosylated species; the concentration was higher in Malawi milk samples from secretors (4.91 ± 1.22 mg/mL) than from nonsecretors (3.42 ± 2.27 mg/mL) (P < 0.05). CONCLUSIONS: HMOs significantly decrease during the course of lactation. Secretor milk contains higher concentrations of total and fucosylated HMOs than does nonsecretor milk. These HMO concentrations can be correlated to the health of breastfed infants in order to investigate the protective effects of milk components. The trials were registered at clinicaltrials.gov as NCT01817127 and NCT00524446.

Quantitation of human milk proteins and their glycoforms using multiple reaction monitoring (MRM).

Human milk plays a substantial role in the child growth, development and determines their nutritional and health status. Despite the importance of the proteins and glycoproteins in human milk, very little quantitative information especially on their site-specific glycosylation is known. As more functions of milk proteins and other components continue to emerge, their fine-detailed quantitative information is becoming a key factor in milk research efforts. The present work utilizes a sensitive label-free MRM method to quantify seven milk proteins (α-lactalbumin, lactoferrin, secretory immunoglobulin A, immunoglobulin G, immunoglobulin M, α1-antitrypsin, and lysozyme) using their unique peptides while at the same time, quantifying their site-specific N-glycosylation relative to the protein abundance. The method is highly reproducible, has low limit of quantitation, and accounts for differences in glycosylation due to variations in protein amounts. The method described here expands our knowledge about human milk proteins and provides vital details that could be used in monitoring the health of the infant and even the mother. Graphical Abstract The glycopeptides EICs generated from QQQ.

Safety and tolerability of Bifidobacterium longum subspecies infantis EVC001 supplementation in healthy term breastfed infants: a phase I clinical trial.

BACKGROUND: Historically, bifidobacteria were the dominant intestinal bacteria in breastfed infants. Still abundant in infants in developing nations, levels of intestinal bifidobacteria are low among infants in developed nations. Recent studies have described an intimate relationship between human milk and a specific subspecies of Bifidobacterium, B. longum subsp. infantis (B. infantis), yet supplementation of breastfed, healthy, term infants with this organism, has not been reported. The IMPRINT Study, a Phase I clinical trial, was initiated to determine the safety and tolerability of supplementing breastfed infants with B. infantis (EVC001). METHODS: Eighty mother-infant dyads were enrolled in either lactation support plus B. infantis supplementation (BiLS) or lactation support alone (LS). Starting with Day 7 postnatal, BiLS infants were fed 1.8-2.8 × 1010 CFU B. infantis EVC001 daily in breast milk for 21 days. Mothers collected fecal samples, filled out health questionnaires, and kept daily logs about their infants' feeding and gastrointestinal symptoms from birth until Day 61 postnatal. Safety and tolerability were determined from maternal reports. RESULTS: There were no differences in the mean gestational age at birth, weight 1 and 2 months postnatal, and breast milk intake between groups. The mean Log10 change in fecal Bifidobacterium from Day 6 to Day 28 was higher (p = 0.0002) for BiLS (6.6 ± 2.8 SD) than for LS infants (3.5 ± 3.5 SD). Daily stool number was higher (p < 0.005) for LS and lower (p < 0.05) for BiLS infants during supplementation than at Baseline. During supplementation, watery stools decreased and soft stools increased by 36% over baseline in BiLS infants (p < 0.05) with no significant changes in stool consistency for the LS infants. None of the safety and tolerability endpoints, including flatulence, bloody stool, body temperature, ratings of gastrointestinal symptoms, use of antibiotics or gas-relieving medications, infant colic, jaundice, number of illnesses, sick doctor visits, or diagnoses of eczema were different for the groups at any point. CONCLUSIONS: The B. infantis EVC001 supplement was safely consumed and well-tolerated. Stools were fewer and better formed in infants in the BiLS group compared with LS group. Adverse events were those expected in healthy infants and not different between groups. TRIAL REGISTRATION: ClinicalTrials.gov NCT02457338 . Registered May 27, 2015.

Lactation and Intestinal Microbiota: How Early Diet Shapes the Infant Gut.

Breast milk is a multifunctional biofluid that provides nutrients along with highly diverse non-nutritive bioactive components such as antibodies, glycans, bacteria, and immunomodulatory proteins. Research over the past decade has confirmed the essential role of breast milk bioactives in the establishment a healthy intestinal microbiota within the infant. The intestinal microbiota of an exclusively breastfed baby is dominated by several species of Bifidobacteria - the most influential member of which is Bifidobacterium longum subspecies infantis (B. infantis) - and is referred to as the milk-oriented microbiome (MOM). MOM is associated with reduced risk of infection in infancy as well as a reduced risk of certain chronic illnesses in adulthood. Establishment and persistence of MOM is dependent on the selective digestion of complex sugar structures in breast milk that are otherwise indigestible to the infant by B. infantis and its relatives. This review focuses primarily on the influence of breast milk glycans and glycosylated proteins on the development of the intestinal microbiome, and how maternal phenotype may influence the development of MOM providing a framework to understand how variation in diet shapes a protective intestinal microbiome.

Site-specific glycosylation of secretory immunoglobulin A from human colostrum.

Secretory immunoglobulin A (sIgA) is a major glycoprotein in milk and plays a key role in mediating immune protection of the gut mucosa. Although it is a highly glycosylated protein, its site-specific glycosylation and associated glycan micro-heterogeneity have still not been fully elucidated. In this study, the site-specific glycosylation of sIgA isolated from human colostrum (n = 3) was analyzed using a combination of LC-MS and LC-MS/MS and in-house software (Glycopeptide Finder). The majority of the glycans found are biantennary structures with one or more acidic Neu5Ac residues; however, a large fraction belonged to truncated complex structures with terminal GlcNAc. Multiple glycosites were identified with nearly 30 glycan compositions located at seven sites on the secretory component, six compositions at a single site on the J chain, and 16 compositions at five sites on the IgA heavy (H) chain. Site-specific heterogeneity and relative quantitation of each composition and the extent of occupation at each site were determined using nonspecific proteases. Additionally, 54 O-linked glycan compositions located at the IgA1 hinge region (HR) were identified by comparison against a theoretical O-glycopeptide library. This represents the most comprehensive report to date detailing the complexity of glycan micro-heterogeneity with relative quantitation of glycoforms for each glycosylation site on milk sIgA. This strategy further provides a general method for determining site-specific glycosylation in large protein complexes.

Comparative Proteomics of Human and Macaque Milk Reveals Species-Specific Nutrition during Postnatal Development.

Milk has been well established as the optimal nutrition source for infants, yet there is still much to be understood about its molecular composition. Therefore, our objective was to develop and compare comprehensive milk proteomes for human and rhesus macaques to highlight differences in neonatal nutrition. We developed a milk proteomics technique that overcomes previous technical barriers including pervasive post-translational modifications and limited sample volume. We identified 1606 and 518 proteins in human and macaque milk, respectively. During analysis of detected protein orthologs, we identified 88 differentially abundant proteins. Of these, 93% exhibited increased abundance in human milk relative to macaque and include lactoferrin, polymeric immunoglobulin receptor, alpha-1 antichymotrypsin, vitamin D-binding protein, and haptocorrin. Furthermore, proteins more abundant in human milk compared with macaque are associated with development of the gastrointestinal tract, the immune system, and the brain. Overall, our novel proteomics method reveals the first comprehensive macaque milk proteome and 524 newly identified human milk proteins. The differentially abundant proteins observed are consistent with the perspective that human infants, compared with nonhuman primates, are born at a slightly earlier stage of somatic development and require additional support through higher quantities of specific proteins to nurture human infant maturation.

The human colostrum whey proteome is altered in gestational diabetes mellitus.

Proteomics of human milk has been used to identify the comprehensive cargo of proteins involved in immune and cellular function. Very little is known about the effects of gestational diabetes mellitus (GDM) on lactation and breast milk components. The objective of the current study was to examine the effect of GDM on the expression of proteins in the whey fraction of human colostrum. Colostrum was collected from women who were diagnosed with (n = 6) or without (n = 12) GDM at weeks 24-28 in pregnancy. Colostral whey was analyzed for protein abundances using high-resolution, high-mass accuracy liquid chromatography tandem mass spectrometry. A total of 601 proteins were identified, of which 260 were quantified using label free spectral counting. Orthogonal partial least-squares discriminant analysis identified 27 proteins that best predict GDM. The power law global error model corrected for multiple testing was used to confirm that 10 of the 27 proteins were also statistically significantly different between women with versus without GDM. The identified changes in protein expression suggest that diabetes mellitus during pregnancy has consequences on human colostral proteins involved in immunity and nutrition.

Breast milk oligosaccharides: structure-function relationships in the neonate.

In addition to providing complete postnatal nutrition, breast milk is a complex biofluid that delivers bioactive components for the growth and development of the intestinal and immune systems. Lactation is a unique opportunity to understand the role of diet in shaping the intestinal environment including the infant microbiome. Of considerable interest is the diversity and abundance of milk glycans that are energetically costly for the mammary gland to produce yet indigestible by infants. Milk glycans comprise free oligosaccharides, glycoproteins, glycopeptides, and glycolipids. Emerging technological advances are enabling more comprehensive, sensitive, and rapid analyses of these different classes of milk glycans. Understanding the impact of inter- and intraindividual glycan diversity on function is an important step toward interventions aimed at improving health and preventing disease. This review discusses the state of technology for glycan analysis and how specific structure-function knowledge is enhancing our understanding of early nutrition in the neonate.

Infant Maturity at Birth Reveals Minor Differences in the Maternal Milk Metabolome in the First Month of Lactation.

BACKGROUND: Human milk is the gold standard of nutrition for infants, providing both protective and essential nutrients. Although much is known about milk from mothers giving birth to term infants, less is known about milk from mothers giving birth to premature infants. In addition, little is known about the composition and diversity of small molecules in these milks and how they change over the first month of lactation. OBJECTIVE: The objective was to understand how milk metabolites vary over the first month of lactation in mothers giving birth to term and preterm infants. METHODS: (1)H nuclear magnetic resonance (NMR) metabolomics was used to characterize metabolites that were present in micromolar to molar concentrations in colostrum (day 0-5 postpartum), transition milk (day 14), and mature milk (day 28) from mothers who delivered term (n = 15) and preterm (n = 13) infants. Principal components analysis, linear mixed-effects models (LMMs), and linear models (LMs) were used to explore the relation between infant maturity and the postpartum day of collection of milk samples. RESULTS: By using a standard NMR metabolite library, 69 metabolites were identified in the milks, including 15 sugars, 23 amino acids and derivatives, 11 energy-related metabolites, 10 fatty acid-associated metabolites, 3 nucleotides and derivatives, 2 vitamins, and 5 bacteria-associated metabolites. Many metabolite concentrations followed a similar progression over time in both term and preterm milks, with more biological variation in metabolite concentrations in preterm milk. However, although lacto-N-neotetraose (LMM, P = 4.0 × 10(-5)) and lysine (LM, P = 1.5 × 10(-4)) significantly decreased in concentration in term milk over time, they did not significantly change in preterm milk. CONCLUSION: Overall, the metabolic profile of human milk is dynamic throughout the first month of lactation, with more variability in preterm than in term milk and subtle differences in some metabolite concentrations. This trial was registered at clinicaltrials.gov as NCT01841268.