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Scientific Evidence for Breastfeeding

Alan Lucas

The global drive to promote breastfeeding targeted at all 134 mil-lion infants born/year on the planet is one of the most pervasive public health interventions. It is therefore critical that the breastfeeding field is evidence based. Three key scientific pillars of breastfeeding have been: (I) that human milk (HM) is ideally adapted for babies since it is the product of 200 million years of mammalian evolution; (2) that HM composition should be seen as the gold standard for infant nutritional requirements; and (3) that HM has numerous clinical benefits for the infant. All of these 3 contentions can be challenged on the grounds that the underlying evi-dence and thinking has been significantly flawed. I shall identify these flaws to help pave the way to a more solid basis for modern breastfeeding medicine.

Firstly, the incorrect use of the evolutionary theory for human breastfeeding is dissected, notably the evidence in humans for a mismatch between our rapidly changing environment and our ancient genes (evolu-tionary discordance). The possible evidence for evolutionary discordance in relation to breastfeeding is broad-based and includes consideration of the risk of vitamin K deficiency bleeding, vitamin D deficiency, iron defi-ciency, n-3 fatty acid intake, and cardiovascular disease risk. The practical implications of evolutionary discordance for optimal nutritional care of breastfed infants are discussed.

Secondly, I shall show how HM composition has been incorrectly translated into dietary intake in a large body of past flawed work that resulted in misleading data. By the 1960s, there had been 1,500 publica-tions on breast milk composition; yet, it is difficult to obtain representa-tive samples of breast milk because milk fat changes during a feed. The past studies greatly overestimated fat and energy in breast milk. Also, breast milk protein content was often estimated from nitrogen content as employed in the dairy industry. However, because of the high non-protein nitrogen content of human milk, this methodology led to over-estimation of breast milk protein content. Unfortunately, these incorrect compositional data were used as a model for the design of infant formulas

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resulting in more rapid growth. Evidence is presented to show that accel-erated early growth contributes to the modern epidemic of obesity and cardiovascular disease risk (“postnatal growth acceleration hypothesis” [1]). Better approaches to the determination of breast milk nutrient con-tent (for instance using stable isotopes) have helped generate more appro-priate data to underpin infant nutrition requirements, guide design future formulas, and provide understanding of the beneficial effects of breast-feeding in relation to reduced later risk of obesity and cardiovascular disease.

Finally, most studies purporting to show benefits of HM are observa-tional and potentially confounded, so causation cannot be proven. Thus, hard experimental evidence is required. However, in term infants, ran-domized trials of breast versus formula feeding are ethically difficult and seldom done. Here, I shall use preterm infants as a model, since numerous randomized controlled trials and physiological studies over 40 years have compared exclusive HM feeding versus cow’s milk exposure. Unexpect-edly diverse immediate beneficial effects span the field of neonatology, and long-term programmed effects have been shown for cognition, brain structure, risk factors for cardiovascular disease, structural development of the heart and lungs, bone health, and atopy. These data add much weight to the evidence obtained in full-term infants using weaker study designs that HM feeding in early life may fundamentally and permanently change the biology, health, and developmental outcomes of the organism.

With these advances in science and thought, breastfeeding is emerg-ing as a major evidence-based field of medical and public health practice.

Reference

1 Singhal A, Lucas A: Early origins of cardiovascular disease: is there a unifying hypothesis? Lancet 2004;363:1642–1645.

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The Biomechanics of Breastfeeding: Bridging the Gap between Engineering-Based Studies and Clinical Practice

Mike Woolridge

Understanding how a baby extracts milk from the breast is the vital cornerstone to practicing sound, effective breastfeeding management in order to optimize milk transfer from mother to baby. In turn, this allows one to maximize the transfer of calorie-rich nutrients, predominantly of breast milk fat.

For several centuries, received wisdom was that babies extract milk from the breast by a combination of baseline suction, compression, and relaxation of the baby’s jaws against the breast, and rhythmical waves of pressure applied to the underside of the breast/nipple held within the baby’s mouth by the tongue [1]. Based on this premise, the core principles of WHO/UNICEF training were established, focusing on optimizing the positioning and attachment of the baby at the breast in order to maximize the effectiveness of milk transfer.

In the past decade, however, this received wisdom has been chal-lenged both by the use of modern ultrasound equipment [2, 3] and engi-neering-based modeling of breast anatomy (specifically the milk duct system) and the baby’s sucking action [4, 5].

A key novel claim was that the baby can generate localized, added suction with its tongue to enhance milk transfer [2, 6]; this has since been confirmed [7], although the evidence is that this novel mechanism remains secondary to the core process of peristaltic expression by the tongue.

In contrast, engineering-based studies [4, 5] have proven both con-troversial and contradictory, providing new insights yet posing fresh chal-lenges. To date, however, they have not altered the core underpinnings of best breastfeeding practice and management.

In the field of medicine, it is recognized that the validity of ran-domized controlled trials should be evaluated by a set of quality control

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standards, and the framework of critical appraisal skills is a way of achiev-ing this. No such quality standards or guidelines exist for evaluating engineering-based models of a physiological process. A comparable framework is needed if the validity of engineering-based models is to be effectively assessed. In practice, in order to address the veracity of the conclusions drawn, it is essential to be able to evaluate several of the assumptions made in these models: whether or not they are valid, and whether specific elements are missing from current models which might affect their outcome.

Certain physical assumptions, made during the modeling process, are known to be incorrect, but have been made in order to simplify the modeling process – for example, that the milk duct walls are rigid. Fur-ther ways in which the modeling process departs from known physiol-ogy include: (i) the view that negative suction pressure is the primary force in these models, without any contribution being made by the pro-gressive peristaltic pressure exerted by the baby’s tongue [8], and (ii) the core assumption that the milk duct system remains patent throughout a feed, thereby ignoring the occlusive impact of the baby’s jaw closure with each suck. The inclusion of any of these natural processes would radi-cally alter the conclusions from modeling, thereby disproving the claim that suction alone can explain milk extraction [4] while giving greater credence to the suggestion that suction alone may not fully explain milk extraction [5].

One feature consistently missing from such analyses is the clinical implications arising from them, and what they add to our understanding in terms of how to help mothers and babies breastfeed more effectively. To this end, a pivotal role played by peristaltic tongue movements, essential to effective breastfeeding, will be identified and elaborated, so providing evidence as to why the core management principles of positioning and attachment are so central to breastfeeding success.

References

1 Woolridge MW: The “anatomy” of infant sucking. Midwifery 1986;2:164–171.2 Geddes DT, Kent JC, Mitoulas LR, Hartmann PE: Tongue movement and intra-oral

vacuum in breastfeeding infants. Early Hum Dev 2008;84:471–477.3 Burton P, Ding J, McDonald D, Fewtrell MS: Real-time 3D ultrasound imaging of

infant tongue movements during breast-feeding. Early Hum Dev 2013;89:635–641.4 Elad D, Kozlovsky P, Blum O, et al: Biomechanics of milk extraction during breast-

feeding. Proc Natl Acad Sci USA 2014;111:5230–5235, supporting information: www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319798111/-/DCSupplemental.

5 Mortazavi SN, Geddes D, Hassanipour F: Lactation in the human breast from a fluid dynamics point of view. J Biomech Eng 2017;139:011009.

6 Sakalidis VS, Hepworth AR, Hartmann PE, Geddes D: Ultrasound imaging of infant sucking dynamics during the establishment of lactation. J Hum Lact 2013;29:205–213.

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7 Monaci G, Woolridge MW: Ultrasound video analysis for understanding infant breastfeeding. Proc 18th IEEE Int Conf Image Processing (ICIP), September 11–14, 2011, pp 1765–1768.

8 Grassi A, Cecchi F, Sgherri G, et al: Sensorized pacifier to evaluate non-nutritive sucking in newborns. Med Eng Phys 2016;38:398–402.

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Physiological Effects of Feeding Infants and Young Children Formula Supplemented with Milk Fat Globule Membranes

Olle Hernell, Magnus Domellöf, Tove Grip, Bo Lönnerdal, and Niklas Timby

An increasing number of studies have reported different health ben-efits from oral supplementation with bovine milk fat globule membrane (MFGM) to infants and children (Table 1) [1, 2]. MFGM is a biologically active milk fraction that contains a large proportion of milk phospho-lipids, sphingomyelins, and gangliosides together with several hundred identified proteins, including mucins, butyrophilin, lactoferrin, and lacta-dherin (Fig. 1). Formula-fed infants are of special interest with respect to MFGM supplementation since they have a lower intake of MFGM com-ponents compared to breast-fed infants because, traditionally, the MFGM fraction is discarded with the milk fat when this is replaced by vegetable oils as the fat source in infant formulas.

Clinical Studies on the Effects of MFGM Concentrates Fed to Infants and Children

In the first double-blind, randomized, controlled trial (DBRCT) in 550 healthy, primarily breast-fed 6- to 11-month-old infants, supplemen-tation with an MFGM-enriched protein fraction reduced diarrheal mor-bidity [3]. In another DBRCT in 70 infants, supplementation with bovine milk gangliosides, provided as a complex bovine milk lipid fraction from 2–8 until 24 weeks of age, increased hand-eye coordination, performance, and general IQ after adjustment for socioeconomic background variables [4]. A third DBRCT including 253 preschool children aged 2.5–6 years evaluated a daily intake of a formula enriched with 500 mg of phospho-lipids with the addition of a phospholipid-rich MFGM concentrate for 4 months, and found reduced days with fever and less behavioral problems during the intervention [5]. In an Indian DBRCT, 450 infants between 8

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and 24 months of age were randomized to a daily dose of milk powder supplemented with 2 g of a spray-dried ganglioside concentrate or milk powder only for 12 weeks [6]. There was no difference between the groups either in the primary outcome rotavirus diarrhea or in the secondary out-comes, including all-cause diarrhea. However, the authors noted that the incidence of rotavirus diarrhea during the study period was lower than expected, making the study underpowered compared to the intention of the design. In a Swedish DBRCT in 160 formula-fed healthy term infants, supplementation with a protein-rich MFGM fraction from <2 until 6 months of age improved cognitive scoring in Bayley III [7]. Further, a reduced incidence of acute otitis media, a reduced antipyretic use, lower concentrations of serum IgG against pneumococci after vaccination, and a lower prevalence of Moraxella catarrhalis in the oral microbiota suggested

Table 1. Double-blind, randomized controlled trials exploring the effects of milk fat globule membrane (MFGM) supplementation to the diet of infants or chil-dren

Study Age Supplementation Main results for MFGM groups

Zavaleta et al. [3]

6–11 months MFGM (Lacprodan® MFGM-10, Arla Foods Ingredients)

Lower longitudinal prevalence of diarrhea Lower incidence of bloody diarrhea

Gurnida et al. [4]

2–8 to 24 weeks Complex milk lipids (AnmumInfacare, Fonterra Cooperative Group)

Higher hand-eye coordination, performance, and general IQ.

Veereman-Wauters et al. [5]

2.5–6 years, for 4 months

MFGM (INPULSE®, Bullinger SA)

Fewer days with fever and lower parental scoring of internal, external, and total behavioral problems

Poppitt et al. [6]

8–4 months, for 12 weeks

Complex milk lipids (Fonterra Cooperative Ltd)

No difference between groups

Timby et al. [7–9, 11]

<2 to 6 months MFGM (Lacprodan® MFGM-10, Arla Foods Ingredients)

Higher cognitive score Lower incidence of otitis media Higher serum cholesterol

Billeaud et al. [10]

14 days to 4 months

Lipid-rich MFGM fraction (Fonterra Cooperative Group)Protein-rich MFGM fraction (Lacprodan® MFGM-10, Arla Foods Ingredients)

Weight gain was noninferior Higher rate of eczema in the protein-rich MFGM group

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an infection-protective effect of MFGM supplementation [8, 9]. In a non-inferiority DBRCT in 199 healthy term infants from 14 days to 4 months of age, a formula enriched with lipids and a formula with a protein-rich bovine MFGM fraction yielded a noninferior weight gain with no serious adverse events compared with a standard formula [10].

Conclusions

Studies investigating the effect of bovine MFGM-supplemented diets on infants and children have shown promising results regarding both neurodevelopment and defense against infections. However, the scientific base of knowledge for MFGM supplementation to infants and children

Milk fat globule

MFGMporteins

MFGM

Glycerophospholipids

Sphingolipids

Cholesterol

Glycosphingolipids

Fig. 1. Schematic drawing of the release of the milk fat globules and compo-sition of the MFGM. Illustration by Erik Domellöf. Reproduced from Hernell et al. [1] with permission.

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is still limited. The number of studies published on MFGM provided to infants and children is small, and the interventions are heterogeneous: different MFGM concentrates have been given for different durations at different infant/child ages and with different main outcomes. However, MFGM supplementation seems safe down to the age of the first week of life in term infants, as no serious adverse effects have been reported.

Infant formulas supplemented with bovine MFGM concentrates have already been launched on many markets, but before firm conclusions can be drawn on the likely health benefits of supplementing the diet of infants and children with MFGM ,more high quality DBRCTs are needed.

References

1 Hernell O, Timby N, Domellöf M, Lönnerdal B: Clinical benefits of milk fat globule membranes for infants and children. J Pediatr 2016;173(suppl):S60–S65.

2 Timby N, Domellöf M, Lönnerdal B, Hernell O: Supplementation of infant formula with bovine milk fat globule membranes. Adv Nutr 2017;8:351–355.

3 Zavaleta N, Kvistgaard AS, Graverholt G, et al: Efficacy of an MFGM-enriched com-plementary food in diarrhea, anemia, and micronutrient status in infants. J Pediatr Gastroenterol Nutr 2011;53:561–568.

4 Gurnida DA, Rowan AM, Idjradinata P, et al: Association of complex lipids contain-ing gangliosides with cognitive development of 6-month-old infants. Early Hum Dev 2012;88:595–601.

5 Veereman-Wauters G, Staelens S, Rombaut R, et al: Milk fat globule membrane (INPULSE) enriched formula milk decreases febrile episodes and may improve behavioral regulation in young children. Nutrition 2012;28:749–752.

6 Poppitt SD, McGregor RA, Wiessing KR, et al: Bovine complex milk lipid containing gangliosides for prevention of rotavirus infection and diarrhoea in northern Indian infants. J Pediatr Gastroenterol Nutr 2014;59:167–171.

7 Timby N, Domellöf E, Hernell O, et al: Neurodevelopment, nutrition, and growth until 12 mo of age in infants fed a low-energy, low-protein formula supplemented with bovine milk fat globule membranes: a randomized controlled trial. Am J Clin Nutr 2014;99:860–868.

8 Timby N, Hernell O, Vaarala O, et al: Infections in infants fed formula supple-mented with bovine milk fat globule membranes. J Pediatr Gastroenterol Nutr 2015;60:384–389.

9 Timby N, Domellöf M, Holgerson PL, et al: Oral microbiota in infants fed a formula supplemented with bovine milk fat globule membranes – a randomized controlled trial. PLoS One 2017;12:e0169831.

10 Billeaud C, Puccio G, Saliba E, et al: Safety and tolerance evaluation of milk fat glob-ule membrane-enriched infant formulas: a randomized controlled multicenter non-inferiority trial in healthy term infants. Clin Med Insights Pediatr 2014;8:51–60.

11 Timby N, Lönnerdal B, Hernell O, Domellöf M: Cardiovascular risk markers until 12 mo of age in infants fed a formula supplemented with bovine milk fat globule mem-branes. Pediatr Res 2014;76:394–400.

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Human Milk Oligosaccharides: Factors Affecting Their Composition and Their Physiological Significance

Norbert Sprenger, Aristea Binia, and Sean Austin

Breastfeeding is related to a lower risk of infections and possibly dia-betes and overweight in later life, while the situation for allergies is less clear [1], which suggests that breast-milk-specific components may con-tribute to such benefits. Among them are the nondigestible human milk oligosaccharides (HMOs), the third largest solid breast milk component. HMOs are elongations of the milk sugar lactose by galactose, N-acetyl-glucosamine, fucose, and sialic acid, which results in structures similar to those on the mucosa. Most HMOs are not present in farmed-animal milks and are different from generic prebiotics such as galacto- and plant-derived fructo-oligosaccharides. Maternal fucosyltransferases FUT2 and FUT3, encoded by the Secretor and Lewis genes, respectively, followed by lactation stage, have the most striking impact on the HMO compo-sition [2]. The presence or absence of functional FUT2 and FUT3 not only affects the abundance of individual fucosyl-HMOs, but also the total HMO concentration in breast milk. The maternal nutritional and health status might influence HMO composition in the breast milk; however, today there are only circumstantial data to this end. Clinical observa-tional studies in breastfed infant-mother dyads associate specific HMOs with infant gut microbiota, morbidity, infectious diarrhea, and allergies. Although observational studies do not establish causality, together with experimental data they suggest possible biological roles for HMOs. In particular, it is believed that they affect the (i) establishment of the early-life microbiota dominated by bifidobacteria, (ii) resistance to pathogens, and (iii) intestinal mucosal barrier and immunity, thereby contributing to immune protection (Fig. 1a) [3].

Clinical intervention trials with infant formula supplemented with 1 HMO (2′fucosyllactose, 2′FL) or 2 HMOs (2′FL with lacto-N-neote-traose) demonstrated that they allow for age-appropriate growth and are well tolerated [4, 5]. A priori defined secondary outcomes suggested that

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≈1%

PromoteBifidobacterium

dominatedmicrobiome

Deflectpathogens

Strengthengut barrierfunction

Educate the developingimmune system

Favoring 2-HMO Favoring control

AntibioticsAntipyretics

Infections and infestationsGastrointestinal disorders

Ear related infection clusterURT infection clusterLRT infection cluster

Bronchitis

0.1 1 10Odds ratio (95% CI)

*

***

a

b

Fig. 1. Illustration of the different biological functions of HMO (a) and risk for infection-related illnesses and medication use in infants fed a formula supple-mented with 2 HMOs (redrawn from Puccio et al. [5]). Illnesses and medication use were reported by parents and verified by a study physician (b). URT, upper respira-tory tract; LRT, lower respiratory tract. Odds ratios with 95% confidence intervals are shown based on percent of infants with at least 1 event at 1 year of age (Fisher’s exact test: * p < 0.05, ** p < 0.001) [6].

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feeding an infant formula with 2 HMOs relates to fewer reported lower respiratory tract illnesses and reduced requirement for related medication (antibiotics and antipyretics) during the first year of life [5]. In parallel, the early-life microbiota composition and community structure in infants fed the 2-HMO formula shifted towards that of breastfed infants. Formula containing 2 HMOs shifted the global microbiota profile towards that of breastfed infants, characterized by a Bifidobacterium dominance and lower abundance of Escherichia, for example. Interestingly, infants with a microbiota community structure typical for control-formula-fed infants had a 2 times higher risk to use antibiotics during the first year of life than those with a microbiota community typical for breastfed infants.

Together, clinical observational studies corroborated by preclinical experimental data and clinical intervention trials support a role for spe-cific HMOs in immune protection leading to the reduced use of antibi-otics. Further clinical studies, well-designed observational and especially placebo-controlled interventions, are warranted to further substantiate and grow our understanding of the HMO biology and significance for infant nutrition.

References

1 Victora CG, Bahl R, Barros AJ, et al: Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet 2016;387:475–490.

2 Kunz C, Meyer C, Collado MC, et al: Influence of gestational age, secretor, and Lewis blood group status on the oligosaccharide content of human milk. J Pediatr Gastroenterol Nutr 2017;64:789–798.

3 Bode L: The functional biology of human milk oligosaccharides. Early Hum Dev 2015;91:619–622.

4 Marriage BJ, Buck RH, Goehring KC, et al: Infants fed a lower calorie formula with 2′FL show growth and 2′FL uptake like breast-fed infants. J Pediatr Gastroenterol Nutr 2015;61:649–658.

5 Puccio G, Alliet P, Cajozzo C, et al: Effects of infant formula with human milk oli-gosaccharides on growth and morbidity: a randomized multicenter trial. J Pediatr Gastroenterol Nutr 2017;64:624–631.

6 Ferrer-Admetlla A, Sikora M, Laayouni H, et al: A natural history of FUT2 polymor-phism in humans. Mol Biol Evol 2009;26:1993–2003

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Fatty Acids and Fat-Soluble Vitamins in Breast Milk: Physiological Significance and Factors Affecting Their Concentrations

Ardythe L. Morrow and Adekunle Dawodu

The lipid fraction is the second-most abundant solid constituent of human milk, and the most important source of dietary energy. Major constituents of the lipid fraction are fatty acids and fat-soluble vitamins, which are critical contributors to infant health and development. Fatty acids have a critical role in infant neurodevelopment, cardiovascular health, and immune regulation. The fat-soluble vitamins – A, D, E, and K – are critical for infant immune health, neurodevelopment, vision, and modulation of coagulation, and provide antioxidants to minimize cellular damage. Thus, these components are highly bioactive and contribute to infant health and development.

Fatty Acids

The fatty acids of human milk are diverse in length and include satu-rated, monounsaturated, polyunsaturated, and branched-chain structures. The three most abundant fatty acids of human milk – oleic, palmitic, and linoleic acid – comprise about two-thirds of the fatty acid fraction. While there are core fatty acids common to diverse global populations, fatty-acid composition is otherwise highly variable across populations and between mothers, depending on maternal diet and genetics. Well-documented dif-ferences in the fatty-acid profile of human milk across populations include linoleic acid, docosahexaenoic acid (DHA), and other n-3 fatty acids, the trans-fatty acids, and branched-chain fatty acids. DHA and other n-3 fatty acids tend to be higher in fish-eating populations; branched-chain fatty acids tend to be found in higher concentrations among mothers who con-sume more daily servings of dairy and beef; and trans-fats occur signifi-cantly more often in the milk of mothers consuming typical western diets but are very low in the milk of women in traditional societies.

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The impact of differences in the fatty-acid profile of human milk on infant health is understudied and an important domain of research. The strongest evidence of impact has been shown in preterm infants. The milk of preterm infants, whether mother’s own milk or donor milk, is typi-cally lacking DHA, and infant body stores are limited. Maternal supple-mentation with preformed DHA could provide an important strategy for improving maternal and infant health.

Table 1. Fatty acids and fat-soluble nutrients in human milk

Fatty acid associated

Docosahexaenoic acidIncreased with intake of fish and other DHA-rich foodsDiffers by populationLower in milk of mothers who deliver preterm

Branched-chain fatty acidsDairy and beef consumption associated with higher levels of specific BCFADiffers by population

Trans-fatty acidsHigher in westernized populations

n-6:n-3 ratioHigher in westernized populationsFat-soluble vitamins

Vitamin ATypically adequate levelsLow in low-resource regions (Africa and Southeast Asia) and mothers with low intake of animal foodsLower with premature delivery

Vitamin DTypically below detectable levelsInfant supplementation is recommendedMilk levels can be increased with dietary maternal supplementation of 6,400 IU/day

Vitamin ETypically adequate levelsLower levels with premature delivery

Vitamin KTypically low levelsInfant status depends on bacterial synthesis or supplementation/injectionLevels can be increased with maternal supplementation

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Fat-Soluble Vitamins

Fat-soluble vitamins A, D, E, and K are vital for infant nutrition. They perform important health functions and can be stored in the liver and fat tissue until required. While human milk typically has adequate levels of vitamins A and E to meet infant needs, there is variation between populations, and levels can be limited in the circumstance of preterm birth. In human milk, vitamin D and K levels are typically limited. The global public health consensus is to supplement all infants with vitamin D for the prevention and management of nutritional rickets. Recent data indicate, however, that supplementation of sufficiently high doses of vita-min D to lactating women (6,400 IU/day) can safely produce clinically relevant levels of milk vitamin D to satisfy the requirement of her nursing infant. Vitamin K is also low in human milk, and direct vitamin K admin-istration to newborns is recommended practice.

Factors associated with human milk concentration are shown in Table 1.

Commentary

Fatty acids and fat-soluble vitamins are the subject of increased attention in public health nutrition. The health of the infant and the impact of natural variation on development observed between popula-tions and mothers is not known. Nutrient deficiencies in human milk may be managed by supplementation of pregnant or lactating mothers or direct supplementation of infants depending on the nutrient. Dietary supplementation with DHA to pregnant mothers is under study. Preterm infants should be considered a nutritionally needy population worldwide. The milk of mothers who deliver preterm infants may be deficient in n-3 fatty acids and fat-soluble vitamins. Focused attention to the fat-soluble nutrient needs and intake of breastfed infants is warranted.

References

1 Ballard O, Morrow AL: Human milk composition: nutrients and bioactive factors. Pediatr Clin North Am 2013;60:49–74.

2 Hollis BW, Wagner CL, Howard CR, et al: Maternal versus infant vitamin D supplementation during lactation: a randomized controlled trial. Pediatrics 2015;136:625–634.

3 Munns CF, Shaw N, Kiely M, et al: Global consensus recommendations on prevention and management of nutritional rickets. J Clin Endocrinol Metab 2016;101:394–415.

4 Robinson DT, Martin CR: Fatty acid requirements for the preterm infant. Semin Fetal Neonatal Med 2017;22:8–14.

5 Van Winckel M, De Bruyne R, Van De Velde S, et al: Vitamin K, an update for the paediatrician. Eur J Pediatr 2009;168:127–134.

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Water-Soluble Vitamins in Human Milk: Factors Affecting Their Concentration and Their Physiological Significance

Lindsay H. Allen and Daniela Hampel

Water-soluble vitamins are essential for the breastfed infant’s health and development, yet they are among the most susceptible to depletion in human milk when maternal status and/or intake is low. B vitamins play essential roles in cell metabolism, including DNA synthesis and regula-tion, fatty acid and amino acid metabolism, and gluconeogenesis, either as cofactors/coenzymes for or as precursors of these cofactors. Vitamin C serves as an antioxidant involved in tissue repair, immune system sup-port, and interferon production. Inadequate supply of one or more water-soluble vitamins to breastfed infants can result in growth retardation, DNA damage, or metabolic defects, and affect the cardiovascular, muscu-lar, gastrointestinal, and nervous systems. Typical deficiency syndromes in infants are beriberi (B1), ariboflavinosis (B2), pellagra (B3), neural tube defects (folic acid), and megaloblastic anemia, growth retardation, and impaired development (B12)[1].

There are natural changes in the concentrations of water-soluble vitamins in human milk over the course of lactation. While vitamins B1 (thiamine), B3 (niacin), and B5 (pantothenic acid) increase throughout the course of lactation, concentrations of vitamin B6, B12, and C decrease. In contrast, vitamin B2 (riboflavin) remains constant, as does choline after an initial increase during the first months of lactation. Folate has a unique pattern with increasing and decreasing concentrations until stabilization in late lactation.

The concentrations of most of the water-soluble vitamins are influ-enced by maternal status and/or supplementation. While vitamins B1, B2, B3, B5, B6, B12, choline, and vitamin C in milk are quite strongly cor-related with maternal status, folate is not. A low maternal intake of ani-mal source foods causes depletion of B12 in the milk although milk B12 appears to be more dependent on maternal liver stores and accumulation in the liver of the fetus. Low intake of riboflavin will also rapidly reduce

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its concentration in milk since humans do not have excessive stores of this vitamin. Maternal supplementation positively affects milk concentrations of vitamins B1, B2, B3, B6, and B12 (Fig. 1) but has no effect on folate. How-ever, the efficacy of maternal supplementation postpartum is somewhat limited [2]. Other factors affecting concentrations of some water-soluble vitamin concentrations in human milk include parity, preterm delivery, diurnal variation, smoking, medication, and maternal illness.

Existing data on the concentrations of water-soluble vitamins in human milk are very limited. Most studies had few participants, some analytical methods were invalid, the vitamin status or intake of the mother was often unknown, and few studies measured concentrations longitu-dinally during lactation. As a result, there is substantial variation in the reported concentrations that were used to set the adequate intakes for infants and recommended intakes for lactating women. We have devel-oped more efficient, validated methods that can now measure most of the B vitamins and their vitamers simultaneously in small volumes of milk [3]. These have revealed the large differences in concentrations among population groups around the world and enabled the efficient deter-mination of the effects of multiple micronutrient supplements on milk vitamins. This raises the question of how to define a “low” value and is

0

50

100

150

200Re

lativ

e m

edia

n ch

ange

, %

*

Thiamine

*** ***

Riboflavin

****

Nicotinamide

*

***

Pyridoxal

*****

Vitamin B12

Vitamins

2 weeks6 weeks24 weeks

Fig. 1. Percent changes in the concentrations (means ± SEM) of water-soluble vitamins in human milk after maternal supplementation compared to nonsupple-mented women (100% value) at 2, 6, and 24 weeks [2]. * p < 0.05, *** p < 0.001, control vs. supplemented groups.

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the goal of our ongoing study to evaluate the concentrations of vitamins (and other nutrients) in milk from well-nourished but unsupplemented women in 4 countries during the first 9 months of lactation. This Moth-ers, Infants and Lactation Quality (MILQ) study will establish reference values that will improve estimates of the nutrient requirements of infants and lactating women and enable the adequacy of milk nutrient concentra-tions to be evaluated and compared across populations.

References

1 Allen LH: B vitamins in breast milk: relative importance of maternal status and intake, and effects on infant status and function. Adv Nutr 2012;3:362–369.

2 Allen LH, Hampel D, Shahab-Ferdows S, et al: Antiretroviral therapy provided to HIV-infected Malawian women in a randomized trial diminishes the positive effects of lipid-based nutrient supplements on breast-milk B vitamins. Am J Clin Nutr 2015;102:1468–1474.

3 Hampel D, York ER, Allen LH: Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk. J Chromatog B Anal Technol Biomed Life Sci 2012;903:7–13.

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Human Milk MicroRNAs/Exosomes: Composition and Biological Effects

Bo Lönnerdal

Human milk provides many benefits to the breastfed infant result-ing in significantly better short- and long-term outcomes compared to formula-fed infants. These benefits are likely achieved by a well-balanced supply of nutrients and wide variety of bioactive components in breast milk. These components include long-chain fatty acids (e.g., DHA), com-plex oligosaccharides, bioactive proteins (e.g. immunoglobulins, lactofer-rin, and osteopontin), nucleotides, and lutein. By various mechanisms that have been extensively studied, they protect the infant against infec-tions and stimulate brain development and visual function.

More recently, it was discovered that breast milk also contains exo-somes, i.e., microvesicles consisting of microRNAs (miRNAs) with sizes of ~22 nucleotides [1]. Exosomes are small extracellular vesicles about 30–100 nm in size and are produced by a variety of cells, including mac-rophages, lymphocytes, dendritic cells, epithelial cells, and tumor cells. They are found in physiological fluids such as plasma, urine, and malig-nant effusions. It is well known that exosomes are important in cell-cell signaling, but their physiological significance in vivo is less known. Early studies suggested promising roles in immunotherapy and cancer therapy, but this is a rapidly advancing field, and many clinical trials are ongoing. It was shown that isolated milk exosomes could affect immune responses of PBMCs and T-regulatory cells [1]. A subsequent study on miRNA expres-sion in breast milk found large numbers of miRNAs (281 of 723 human miRNAs known at that time) and, in particular, high levels of immune-related miRNAs during the first 6 months of lactation [2]. Several breast milk miRNAs have been shown to originate from the mammary gland [3], and many of them are involved in cellular development and immune function.

Exosome-mediated transfer of miRNAs is a novel mechanism of genetic exchange between cells. It is therefore possible that exosomes in milk may survive digestion and deliver miRNAs to intestinal cells, or, if transferred into the blood stream, to cells in other tissues. In vitro

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digestion experiments mimicking conditions in the infant gut have shown that exosomes and their miRNA cargo can survive proteolytic diges-tion [4]. Further, when human intestinal epithelial cells in culture were exposed to exosomes isolated from breast milk that was undigested or subjected to in vitro digestion, it could be shown by confocal microscopy that the cells could take up exosomes from both untreated and in vitro digested breast milk and that they migrated to the nucleus [4]. Therefore, it is possible that breast milk exosomes may affect gene transcription in the small intestine. Research on human adults consuming cow milk in various amounts has shown that major bovine milk miRNAs are found in the circulation postprandially and in a dose-dependent manner, fur-ther suggesting that exosomes can resist conditions in the gastrointestinal tract and be delivered to the systematic circulation [5]. Thus, it is pos-sible that milk miRNAs may transfer genetic material to the infant and thereby affect gene transcription and regulation of cellular events in sev-eral tissues.

References

1 Admyre C, Johansson SM, Qazi KR, et al: Exosomes with immune modulatory fea-tures are present in human breast milk. J Immunol 2007;179:1969–1978.

2 Kosaka N, Izumi H, Sekine K, Ochiya T: MicroRNA as a new immune-regulatory agent in breast milk. Silence 2010;1:7.

3 Alsaweed M, Lai CT, Hartmann PE, et al: Human milk miRNAs primarily originate from the mammary gland resulting in unique miRNA profiles of fractionated milk. Sci Rep 2016;6:20680.

4 Liao Y, Du X, Li J, Lönnerdal B: Human milk exosomes and their microRNAs survive digestion in vitro and are taken up by human intestinal cells. Mol Nutr Food Res 2017;61, DOI: 10.1002/mnfr.201700082.

5 Baier SR, Nguyen C, Xie F, Wood JR, Zempleni J: MicroRNAs are absorbed in bio-logically meaningful amounts from nutritionally relevant doses of cow milk and affect gene expression in peripheral blood mononuclear cells, HEK-293 kidney cell cultures, and mouse livers. J Nutr 2014;144:1495–1500.

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Human Milk Proteins: Composition and Physiological Significance

Sharon M. Donovan

Human milk (HM) is the ideal food that ensures optimal growth and development of infants [1]. In addition, HM contains a wide vari-ety of bioactive components, including lipids, oligosaccharides, and pro-teins. Over the past 30 years, infant formulas have undergone dramatic changes in nutritional composition to more closely mimic that of HM [2]. However, clinical and epidemiological studies show that differences in short- and long-term health outcomes still persist between breastfed and formula-fed infants, including growth patterns, nutritional status, gut microbiota composition, prevalence of infection, and health out-comes [1]. HM contains over 400 proteins that can be broadly classified into 3 categories: caseins, whey proteins, and mucins, which are present in the milk fat globule membrane (MFGM). HM is whey predominant, but the whey/casein ratio of HM changes during the course of lactation, being 90/10 in colostrum and changing to 60/40 in mature HM. The pre-dominant caseins in HM are b and k, whereas bovine milk contains a, b, g, and k caseins. The proteins present in significant quantities in the whey fraction are α-lactalbumin, lactoferrin, IgA, osteopontin (OPN), and lysozyme. The predominant whey protein in bovine milk is b-lac-toglobulin, although low concentrations of α-lactalbumin, lactoferrin, and OPN in bovine milk have enabled their isolation and utilization in preclinical and clinical trials. Additionally, bioactive peptides are formed during the digestion of casein and whey, and glycans from glycoproteins are bifidogenic, adding further complexity to the functional properties of HM proteins. These functions include: serving as a source of amino acids; improving the bioavailability of micronutrients, including vitamins, minerals, and trace elements, providing stimulation of intestinal growth and maturation; supporting immunologic defense; shaping the microbi-ome; and enhancing learning and memory (Fig. 1) [2, 3]. Recent advances in dairy technology have enabled the isolation of bioactive milk proteins from bovine milk in sufficient quantities for clinical investigations and, in some cases, addition to commercially available infant formulas [2].

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Herein, the current evidence on HM protein composition and bioactiv-ity of HM proteins will be reviewed, with a focus on lactoferrin, OPN, and the MFGM [4]. Lactoferrin is a non-heme iron-binding protein that has been shown to beneficially impact iron absorption in the breastfed infant and exert bacteriostatic effects. In the piglet model, bovine lacto-ferrin stimulated intestinal cell proliferation. In randomized controlled clinical trials, bovine lactoferrin reduced diarrhea and respiratory ill-nesses in term infants and sepsis and necrotizing enterocolitis in preterm infants [5]. OPN is an acidic, glycosylated, and highly phosphorylated protein. It interacts with cell surface integrins and the CD44 receptor to influence biomineralization, tissue remodeling, and immune regulation. Bovine OPN supplemented to formula at the concentration present in HM changed intestinal gene expression in rhesus monkeys to be more similar to breastfed monkeys. In a randomized controlled clinical trial, bovine OPN reduced fever incidence and serum TNF-a concentrations [4]. Lastly, MFGM is the triple membrane system that encapsulates milk fat. It contains cellular components, including cholesterol, glycerol phos-pholipids, sphingolipids, and proteins, including mucin 1, butyrophilin, CD36, adipophilin, and lactadherin. These bioactive components con-tribute to the antiviral and antibacterial activities of MGFM. In random-ized controlled clinical trials, MFGM from bovine milk reduced diarrhea, fever, and antipyretic use and increased IQ [4]. In summary, HM contains many bioactive proteins that act independently and synergistically to

Provide essential aminoacids

Serve as prebiotics and shape microbiome

Support brain development,learning, and memory

Promote host defense and immune development

Stimulate intestinal growth and maturation

Enhance nutrient digestion and absorption

Humanmilk

proteins

Fig. 1. Biological functions of human milk proteins.

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provide multilayer defense against infection, as well as stimulate intesti-nal and cognitive development and shape the microbiome. Purification of bioactive proteins from bovine milk have allowed clinical trials in infants and will ultimately enable modifications in infant formula composition to narrow the differences in health outcomes between breastfed and for-mula-fed infants.

References

1 Victora CG, Bahl R, Barros AJ, et al: Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet 2016;387:475–490.

2 Hernell O: Human milk vs. cow’s milk and the evolution of infant formulas. Nestlé Nutr Workshop Ser Pediatr Program 2011;67:17–28.

3 Lönnerdal B, Erdmann P, Thakkar SK, et al: Longitudinal evolution of true protein, amino acids and bioactive proteins in breast milk: a developmental perspective. J Nutr Biochem 2017;41:1–11.

4 Demmelmair H, Prell C, Timby N, et al: Benefits of lactoferrin, osteopontin and milk fat globule membranes for infants. Nutrients 2017;9:E817.

5 Donovan SM: The role of lactoferrin in gastrointestinal and immune development and function: a preclinical perspective. J Pediatr 2016;173S:S16–S28.

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Early-Life Nutrition, Growth Trajectories, and Long-Term Outcome

Ferdinand Haschke, Christoph Binder, Mercedes Huber-Dangl, and Nadja Haiden

Introduction

It is well established that nutrition during the first 1,000 days can have a long-term effect on growth, metabolic outcome, and long-term health [1, 2]. We review long-term anthropometric follow-up of children with risk of later morbidity: (a) very-low-birthweight (VLBW) infants who have birthweights <10% percentile of weight and receive fortified breast milk, (b) infants from developing countries who are breastfed accord-ing to the present recommendations but have low birthweight and birth length, and (c) children from developed countries who were enrolled in randomized controlled trials (RCTs) to test if breastfeeding and low-pro-tein formulas can prevent from rapid weight gain and childhood obesity.

VLBW Infants

Following international recommendations for nutrition of VLBW infants [3] (birthweight <1,500 g; <32 weeks of gestation) now contrib-utes to better postnatal growth which should be parallel to a percentile line of intrauterine growth charts. The segment of VLBW infants which have a birthweight below the 10th percentile for gestational age (“born too small”) includes growth-restricted VLBW infants who are born con-stitutionally small (SGA) and VLBW infants with intrauterine growth restriction (IUGR) which is caused by a complex antenatal pathology [4]. For VLBW infants who are “born too small” ESPGHAN recommends an “enhanced nutrients strategy” which provides extra nutrients up to 52 weeks [3]. Actually, reliable data are lacking to guarantee that the recom-mended “enhanced nutrients strategy” is safe and effective for both SGA and IUGR infants in terms of long-term growth, i.e. no increased risk of persisting postnatal malnutrition as well as of later obesity, diabetes type 2, and cardiovascular events. We investigated the impact of the “enhanced

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nutrients strategy” up to 52 weeks of postconceptional age on growth of VLBW infants (SGA with no genetic defects, malformations, intrauterine infections, n = 31; IUGR with pathological ultrasound measurements [4], n = 127). Mean birthweights of SGA and IUGR infants were 600 and 688 g (ns), and mean gestational ages were 25 and 29 weeks (<0.001), respec-tively. Enteral feeding of all infants started with breast milk that was then fortified with a human milk fortifier. At discharge, 68% of the infants still received breast milk. IUGR infants showed low weight with downwards crossing of weight percentiles between discharge and 3 months (corrected for GA). Mean weight of the SGA infants crossed the 10th percentile of the WHO standards of weight already at 6 months corrected for GA. A longitudinal analysis indicated higher weight of the SGA group between 3 and 24 months corrected for GA (p < 0.05). BMI of both groups was similar during the observation period. Our data question the ESPGHAN approach [3] that there is no need to develop separate nutrition guidelines for VLBW who are SGA or IUGR, but randomized controlled studies which include body composition and metabolic outcome measurements are necessary to prove the preliminary findings.

Breastfed Infants from Developing Countries – Stunting

The associations of breastfeeding with growth and health in devel-oping countries can be studied by repeatedly analyzing DHS (demo-graphic health surveys; US) datasets that provide information on nutrition, growth, and health. We reviewed data of more than 130,000 infants and small children (0–6, 6–12, 12–24 months) from 20 develop-ing countries that were collected at least twice at intervals of 5–10 years during the last 2 decades [5, and unpubl. data]. Exclusive breastfeeding was associated with significantly higher weight, length, and lower prob-ability of stunting, wasting, and infections. DHS data of infants between 6 and 24 months also reflect the influence of low-quality complemen-tary feedings and poor environmental conditions in developing coun-tries, which contribute to the high stunting and wasting rates. Growth trajectories from 2 well-controlled African cohorts [6, 7] with strong breastfeeding support showed the importance of maternal stature, nutrition, and health as well as maternal nutrition before conception and during pregnancy: growth trajectories of infants who were in the top 10th percentile segment of length at birth grew almost according to the WHO standards until 2 years. However, those infants in the bottom 10th percentile segment at birth (i.e., newborns with disturbed intra-uterine growth) showed poor growth and had mean length at 2 years that was below the –2 z-score of the WHO standards. In addition to breastfeeding support, future key targets should be to improve nutrition

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of adolescent girls, young women, and during pregnancy. Height catch-up in young children, even in the absence of external nutritional inter-ventions, clearly contradicts the widely held impression that a window of opportunity closes at 24 months of age. The extent of catch-up after 24 months is highly context specific and presumably reflects the avail-ability of foods, food-consumption patterns, the composition of diets, and the prevailing burden of infections (especially those affecting gas-trointestinal function).

Is Low-Protein Intake during the Breastfeeding Period and Beyond a Factor That Contributes to Prevention of Obesity?

Infants fed traditional high-protein formulas have higher weight than breastfed infants at least until 24 months. RCTs indicate that infants receiving new low-protein follow-up formulas have lower weight gain during the first 12 months than infants receiving high-pro-tein formulas [8–11]. Follow-up of clinical trials until 5–6 years indi-cates that children who were on low-protein formulas during the first year have BMIs similar to children who were breastfed [12, 13]. Dur-ing longitudinal body composition follow-up, we found that percent-age of body fat is more rapidly decreasing between 6 and 60 months, if children had been exclusively breastfed for 4–6 months or had been fed low-protein formulas until 12 months [13]. Children who received higher protein formulas during infancy showed only marginal decrease in percentage body fat until 60 months (p < 0.05). An RCT indicates that higher protein intake during infancy results in significantly higher BMI at 72 months and a higher percentage of childhood obesity [12]. A longitudinal cohort study that reflects the French childhood popu-lation [14] indicates that higher protein intake (>15% of calories) is associated with higher BMI during school age, adolescence, and young adulthood.

Conclusions

VLBW infants who are IUGR show low weight gain after discharge from hospital when they receive fortified breast milk. RCTs are necessary to confirm the results of our cohort study and to test new fortification strategies of breast milk. Exclusive breastfeeding is important to prevent infants from stunting in developing countries. Further preventive mea-sures include nutritional supplementation of young women before and during pregnancy, promotion of breastfeeding and improvement of qual-ity of complementary foods. RCTs which include follow-up of growth and

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body composition during childhood indicate that breastfeeding and the use of low-protein formulas can contribute to prevention of rapid weight gain during infancy and childhood obesity.

References

1 Barker DJ, et al: Fetal nutrition and cardiovascular disease in adult life. Lancet 1993;341:938–941.

2 Godfrey KM, Costello PM, Lillycrop KA: Development, epigenetics and metabolic programming. Nestle Nutr Inst Workshop Ser 2016;85:71–80.

3 ESPGHAN Committee on Nutrition, Aggett PJ, Agostoni C, et al: Feeding preterm infants after hospital discharge: a commentary by the ESPGHAN Committee on Nutrition. J Pediatr Gastroenterol Nutr 2006;42:596–603.

4 Society for Maternal-Fetal Medicine Publications Committee, Berkley E, Chauhan F, Abuhamad A: Doppler assessment of the fetus with intrauterine growth restriction. Am J Obstet Gynecol 2012;206:300–308.

5 Haschke F, Haiden N, Detzel P, et al: Feeding patterns during the first 2 years and health outcome. Ann Nutr Metab 2013;62(suppl 3):16–25.

6 Prentice AM, Ward KA, Goldberg GR, et al: Critical windows for nutritional inter-ventions against stunting. Am J Clin Nutr 2013;97:911–918.

7 Prendergast AJ, Humphrey JH: Stunting persists despite optimal feeding: are toilets part of the solution? Nestle Nutr Inst Workshop Ser 2015;81:99–110.

8 Singhal A, Kennedy K, Lanigan J, et al: Nutrition in infancy and long-term risk of obesity: evidence from 2 randomized controlled trials. Am J Clin Nutr 2010;92:1133–1144.

9 Koletzko B, von Kries R, Closa R, et al: Lower protein in infant formula is associ-ated with lower weight up to age 2 years: a randomized clinical trial. Am J Clin Nutr 2009;89:1836– 1845.

10 Inostroza J, Haschke F, Steenhout P, at al: Low-protein formula slows weight gain in infants of overweight mothers. J Pediatr Gastroenterol Nutr 2014;59:70–77.

11 Ziegler EE, Fields DA, Chernausek SD, at al: Adequacy of infant formula with protein content of 1.6 g/100 kcal for infants between 3 and 12 months. J Pediatr Gastroenterol Nutr 2015;61:596–603.

12 Weber M, Grote V, Closa-Monasterolo, et al: Lower protein content in infant formula reduces BMI and obesity risk at school age: follow-up of a randomized trial. Am J Clin Nutr 2014;99:1041–1051.

13 Haschke F, Gratwohl D, Haiden N: Metabolic programming: effects of early nutri-tion on growth, metabolism and body composition. Nestle Nutr Inst Workshop Ser 2016;86:87–95.

14 Rolland-Cachera MF, Akrout M, Péneau S: Nutrient intakes in early life and risk of obesity. Int J Environ Res Public Health 2016;13:E564.

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Early-Life Nutrition and Cognitive Development: Imaging Approaches

Weili Lin, Kristine R. Baluyot, Manjiang Yao, Jian Yan, Li Wang, Gang Li, Brittany Howell, Jed T. Elison, and Dinggang Shen

Brain development in the first years of life is the most dynamic and perhaps the most important phase of brain maturation [1]. Brain develop-ment can potentially be categorized into seven basic stages [2], includ-ing neurogenesis, cell migration, cell differentiation, dendrite and axonal growth, synaptogenesis, synaptic pruning, and myelogenesis, respectively. These complex and dynamic cellular processes set the foundation for the remarkable cognitive development and maturation during the first years of life. Any adverse effects leading to deviation from these well-orchestrated processes could result in life-long impacts on the health and development of our brain. It is widely recognized that adequate nutrition is necessary for normal brain development during pregnancy and infancy; particular nutrients most likely affect distinct aspects of brain development. The critical dosage windows and time frames for various nutrients needed at different stages of brain development are likely dissimilar. Long-chain polyunsaturated fatty acids are essential for neurogenesis and synapto-genesis, affecting pre- and postnatal brain development. Sphingomyelin is crucial for white matter myelination, which undergoes rapid develop-mental processes from late third trimester throughout the first years of life. It should also be recognized that different nutrients could contribute to similar aspects of brain development, functioning in a complementary or synergistic manner. For example, iron, choline, and sphingomyelin play important roles in white matter myelination. Table 1 summarizes a list of key nutrients with established and/or emerging roles on different aspects of early brain development. Large amount of knowledge about nutrition and brain is from preclinical investigations and clinical manifes-tations of nutrient deficiencies in humans. It remains a challenge to iden-tify potential associations between intakes of specific nutrients and early brain development in normal healthy population when behavioral and cognitive assessments are employed as the sole outcome measures. Behav-ioral assessments, although robust, suffer from relatively low sensitivity,

29

difficulties assessing higher-order brain functions, particularly during infancy, and lack of ability to provide insights into the neural substrates underlying brain functional maturation. In contrast, magnetic resonance imaging (MRI) is capable of providing detailed anatomical and functional information – an ideal tool to characterize brain functional develop-ment and elucidate the effects of early nutrition. However, MRI is highly

Table 1. Key nutrients and their roles in early brain development

Nutrients Roles

MineralsIron Myelination, neurotransmission, brain growth, cofactor for

brain enzymesZinc Neurogenesis, neuron maturation and migration, cofactor for

>200 enzymesSelenium Component of selenoproteins in brain, antioxidantsIodine Neuron differentiation and maturation, myelination,

neurotransmissionCopper Neurotransmission, brain energy metabolism, antioxidantMagnesium Brain energy metabolism, myelination, neurotransmission

LipidsLCPUFA(DHA, ARA)

Neurogenesis and growth, synaptogenesis, two major lipids of gray matter

Phospholipids Major component of neuronal membrane, precursor for key second messengers

Sphingomyelin Myelination, major component of myelin sheathGangliosides Component of neuronal membrane, signal transduction

B vitaminsFolate Myelination, neural cell proliferation and differentiation, DNA

biosynthesisB12 Myelination, neural cell proliferation and differentiation,

1-carbon metabolismCholine Neurotransmitter synthesis, myelination, DNA methylation

CarotenoidsLutein Major carotenoid in brain, antioxidant

ProteinsLactoferrin Major iron-binding protein, major whey protein in human milkOsteopontin Breast milk protein important for immunity and emerging

research suggests a role in myelination

CarbohydratesHuman milkoligosaccharides

Emerging roles on brain likely via the microbiota-gut-brain connection

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sensitive to motion artifacts. Nonetheless, our team has developed strate-gies that enable imaging of typically developing children from birth to teens without sedation [3].

Table 2 provides the first quantitative evidence (cortical gray matter volume [GM], cortical thickness [CT], and brain surface area [SA]) of early brain structural development from a cohort of typically developing children with a dense sampling scheme during the first 6 years of life. The total GM is almost doubled by the 9th month, followed by a much slower growth pace after the first year of life. Concurrently, CT increases in year 1 and decreases starting from the 18th month. Finally, SA exhibits a simi-lar growth trajectory as that of cortical GM volume during the first 6 years of life, suggesting that SA expansion may play an equally or even more important role when compared to CT during early brain development.

In addition to characterizing brain structural development, resting state fMRI has been employed to discern detailed functional maturation during the first years of life [4]. During year 1, the topologies of the senso-rimotor and auditory networks are highly consistent with those observed in adults. In contrast, higher-order brain functional networks are more primitive. In addition, while the visual networks are topologically similar to adults, they undergo tremendous growth in year 1.

Together, the structural and functional MRI has provided highly critical and quantitative insights into early brain development and serves as an important stepping stone to rigorously determine the potential interplay between nutrient intakes and early brain development. More

Table 2. Brain structural development during the first 6 years of life

Age Gray matter, cm3 Cortical thickness, mm

Surface area, cm2

volumes,mean ± SD

ratio tobirth

thicknessmean ± SD

ratio tobirth

area,mean ± SD

ratio tobirth

2 weeks 92,832.5±10,291 1 2.21±0.14 1 ,621.3±57.3 13 months 124,854.9±11,628.4 1.34 2.31±0.11 1.04 ,797.3±78.7 1.286 months 158,763.8±15,576.3 1.71 2.54±0.17 1.15 ,911.1±82.4 1.479 months 181,632.8±35,636 1.96 2.65±0.17 1.2 1,005.9±106.5 1.62

12 months 199,191.8±17,073 2.15 2.72±0.18 1.23 1,077.2±100.5 1.7318 months 213,185.8±21,634 2.3 2.69±0.19 1.22 1,155.2±82.7 1.8624 months 232,574.2±23,185.5 2.51 2.65±0.1 1.2 1,279.8±109 2.0636 months 249,279.8±23,092.8 2.69 2.62±0.07 1.18 1,375.6±131 2.2148 months 258,033.7±20,339.5 2.78 2.61±0.08 1.18 1,427.3±108.4 2.360 months 262,183±23,958.3 2.82 2.59±0.06 1.17 1,461.4±122.4 2.3572 months 263,971.2±22,352.3 2.84 2.6±0.07 1.17 1,456.4±121.9 2.34

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recently, research regarding brain imaging, cognitive development, and nutrition has intersected in expanded interdisciplinary efforts to under-stand the gut-brain axis, which could further shed light on our under-standing of the complex interaction between brain development and gut microbiome [5].

References

1 Casey BJ, Tottenham N, Liston C, Durston S: Imaging the developing brain: what have we learned about cognitive development? Trends Cogn Sci 2005;9:104–110.

2 Kolb B, Gibb R: Brain plasticity and behaviour in the developing brain. J Can Acad Child Adolesc Psychiatry 2011;20:265–276.

3 Lin W, Meng Y, Li G, et al: Developmental trajectories of cortical thickness and myelin contents from birth to 6 years old (poster). 22nd Annual Meeting of the Organization for Human Brain Mapping, Vancouver, 2017.

4 Gao W, Zhu H, Giovanello KS, et al: Evidence on the emergence of the brain’s default network from 2-week-old to 2-year-old healthy pediatric subjects. Proc Natl Acad Sci USA 2009;106:6790–6795.

5 Collins SM, Surette M, Bercik P: The interplay between the intestinal microbiota and the brain. Nat Rev Microbiol 2012;10:735–742.

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Early-Life Nutrition and Gut Immune Development

Lieke van den Elsen, Akila Rekima, and Valérie Verhasselt

Gut immune function conditions development of diseases that result from defects in immune regulation such as allergic and obesity-related disease [1]. As epidemiological studies support the developmental origin of health and disease, the deciphering of the critical factors modulating gut immune development should allow the advance of primary preven-tion strategies specifically adapted to the early-life immune system. Here, we will emphasize how nutrition can shape microbiota composition and metabolite production with immune-modulatory properties. We will also focus on the role of dietary compounds recently demonstrated to be essential in immune development and function such as dietary antigens, vitamin A, and aryl hydrocarbon receptor (AhR) ligands.

Microbiota is necessary for lymphoid tissue development and immune differentiation such as IgA secretion, regulation of IgE responses, and differentiation of T cells subsets [1]. Besides mode of delivery, nutri-tion is the key factor directing the early microbiota composition and function [2]. Breast milk contains viable bacteria that will contribute to the establishment of the neonatal microbiota, and maternal IgA will alter colonization patterns in the neonate. Breast milk also contains nutrients specific for the growth of commensals, i.e. human milk oligosaccharides (HMO), which stimulate the growth of bifidobacteria and affect their metabolic function. In animals fed solid food, Clostridia can metabolize dietary fibers into short-chain fatty acids (SCFA), while in breastfed neo-nates, SCFA are derived from HMO metabolized by bifidobacteria (Fig. 1a). The role of SCFA in gut immunity in preweaned mice has not been assessed yet. In young weaned mice, they were found to stimulate regula-tory T cell expansion, IgA and mucus secretion, gut epithelium barrier function, ILC3 function, and induce resistance to food allergy and gut inflammatory disease [3] (Fig. 1a). Some commensals, such as Lacto-bacillus, metabolize tryptophan, an essential amino acid that is a com-mon constituent of protein-based foods (Fig. 1b). The metabolites bind AhR expressed in ILC3 and stimulate the postnatal formation of isolated

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lymphoid follicles and IL-22 secretion necessary for gut barrier func-tion and protection from Citrobacter infection and colitis [3] (Fig. 1b). In breastfed infants, AhR ligands could originate from maternal microbiota or from maternal diet (Fig. 1b).

Mice studies have recently highlighted the regulatory function of diet-derived antigens in the small intestine [4] (Fig. 1c). In the human [5],

Allergy, colitis

Allergy

Dietary antigensTGF-β

TGF-β

RALDH+DC

RALDH+DC

TregsTregsTh1

lgGVitamin A

SCFA, vitamin A

Tregs

B cellILC3

IL-22 Paneth cell

Citrobacter infection, colitis

ILC3

ILF

AMPIndole derivatives

IgLactobacillus

Tryptophan

IL-22

MucusslgAAMP

SCFA

ClostridiaBifidobacteria

HMO

Goblet cell

a

c

b

(For legend see next page.)

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food diversification in the first year of life was associated with decreased risk of allergies. The shaping of immune reactivity by induction of oral tolerance to specific antigens during the period of immune ontogeny may be possible in the case of egg (OVA) and peanut antigen. Additional TGF-β, vitamin A, and IgG from maternal milk were critical for toler-ance induction towards OVA transferred through breast milk in rodents [6] (Fig. 1c). TGF-β is a growth factor for epithelium, and both vitamin A and IgG acted on antigen transfer through epithelium. Vitamin A also increased the function of dendritic cells involved in tolerance and Th1 differentiation (Fig. 1c). Our recent data showed that not all the antigens in breast milk induce oral tolerance. Antigen from house dust mite, Der p 1, is present in human breast milk and its presence increased the risk of allergy both in mice and in the humans. This stresses the need to identify how maternal milk factors could be modulated to counteract deleterious action of some allergens [6].

In conclusion, before weaning, the physiological food for mam-mals is providing the neonate with the factors necessary for immune

Fig. 1. Impact of food on immune ontogeny. a Short-chain fatty acids (SCFAs) stimulate regulatory T-cell expansion, IgA, and mucus secretion, gut epithelium barrier function, antimicrobial peptide secretion (AMP), and ILC3 function and induce resistance to food allergy and gut inflammatory disease. Human milk oligo-saccharides (HMO) are present in human milk and stimulate the growth of bifido-bacteria that can metabolize HMO into SCFA. After weaning, metabolic function of bifidobacteria changes, and they become able to metabolize complex sugars from dietary fibers similarly to clostridia found in microbiota of older children. b Aryl hydrocarbon receptor (AhR) ligands bind to AhR receptor expressed on ILC3. They stimulate the postnatal formation of isolated lymphoid follicles (ILF) and IL-22 secretion necessary for gut barrier function and protection from Citrobacter infec-tion and colitis. AhR ligands are found in cruciferous vegetables such as broccoli and cabbage. They can also be produced by some commensals, such as Lactobacillus, which metabolize tryptophan from protein-based foods into indole derivatives. In breastfed infants, AhR ligands can originate from maternal microbi-ota metabolites with maternal milk immunoglobulin helping in the transfer of these metabolites to the neonate. Breast milk contributes also to AhR-mediated immune ontogeny by stimulating the growth of Lactobacillus. c After weaning, antigens derived from solid food are necessary for populating the small intestine with induced Tregs. Tregs specific to dietary antigens can be induced by oral expo-sure. Before weaning, oral tolerance can be induced to antigens from maternal diet present in breast milk. This requires the presence of additional cofactors in breast milk such as TGF-β, vitamin A, and IgG. Vitamin A increases gut barrier function, the capacity of dendritic cells (DCs) to metabolize vitamin A into retinoic acid and Th1 differentiation. Antigens bound to IgG are better transported across the epithe-lium and induce FoxP3 Tregs that are responsible for potent and long-lasting toler-ance. After weaning, Treg induction towards oral antigen is favored by SCFAs. These induce TGF-β secretion from epithelium and stimulate retinoic acid forma-tion from vitamin A by DCs.

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maturation, which the neonate would otherwise miss due to the lack of a diverse microbiota and solid food-derived molecules. Breast milk exposes the infant to a variety of food antigens, and it contains ligands that are critical for lymphoid tissue development and immune function such as AhR ligands and vitamin A. It provides HMO, as surrogates to fibers found in solid food, for commensals to produce SCFA. Breast milk also delivers a microbiota and food for commensal growth in the sterile neo-nate gut. After weaning, solid food-derived antigens and vitamins as well as food metabolites produced by the microbiota will continue to shape the immune system and dictate susceptibility to local and systemic immune-mediated disease.

References

1 Belkaid Y, Harrison OJ: Homeostatic immunity and the microbiota. Immunity 2017;46:562–576.

2 van Best N, Hornef MW, Savelkoul PH, Penders J: On the origin of species: factors shaping the establishment of infant’s gut microbiota. Birth Defects Res C Embryo Today 2015;105:240–251.

3 Rooks MG, Garrett WS: Gut microbiota, metabolites and host immunity. Nat Rev Immunol 2016;16:341–352.

4 Kim KS, Hong SW, Han D, et al: Dietary antigens limit mucosal immunity by induc-ing regulatory T cells in the small intestine. Science 2016;351:858–863.

5 Palmer DJ, Prescott SL, Perkin MR: Early introduction of food reduces food allergy – pro and con. Pediatr Allergy Immunol 2017;28:214–221.

6 Munblit D, Verhasselt V: Allergy prevention by breastfeeding: possible mecha-nisms and evidence from human cohorts. Curr Opin Allergy Clin Immunol 2016;16:427–433.

36

Early-Life Nutrition and Microbiome Development

Erika Isolauri, Samuli Rautava, Seppo Salminen, and Maria Carmen Collado

Recent reports link clinical conditions, phenotypes alternating from inflammatory bowel disease, obesity, and allergic diseases to neurodevel-opmental disorders, to aberrant gut microbiota composition [reviewed in 1]. This has led to a growing interest in host-microbe cross talk, charac-terizing the healthy microbiome and modifying its deviations at an early age. The rationale arises from the recognition of the intimate interrela-tionship between diet, immune system and microbiome, and the origins of human disease.

Before satisfactory preventive measures can be put in practice, impor-tant questions remain to be solved. First, we need more profound under-standing of the complex mechanisms underlying these heterogeneous manifestations of immune-mediated and microbiome-associated chronic conditions. Second, long-term follow-up studies are required to deter-mine whether the changes in the microbiome underlie the pathogenesis of noncommunicable diseases or are merely end results thereof, confront-ing the question of causality. This uncertainty notwithstanding, the com-plex and bidirectional interrelationship of the diet and the gut microbiota is becoming evident. Early exposures by the enteral route induce dynamic adaptive modifications in the microbiota composition and activity, which may carry long-term clinical impacts. Microbiota changes, again, control energy acquisition and storage and may contribute to gut immunological milieu; high-energy Western diet alters the microenvironment of the gut leading to propagation of the inflammatory tone and perturbation of gut barrier function and thereby to systemic low-grade inflammation [2, 3].

The cornerstone of prevention of noncommunicable diseases is breastfeeding [4]. Not only does it provide the infant with nutrients, it may also confer immunologic protection at the portal of entry where major load of antigens is encountered, the gut barrier. A delicate balance of stimulatory, even inflammatory, maturational signals, together with a myriad of anti-inflammatory compounds, is transferred from mother to

37

infant via breastfeeding. Human milk protective compounds also include specific oligosaccharides and fatty acids influencing early microbial colo-nization and gut barrier adherence of pathogens and other microbes, but also specific microbiota and molecules operating in host-microbe interaction.

Breastfeeding provides several health benefits that are likely to be caused by promotion of age-appropriate and environment-adjusted gut colonization. There is abundant evidence that breast milk complements the microbiota transmission to the infant gut: the mother provides the infant with bifidobacteria, lactic acid bacteria, and other microbiota com-ponents in significant quantities during breastfeeding. Several active com-pounds of breast milk accomplish this progression. However, the microbes and other active compounds in breast milk strongly vary according to the mother’s health and weight gain during pregnancy, and the mode of delivery. In general, the infant’s probability of being colonized by bifido-bacteria is lower when the mother has higher BMI, excessive weight gain

Disease risk factors

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Fig. 1. The progression of gut colonization and the child’s risk of developing noncommunicable diseases. Key risk factors during the perinatal period and infancy include unfavorable nutritional environment during pregnancy, being born preterm or by caesarean section or devoid of important immunomodulatory compounds of breast milk. Resilience to unfavorable changes during this critical period of maturation may be achieved by endorsing breastfeeding and introduc-tion of active protective compounds.

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during pregnancy, and the child is delivered via caesarean section, and higher when the mother is of normal weight, has notable bifidobacteria colonization in her own gut and breast milk and is breastfeeding (Fig. 1).

The model of early nutrition for future studies is the healthy breast-fed infant that remains healthy in the long-term. Scientific interest is cur-rently extending from the duration of breastfeeding to the composition of breast milk, and characterization of the key regulatory substances therein. Human milk, rich in bioactive compounds including health-promoting microbes and their optimal growth factors, human milk oligosaccharides, continues to afford tools to study diet-microbiota interactions for research aiming at reducing the risk of noncommunicable diseases.

References

1 Rautava S, Luoto R, Salminen S, Isolauri E: Microbial contact during preg-nancy, intestinal colonization and human disease. Nat Rev Gastroenterol Hepatol 2012;9:565–576.

2 Bäckhed F, Ding H, Wang T, et al: The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci USA 2004;101:15718–15723.

3 Shin NR, Whon TW, Bae JW: Proteobacteria: microbial signature of dysbiosis in gut microbiota. Trends Biotechnol 2015;33:496–503.

4 Victora CG, Bahl R, Barros AJ, et al: Breastfeeding in the 21st century: epidemiology, mechanisms, and lifelong effect. Lancet 2016;387:475–490.

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Human Milk and Clinical Outcomes in Preterm Infants

Paula P. Meier

Human milk from the infant’s own mother (mother’s own milk; MOM) has been linked for decades with beneficial clinical outcomes for infants born prematurely, especially those born extremely preterm (<32 weeks of gestation) and very-low birthweight (VLBW; birthweight <1,500 g). The primary impact of MOM in this population is the reduction of potentially preventable morbidities including: necrotizing enterocolitis, late-onset sepsis (sepsis), bronchopulmonary dysplasia, severe retinopathy of prematurity, neurodevelopmental problems, and rehospitalization after discharge from the neonatal intensive care unit (NICU) [1]. Additionally, MOM supports adequate growth with exogenous fortification and clinical management of lipid variability in pumped and stored MOM. Recently, studies have examined the cost-effectiveness of MOM as a targeted NICU strategy to reduce the incidence and severity of these costly morbidities, which set the stage for lifelong neurodevelopmental and health problems and their associated costs. However, all studies are limited by the inability to randomly assign MOM feedings, so the most common research design is the observational cohort. Additional limitations in studies include: ret-rospective and/or secondary analyses of datasets that were not designed specifically to measure the impact of MOM, inconsistent and imprecise measurement of amounts of MOM and non-MOM received by the infant; inconsistent diagnoses of morbidities; lack of inclusion of minority infants who received MOM; older cohorts (1980–1990s) with different medical care and nutritional options; and combining MOM and donor human milk (DHM) feedings into a single human milk feeding measure.

The LOVE MOM cohort (Longitudinal Outcomes of Very Low Birthweight Infants Exposed to Mothers’ Own Milk; NIH: R010009; Meier PI) enrolled 430 VLBW infants (52% black, 27% Hispanic, 21% Caucasian) between 2008 and 2012, with the objective of determining the health outcomes and costs of differing doses and exposure peri-ods of MOM feedings, while controlling for the limitations in previ-ous studies. In this prospective cohort study, 98% of infants received

40

some MOM; mLs of MOM and formula (no DHM was used) were measured daily, and MOM dose was calculated both as a percentage of total enteral feedings and as a weight-adjusted (mL/kg/day) measure. Morbidities were diagnosed and validated independently by 2 neona-tologists; cost data were actuarial rather than estimated, and propensity scoring was used to control for the risk of morbidities (confounders). Data revealed a dose-response relationship between higher amounts of MOM received during critical exposure periods during the NICU hos-pitalization and a reduction in the risk of specific morbidities and their costs (Table 1). These findings suggest that MOM functions by different mechanisms over the course of critical exposure periods to reduce the risk of potentially preventable morbidities and their associated costs in VLBW infants.

References

1 Meier P, Patel A, Esquerra-Zwiers A: Donor human milk update: evidence, mecha-nisms, and priorities for research and practice. J Pediatr 2017;180:15–21.

Table 1. Relationship between MOM dose and exposure and potentially pre-ventable morbidities and their costs: the LOVE MOM cohort

First author [Ref.], year

Morbidity Cost

Johnson [2], 2015

NEC: Any formula received during days 1–14 after birth increased the risk of NEC 3.5 times

Cost of NEC: USD 43,818 (2012 USD)Value of MOM: Each additional mL/kg/day of MOM during days 1–14 after birth = USD 534 (2012 USD)

Patel [3], 2013

Sepsis: Each additional 10 mL/kg/day of MOM received during days 1–28 after birth reduced the odds of sepsis by 19%

Value of MOM: Differences between the highest (≥50 mL/kg/day) and lowest (<25 mL/kg/day) doses of MOM = USD 31,514 (2012 USD)

Patel [4], 2017

BPD: Each additional 10% increase in the MOM dose (percentage) through to 36 weeks’ gestation reduces the odds of BPD by 9.5%

Cost of BPD: The additional cost of BPD = USD 41,929 (2014 USD)

Patra [5], 2017

Neurodevelopmental outcome: Each 10 mL/kg/day increase in MOM during the NICU hospitalization is associated with 0.35 increase in cognitive index score at 20 months’ corrected age

Value of MOM: publication in review

NEC, necrotizing enterocolitis; BPD, bronchopulmonary dysplasia.

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2 Johnson TJ, Patel AL, Bigger HR, et al: Cost savings of human milk as a strategy to reduce the incidence of necrotizing enterocolitis in very low birth weight infants. Neonatology 2015;107:271–276.

3 Patel AL, Johnson TJ, Engstrom JL, et al: Impact of early human milk on sepsis and health-care costs in very low birth weight infants. J Perinatol 2013;33:514–519.

4 Patel AL, Johnson TJ, Robin B, et al: Influence of own mother’s milk on bronchopul-monary dysplasia and costs. Arch Dis Child Fetal Neonatal Ed 2017;102:F256–F261.

5 Patra K, Hamilton M, Johnson T, et al: NICU human milk dose and 20-month neurodevelopmental outcome in very low birth weight infants. Neonatology 2017;112:330–336.

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Metabolomics in Human Milk Research

Carolyn M. Slupsky

The first years of a child’s life are critical for growth and develop-ment, and despite decades of research, we still do not understand how infant diet shapes a child for both short- and long-term health. The link between food and health is complex, and although breastfeeding is known to have short- and long-term benefits, the relationship between food and the developing neonate is not understood, primarily because in the past there has been a lack of analytical tools. Indeed, most infant studies rely on crude measures of child health such as growth, and absence of obvi-ous disease. While these assessments can reveal rudimentary associations between dietary components and lack of adverse outcomes in the short-term, they do not directly address the impact of food or food components on metabolic health that may have long-term consequences.

Making matters more complex, analysis of food is not trivial. Although decades of research have gone into studying human milk, most research has focused on studying proteins, lipids, and micronutrients. It is now recognized that there are other factors in milk that may be impor-tant for infant health, including small-molecule metabolites that include a unique class of sugars known as oligosaccharides. Human milk oligosac-charides, which are complex in structure, act as both food for beneficial bacteria and decoys for pathogens [1], and it is now being shown that they can help build the immune system through modulating CD14 expression and altering plasma cytokine levels [2, 3]. Additionally, there are other metabolites present in milk, and their function is not fully understood, although their expression appears to be controlled through the mam-mary gland as well [4], and they may have important consequences for the developing neonate. Through the development of modern nuclear magnetic resonance- and mass spectrometry-based metabolomics tech-niques, we are now in an era where we can measure these small molecules in food, and this will help us understand how food impacts health in an unprecedented way.

Analysis of the infant metabolome has led to important revela-tions regarding how infant diet impacts development. Breastfed infants

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have been shown to have lower levels of plasma branched-chain amino acids (isoleucine, leucine, and valine), and urea, as well as higher levels of ketone bodies (acetone), acetate, and myo-inositol [5]. Additionally, breastfed infants have lower insulin levels than their formula-fed coun-terparts 2 h after feeding [5]. High levels of serum branched-chain amino acids and/or insulin activates mechanistic target of rapamycin (mTOR), a serine/threonine kinase that is a master regulator of cell metabolism. mTOR Complex 1 (mTORC1) signaling is particularly important for the control of growth and metabolism of bone, skeletal muscle, the central nervous system, the gastrointestinal tract, blood cells, and other organs. For formula-fed infants, enhanced activation of this pathway may have lasting impacts on overall metabolism and potentially health.

More study of human milk and infant metabolism that incorpo-rates metabolic phenotype (measured through the metabolome of blood, urine, and feces), gut microbial composition and function, as well as genetic (and epigenetic) data will help us understand the purpose of spe-cific milk components, the individual responses to diet, as well as how diet and genetics work together with the gut ecosystem to guide cognitive and metabolic development.

References

1 Underwood MA, German JB, Lebrilla CB, et al: Bifidobacterium longum subspecies infantis: champion colonizer of the infant gut. Pediatr Res 2015;77:229–235.

2 Goehring KC, Marriage BJ, Oliver JS, et al: Similar to those who are breastfed, infants fed a formula containing 2′-fucosyllactose have lower inflammatory cytokines in a randomized controlled trial. J Nutr 2016;146:2559–2566.

3 He Y, Liu S, Kling DE, et al: The human milk oligosaccharide 2′fucosyllactose mod-ulates CD14 expression in human enterocytes, thereby attenuating LPS-induced inflammation. Gut 2016;65:33–46.

4 Smilowitz JT, O’Sullivan A, Barile D, et al: The human milk metabolome reveals diverse oligosaccharide profiles. J Nutr 2013;143:1709–1718.

5 Slupsky CM, He X, Hernell O, et al: Postprandial metabolic response of breast-fed infants and infants fed lactose-free vs regular infant formula: a randomized con-trolled trial. Sci Rep 2017;7:3640.

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Human Milk Oligosaccharides: Next-Generation Functions and Questions

Lars Bode

The past decade has experienced an immense increase in research on human milk oligosaccharides (HMOs), mostly driven by (a) advances in high-throughput glycan analysis and (b) large-scale glycan synthesis as well as (c) the advent of modern microbiome research.

Advances in high-throughput glycan analysis have enabled the research community to analyze HMO composition in hundreds and sometimes thousands of milk samples from large mother-infant cohorts to investigate associations between maternal factors and HMO com-position as well as between HMO composition and infant outcomes. However, the identified associations cannot prove cause-and-effect rela-tionships. Cohesive and consistent results from suitable preclinical in vitro, tissue culture and animal models, human cohort associations, as well as randomized controlled trials (RCTs) will be required to make con-clusive claims about specific HMO functions. As an example from our own work, we identified a specific HMO called disialyllacto-N-tetraose (DSLNT) that reduces incidence and severity of necrotizing enterocoli-tis (NEC) in a rodent model [1]. While the results are encouraging, the validity of data from available preclinical NEC models in rodents or pig-lets is limited [2]. Animals are exposed to external hypoxic and/or hypo-thermic insults that are rather artificial, and the use of animals itself is a limitation due to interspecies differences in gastrointestinal development, anatomy, and physiology. Thus, advancing a potential therapeutic like DSLNT from controversial preclinical models to clinical treatment trials carries a tremendous risk of failure. However, in parallel, we conducted a multicenter clinical cohort study on 200 mothers and their preterm, very low-birthweight infants that were predominantly human milk-fed and showed that infants who developed NEC received less DSLNT with the milk than infants who did not develop NEC [3]. The latter results do not prove cause-and-effect, but the combination of preclinical testing and human cohort associations raises the confidence towards clinical applica-tion. Future studies in the preclinical model as well as in tissue culture

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are going to help us elucidate the underlying mechanism, and a carefully designed RCT is going to be needed to ultimately prove the use of DSLNT in NEC prevention.

Advances in glycan synthesis have made individual HMOs available for research as well as commercial application. Chemoenzymatic synthe-sis is extending our repertoire of available HMOs at smaller scale for in vitro and preclinical research [4]. The help of bioengineered microbes allows the synthesis of individual HMOs at large scale for commercial application [5]. However, knowledge about specific functions and poten-tial adverse effects of individual HMOs remains very limited at best. Human milk contains a personalized mixture of hundred or more dif-ferent and structurally distinct HMOs, which raises questions whether the application of single individual HMOs instead of complex mixtures causes imbalances in infant gut microbial communities or in the infant immune system with potential short- and long-term health consequences.

Advances in microbiome research and the associated analytical and bioinformatics tools further sparked an interest in HMOs. The compo-sition of microbial communities in the gut and other organs and their metabolic and functional capabilities have been associated with numer-ous conditions, including obesity, inflammatory bowel disease, autism, asthma, and allergies – to only name a few. The concept that HMOs help shape microbial communities early on in life and affect short- and long-term health is indeed striking. Future research is going to apply a combination of preclinical and clinical studies to systematically elucidate HMO structure-function relationships and identify whether individual HMOs like DSLNT or mixtures of HMOs (the way they naturally occur in human milk) provide short- or long-term benefits to infants and poten-tially adults.

References

1 Jantscher-Krenn E, Zherebtsov M, Nissan C, et al: The human milk oligosaccha-ride disialyllacto-N-tetraose prevents necrotizing enterocolitis in neonatal rats. Gut 2011;61:1417–1425.

2 Tanner SM, Berryhill TF, Ellenburg JL, et al: Pathogenesis of necrotizing enterocoli-tis: modeling the innate immune response. Am J Pathol 2015;185:4–16.

3 Autran CA, Kellman BP, Kim JH, et al: Human milk oligosaccharide composition predicts risk of necrotizing enterocolitis in preterm infants. Gut 2018;67:1064–1070.

4 Prudden AR, Liu L, Capicciotti CJ, et al: Synthesis of asymmetrical multiantennary human milk oligosaccharides. Proc Natl Acad Sci USA 2017;114:6954–6959.

5 Bode L, Contractor N, Barile D, et al: Approaches to overcome the limited availability of human milk oligosaccharides: challenges and opportunities for research and appli-cation. Nutr Rev 2016;74:635–644.

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Guiding Development of the Neonate: Lessons from Mammalia

Kevin R. Nicholas, Vengama Modepalli, Ashalyn P. Watt, Lyn A. Hinds, Amit Kumar, Christophe Lefevre, and Julie A. Sharp

Significantly premature and low-birthweight babies have acute chal-lenges for survival, largely due to limited development of their lungs and gut. Furthermore, disruption to the timing of setting developmen-tal clocks in the neonate often results in increased frequency of mature-onset disease, and this is exacerbated if growth rates are accelerated too aggressively [1]. The cost to manage these babies in hospitals is consider-able, and there is an increased prevalence of the problem in the develop-ing world. Mothers often cannot breastfeed and the only option available is providing either formula or pasteurized donor milk to improve the growth of these babies. New approaches are required to manage the early stages of treatment, particularly a focus on development of tissues with-out an accompanying large increment in growth.

New innovative approaches for therapy are emerging with the oppor-tunity to exploit the unusual reproductive strategy of the tammar wallaby, an Australian marsupial. This animal has a short 26-day gestation which is associated with a poorly developed placenta, and gives birth to an altri-cial young that is equivalent to a mid-late pregnant human embryo. Lac-tation extends to 300 days, and consequently there is greater investment in postnatal development of the young.

The pouch young are born with immature organs, and during early lactation the mother limits growth while organs necessary for their sur-vival such as gut, lung, lymphoid tissues, and nervous system (including brain and spinal cord) are rapidly developed [2]. The tammar neonate remains in the pouch and attached to the teat for 100 days, and the mother progressively changes the composition of the milk to deliver sig-nals for this development; this closely resembles the relationship between the human fetus and placenta. Fostering experiments demonstrated that transferring the early-phase tammar pouch young to a later-phase lactat-ing tammar accelerated the growth and physical development of pouch young [3] (Fig. 1C). Therefore, examining timed delivery of bioactives

47

-26

Days of lactation

20

40

60

80

100

0Chan

ges i

n in

dica

ted

para

met

ers

(arb

itrar

y un

its)

Eating grass

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Protein

Carbohydrate

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Day 220Day 185Day 70Day 6

0 50 100 150 200 250 300 350

a

b

c

Fig. 1. a The tammar as a model system for premature and low-birthweight babies. The tammar young is 6 days of age, the human neonate is 26 weeks of age. b Tammar wallaby lactation strategy. Progressive changes in milk composition and growth of the young during the three phases of the lactation cycle in the tammar wallaby. Total protein concentration does not change significantly during early lac-tation, but there is a progressive change in the kinds of proteins secreted. c Fostered pouch young. Pouch young at day 120 of age were either cross-fostered to host mothers at day 170 of lactation or retained on mothers at day 120 of lacta-tion. After 50 days, both pouch young were removed. The more mature animal (shown on the right) was fostered to a mother at a more advanced stage of lactation.

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in the milk may provide a better understanding of the signaling pro-gram of the placenta, and other tissues, required for normal eutherian development.

The composition of milk in eutherians does not change significantly during lactation apart from the transition from colostrum to mature milk. However, the signaling factors from the human placenta, amniotic fluid, and potentially colostrum involved in the development of the fetus/neo-nate are most likely delivered in the milk of marsupials in the early phase of lactation. For example, the lung in newborn marsupials is so immature at birth, the neonate respires through the skin for the first 2 weeks. Stud-ies using in vitro models have shown that milk collected from marsupials during early lactation (day 20–100), but not late lactation (day 100–300) stimulated proliferation and differentiation of cultured whole lung from

Culture media(DMEM) plus 10%Tammar milk protein  

Lungs transferred toculture media 

Day 12 mouseembryo 

a

D 120 milkD 60 milkD 20 milk

0 h

24 h

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72 h

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bDay 60milk  Control

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0 h24 h48 h72 h84 h

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No.

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inal

 end

 bud

s

010203040506070

e DMEMDay 120Day 60

Fig. 2. Effect of early-phase tammar wallaby milk on lung branching morpho-genesis. a Lungs were removed from embryonic mice and cultured with 10% tam-mar milk. b–e Lungs showed extensive branching morphogenesis and increased volume after 3 days of culture with milk collected at day 60 of lactation. In contrast, embryonic lungs cultured in media with either 10% PBS (control) or day 120 milk showed a comparative delay in branching morphogenesis, and the lung was smaller after 3 days of culture. Sections of lung stained with HE confirmed that the morphology of embryonic lungs treated with day 60 milk showed increased branching.

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mouse embryos, and these signaling molecules were directed to differen-tiation of both lung epithelial and mesenchymal cells [4]. This temporal delivery of bioactivity provides a window to search for the signaling mol-ecules in the milk (Fig. 2).

A second approach has exploited the comparison of databases of dif-ferentially expressed genes in the tammar mammary gland in early lac-tation, human milk, colostrum, placenta, and the amniote. A focus only on genes coding for secreted proteins has allowed for a comparison of potential protein-signaling molecules secreted by these tissues. This latter analysis has identified a number of proteins of interest in placenta and amniotic fluid, and unexpectedly identified signaling candidates in colos-trum, prompting the need to reexamine the role of colostrum in develop-ment of both term and preterm babies.

Exploiting comparative approaches provides new options to under-stand the role of milk in acute and chronic development of the baby. Stud-ies using the tammar wallaby may lead to a new range of human milk fortifiers that include bioactives to specifically target tissue development in the human neonate to improve outcomes for premature and low-birth-weight babies with application in the developed and developing world.

References

1 Sharp JA, Watt A, Lefevre CM, Nicholas KR: Human milk bioactivity: lessons from the evolution of lactation. Aust Biochem 2017; 48:13–18.

2 Sharp JA, Wanyonyi S, Modepalli V, et al: The tammar wallaby: a marsupial model to examine the timed delivery and role of bioactives in milk. Gen Comp Endocrinol 2017; 244:164–177.

3 Trott JF, Simpson KJ, Moyle RLC, et al: Maternal regulation of milk composition, milk production and pouch young development during lactation in the tammar wal-laby (Macropus eugenii). Biol Reprod 2003;68:929–936.

4 Modepalli V, Hinds LA, Sharp JA, Lefevre C, Nicholas KR: Role of marsupial tammar wallaby milk in lung maturation of pouch young. BMC Dev Biol. 2015;15:16.

50

Milk Lipids: A Complex Nutrient Delivery System

J. Bruce German

The evolutionary origin of lactation and the composition, struc-tures, and functions of milk’s biopolymers illustrates that the Darwinian pressure on lactation selected for biopolymers with considerable struc-tural complexity and discrete functions within the digestive system [1]. For example, complex sugar polymers, oligosaccharides, possess unique properties in guiding the growth of intestinal bacteria that are not pos-sible by feeding their simple sugars; proteins exhibit enzymatic activities towards other milk components rendering those components both more digestible but also releasing biologically active products. To date, however, the most complex structure in mammalian milk, the fat globule, has not been effectively examined beyond its simple composition. The globules of milk are heterogeneous in size, composition and function, and new research tools and models are beginning to understand the mechanisms that control the assembly of globules in the mammary gland and the dis-assembly within the infant.

Assembly

Milk globules represent a unique biological particle class composed of a triglyceride core bounded by a phospholipid monolayer, assembled in the endoplasmic reticulum, and a complete bilayer structure enrobed around the globule by the mammary epithelial plasma membrane dur-ing globule secretion [2, 3]. The size, diversity, and composition of milk fat globules changes during lactation and as a function of genetics, diet, and mammary gland metabolism [4]. The size of fat globules is directed in part by the availability of specific precursor lipids [5]. Phosphatidyl choline and phosphatidyl ethanolamine compete for occupancy of the globule surface, and their distinct physical properties guide the fusion events that ultimately determine the size of globules as they form and are secreted into milk. The proportion of these two complex phospholipids is thus a vital determinant of globule assembly. Lipid trafficking within the

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mammary epithelial cells is determined in part by available fatty acids and by mitochondria. These research breakthroughs not only provide a mech-anistic understanding for these processes but offer the possibility to guide globule size, composition, and function with exogenous treatment [5].

Disassembly

Fat globules undergo complex disruption during digestion within the intestine of the infant. The importance of the digestion process has been implied by the discoveries of endogenous lipase enzymes within the milk itself that guide lipid digestion even in the absence of digestion capacity within early infants. Complex lipids interact with the aqueous phase to produce a diverse tableau of possible structures that exhibit considerable organization within the nanometer and micrometer length scales. The biological membrane is just one example of a three-dimen-sional lipid phase that forms spontaneously depending on the concentra-tion and composition of lipids. As the lipid composition of fat globules changes within the intestine due to changes in the molecular structure of complex lipids resulting from hydrolysis, new three-dimensional phases/structures can form spontaneously. However, these phases are dynamic and change spontaneously as a function of concentration. Dynamic lipid structures have been ostensibly impossible to follow scientifically due to their ephemeral nature. High-intensity, coherent X-rays are formed by synchrotron accelerators. It is thus possible to introduce complex lipid mixtures into these beams and follow the structures formed in real time. Recent studies on the phase changes during lipolysis have demonstrated that the ensemble of complex lipids of mammalian milks forms distinct cubic structures in real time [6]. The cubic phase is of particular inter-est since it is a three-dimensional bicontinuous phase capable of dissolv-ing and transporting both water-soluble and lipid-soluble components in all directions. The cubic phase presence has been linked to successful absorption of a variety of fat-soluble nutrients, yet it has not been possible previously to determine if milk lipids form cubic phases. Thus, the lipid globule constitutes the precursors for a complex, higher-order, structured delivery system that self-assembles within the infant’s intestine, facilitat-ing absorption by the infant.

Composition

The composition of mammalian milk lipids is a perplexingly sensitive process. Unlike the other biopolymer classes (proteins, glycans, polynu-cleotides), lipid composition is significantly altered by the composition of fatty acids within the maternal diet. As a result of the widespread observa-tions of the environmental influences on milk composition, considerable

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research has focused on this aspect. Much less interest has focused on the fatty acids that are synthesized by the mammary gland itself. Some fatty acids are relatively constant in milk across species, and new research is beginning to reveal functions of these fatty acids beyond simple fuel provision. The most abundant single fatty acid in milk, palmitic acid, has been identified as a potent ligand for the PGC-1β transcription coacti-vator within the liver [7]. This nuclear regulator orchestrates the tran-scription of the complex machinery necessary to synthesize and assemble very-low-density lipoproteins within the liver, thus guiding not only liver lipid secretion but whole-body energy metabolism. The fatty acid deriva-tive of desaturation of palmitic acid, palmitoleic acid, has been identified as possessing an alternative signaling function. Characterized as the first of a class of lipid signaling lipids “lipokines,” palmitoleic acid controls hepatic gluconeogenesis, lipid uptake in the muscle, and potentially food intake systemically [8]. The presence of these fatty acids within milk sug-gests that various aspects of fuel metabolism within the infant are under direct control of the composition of milk’s constitutive fatty acids.

This growing body of evidence argues for a broader view of milk composition that includes the complex structures of large biopolymers, their structures as ensembles, their distinct activities within the milk as it is digested, and the influence of this structural dimension across the nanometer and micrometer length scales on the health value of milk within the entire diet.

References

1 Hinde K, German JB: Food in an evolutionary context: insights from mother’s milk. J Sci Food Agric 2012;92:2219–2223.

2 German JB: Dietary lipids from an evolutionary perspective: sources, structures and functions. Matern Child Nutr 2011;7:2–16.

3 Argov N, Lemay DG, German JB: Milk fat globule structure and function: nanosci-ence comes to milk production. Trends Food Sci Technol 2008;19:617–623.

4 Mesilati-Stahy R, Mida K, Argov-Argaman N: Size-dependent lipid content of bovine milk fat globule and membrane phospholipids. J Agric Food Chem 2011;59:7427–7435.

5 Cohen B-C, Shamay A, Argov-Argaman N: Regulation of lipid droplet size in mam-mary epithelial cells by remodeling of membrane lipid composition – a potential mechanism. PLoS One 2015;10:e0121645.

6 Salentinig S, Phan S, Hawley A, Boyd BJ: Self-assembly structure formation during the digestion of human breast milk. Angew Chem Int Ed Engl 2015;54:1600–1603.

7 Lin J, Yang R, Tarr PT, et al: Hyperlipidemic effects of dietary saturated fats mediated through PGC-1beta coactivation of SREBP. Cell 2005;120:261–273.

8 Cao H, Gerhold K, Mayers JR, et al: Identification of a lipokine, a lipid hormone link-ing adipose tissue to systemic metabolism. Cell 2008;134:933–44.

List of Speakers

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Prof. Lindsay H. AllenWestern Human Nutrition Research CenterDepartment of NutritionUniversity of California, Davis430 W. Health Sciences DriveDavis, CA95616USAE-Mail Lindsay.Allen@ARS.USDA.GOV

Prof. Lars BodeDivision of Neonatology andDivision of Gastroenterology and NutritionUniversity of California, San Diego 9500 Gilman Drive – MC0715La Jolla, CA 92093USAE-Mail lbode@ucsd.edu

Prof. Sharon M. DonovanDepartment of Food Science and Human NutritionCarl R. Woese Institute for Genomic BiologyUniversity of Illinois449 Bevier Hall 905 S. Goodwin AvenueUrbana, IL 61801USAE-Mail sdonovan@illinois.edu

Prof. J. Bruce GermanFood Science and TechnologyUniversity of California, DavisRMI North Building1 Shields AvenueDavis, CA 95616USAE-Mail jbgerman@ucdavis.edu

Prof. Ferdinand HaschkeParacelsus Medical UniversitySalzburg and Medical University ViennaWähringer Gürtel 18–20AT–1090 ViennaAustriaE-Mail fhaschk@gmail.com

Prof. Olle HernellPediatrics, Department of Clinical SciencesUmeå UniversitySE–901 85 UmeåSwedenE-Mail olle.hernell@umu.se

Prof. Erika IsolauriDepartment of Pediatrics and Adolescent MedicineTurku University HospitalKiinamyllynkatu 4–8FI–20520 TurkuFinlandE-Mail eriiso@utu.fi

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Prof. Weili LinBiomedical Research Imaging CenterCB#7513 University of North Carolina at Chapel HillChapel Hill, NC 27599USAE-Mail weili_lin@med.unc.edu

Prof. Bo LönnerdalDepartment of Nutrition and Internal MedicineUniversity of California, Davis3217C Meyer Hall One Shields AvenueDavis, CA 95616USAE-Mail bllonnerdal@ucdavis.edu

Prof. Alan LucasInstitute of Child HealthUniversity College London30 Guilford Street London WC1N 1EHUKE-Mail a.lucas@ucl.ac.uk

Prof. Paula MeierNeonatal Intensive Care Section of Neonatology, Department of Pediatrics Rush University Medical Center1653 West Congress ParkwayChicago, IL 60612USAE-Mail Paula_Meier@rush.edu

Prof. Ardythe L. MorrowCincinnati Children’s Hospital Medical CenterCollege of Medicine, University of Cincinnati 3333 Burnet AvenueCincinnati, OH 45229USAE-Mail ardythe.morrow@cchmc.org

Prof. Kevin Roy NicholasSchool of BioSciencesThe University of MelbourneGrattan StreetParkville, VIC 3010AustraliaE-Mail kevin.nicholas@unimelb.edu.au

Prof. Carolyn M. SlupskyDepartment of NutritionUniversity of California, DavisOne Shields AvenueDavis, CA 95616USAE-Mail cslupsky@ucdavis.edu

Dr. Norbert SprengerInstitute of Nutritional Science Nestlé Research Center Nestec Ltd.Vers-Chez-Les-Blanc CH–1000 Lausanne 26 SwitzerlandE-Mail norbert.sprenger@rdls.nestle.com

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Prof. Valerie VerhasseltSchool of Molecular ScienceUniversity of Western Australia53 Stirling HighwayPerth, WA 6009 AustraliaE-Mail verhasseltvalerie@gmail.com

Dr. Mike WoolridgeGreat Ormond Street & Institute of Child HealthUniversity College London30 Guilford Street London WC1N 1EHUKE-Mail m.woolridge@ucl.ac.uk