human milk oligosaccharides: benefits for the breastfed
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COMMUNITIES AND HOSPITALS ADVANCING MATERNITY PRACTICES
Wednesday Webinar: February 2019
Human Milk Oligosaccharides: Benefits for the breastfed infant and beyond
Presented byDr. Lars Bode, Associate Professor of Pediatrics
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COMMUNITIES AND HOSPITALS ADVANCING MATERNITY PRACTICES
Upcoming CHAMPS 4-Hour Clinical SkillsTrainings
• (4- hour) Anderson Regional Medical Center• Wednesday, February 20th, 2019 (morning session)
• Trainings are open to all CHAMPS hospitals and CHAMPS community partners. You can register for the trainings at CHEERequity.org/trainings
2/14/19
Human Milk OligosaccharidesBenef i ts for the breast fed infant and beyond
Lars Bode, PhD
L a r s s o n - R o se n q u i s t F o u n d a t i o nM o t h er - M i l k - I n f a n t C e n t e r o f R e s e a r c h E x c e l l e n c e
U n i v e r s i t y o f C a l i f o r n ia , S a n D i e g o
DisclosuresResearch in the Bode lab and the UC San Diego Mother-Milk-Infant Center of Research Excellence (LRF MOMI CORE) is currently supported by
• National Institutes of Health (NIH)• National Science Foundation (NSF)• Bill & Melinda Gates Foundation (BMGF)• Family Larsson-Rosenquist Foundation (FLRF)• Gerber Foundation (PI: Dr. Goran)
Dr. Bode receives travel reimbursements from Universities, foundations, and societies to speak at national and international grand rounds and conferences.
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DisclosuresNone of the work would be possible without amazing lab members as well as collaborators from across the US and around the world!
The Power of Human Milk
Saving Lives
“The deaths of 823,000 children and 20,000 motherseach year could be averted through universal breastfeeding, along with economic savings of US$300 billion.”
Editorial Commentary, The Lancet - Breastfeeding Series 2016
WHY?HOW?
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Fat
Protein
Lactose
Oligosaccharides
12
35
65
5-15 !
35
35
450.05
[g/L]
Macromolecules in Human Milk
What areHuman Milk Oligosaccharides
(HMOs)?
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≠
Glucose (Glc)
Galactose (Gal) N-acetylneuraminic acid (Neu5Ac) sialic acid
N-acetyl-glucosamine Fucose (Fuc) (GlcNAc)
Human Milk Oligosaccharides (HMOs) are Complex Sugars
β1-6
n=0-15
β1-3
β1-4
β1-3 β1-4
α1-2α1-3α1-4
α2-3α2-6
lactoselacto-N-biose
N-acetyllactosamineβ1-4 β1-4
α1-2 α1-3
2’-fucosyllactose (2’FL)
β1-4
3-fucosyllactose (3FL)
α2-3α2-6 β1-4
3’-sialyllactose (3’SL)
6’-sialyllactose (6’SL)
β1-4
β1-3
β1-3
Lacto-N-tetraose (LNT) Lacto-N-neotetraose(LNnT) (type 1 chain) (type 2 chain)
β1-4
β1-3β1-4 β1-4
β1-3
β1-3
β1-4
β1-6
Lacto-N-hexaose(LNH)
β1-4
β1-3
β1-3
β1-4
β1-6
β1-3
iso-Lacto-N-octaose
β1-4
β1-3β1-4
β1-3
para-Lacto-N-octaose
β1-3
β1-3
β1-4
β1-3
β1-4
β1-3
β1-3
β1-4
β1-6β1-4 β1-3
iso-Lacto-N-neooctaose
α1-2
Lacto-N-fucopentaoseI (LNFP I)
β1-4
β1-3
β1-3
β1-4
β1-3
β1-3
Lacto-N-fucopentaoseII (LNFP II)
α1-4
β1-4
β1-3
α1-3
Lacto-N-fucopentaoseIII (LNFP III)
β1-4
β1-4
β1-3
β1-3
Lacto-N-fucopentaoseV (LNFP V)
α1-3
Glucose (Glc) Galactose (Gal) N-acetylglucosamine (GlcNAc) Fucose (Fuc) N-acetylneuraminic acid (NeuAc)
α2-3
β1-4
β1-3
β1-3
LS-Tetrasaccharide a (LST a)
α2-6
β1-4
β1-3
β1-3
LS-Tetrasaccharide b (LST b)
α2-6β1-3
β1-4
β1-4
LS-Tetrasaccharide c (LST c)
α2-6
β1-4
β1-3
β1-3
α2-3
Disialyllacto-N-tetraose (DSLNT)
150 – 200 different HMO
HMO Blueprint and Structural Diversity
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A) Absolute HMO concentration (nmol/mL)
Personalized complex HMO composition(HMO ”fingerprint”)
preliminary data from CHILD study cohort (in collaboration with Dr. Meghan Azad)
n=1,206
B) Relative HMO composition (% of total HMOs)
Personalized complex HMO composition(HMO ”fingerprint”)
preliminary data from CHILD study cohort (in collaboration with Dr. Meghan Azad)
n=1,206
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Human milk oligosaccharides (HMO)
! 1 - 3 ! 1-4
! 1 -6
n=0-15
! 1-3
! 1-4″ 1-2/3/4
″ 2-3/6n=1-15
Glucose (Glc)
Galactose (Gal) N-acetylneuraminic acid (Neu5Ac) sialic acid
N-acetyl-glucosamine Fucose (Fuc) (GlcNAc)
!!! #
!
#$% !$%
Oligosaccharides currently added to infant formula
…are structurally distinct from HMOs
HMO MetabolismHMO
resistant to• low pH• pancreatic and
brush border enzymes
small intestine:>1% absorbed,appear in the urine
colon:bacterial degradation,10-80% excreted w/ feces
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SummaryWhat are HMOs?
• third most abundant component in human milk
• composition follows basic blueprint
• more than 150 different HMOs identified
• inter- and intrapersonal differences in HMO composition
• infant formula oligosaccharides (GOS/FOS) structurally (and functionally) different from HMOs
• reach distal GI tract intact, but are also absorbed and reach systemic circulation and organs other than the gut
Which maternal factors contribute to variation in
Human Milk Oligosaccharides (HMOs) composition?
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Maternal Drivers
Genetics
>5,000 milk samples
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Study nCHILD 1,143
STEPS 803
UCSD Biorep 609
Botswana 498
MACS 477
INSPIRE 411
India 298
Uganda 126
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>5,000 milk samples
~95% explained by one SNP
GWAS reveals genes involved inHMO biosynthesis
CHILD cohort data - In collaboration with Dr. Meghan Azad (Manitoba), Dr. Qingling Duan, Le Chang (Ontario)
2’FL
-Log
10(p
)
Chromosomes
Chromosome 19FUT2
10
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Study nCHILD 1,143
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α1-4 β1-R
Lewis b (Leb)
Structural Diversity and GeneticsGroup 1: Lewis-positive Secretor (Se+Le+)Lewis a-b+
β1-R FUT2 β1-R FUT3
Group 2: Lewis-positive Nonsecretor (Se-Le+)Lewis a+b-
α1-4 β1-Rβ1-R F✗UT2
FUT3
Lewis a (Lea)
Group 3: Lewis-negative Secretor (Se+Le-)Lewis a-b-
β1-R FUT2 β1-RF✗UT3
Group 4: Lewis-negative Nonsecretor (Se-Le-)Lewis a-b-
β1-R F✗UT2 F✗UT3
inte
nsity
[mV
]
Secretor Non-Secretor
α1-2
(2) 2’-fucosyllactose
α1-2
(7) Lacto-N-fucopentaose I
Structural Diversity and Genetics
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GWAS reveals genes involved inHMO biosynthesis
2’FL LNFP1FUT2_rs601338 (“Caucasian SNP”)
wt het homo
α1-2β1-3
α1-2
P<0.0001 P<0.000115,000 4,000
3,00010,000
2,0005,000
1,000
0 00 1 2 0 1 2
LNFP2 LNFP3
2’FL
[nm
ol/m
L]
β1-4β1-4
β1-3
LNFP
1[n
mol
/mL]
β1-4 β1-4
α1-4 β1-4β1-3
β1-3
0 1 2 0 1 20
2,000
4,000
6,000 300
LNFP
2[n
mol
/mL]
P<0.0001
β1-3
α1-3
0
100
200
LNFP
3[n
mol
/mL]
P<0.0001
GWAS reveals genes involved inHMO biosynthesis
FUT2_rs601338 (“Caucasian SNP”)
wt het homo
0 1 2
1,000
0
3,0002,000
2,000
4,000
LNT
[nm
ol/m
L]
β1-4
β1-3
β1-3
P<0.0001
LNT LNnT
0 1 20
1,000
3,000
LNnT
[nm
ol/m
L]
β1-4
β1-3β1-4
P<0.04
400 400
300 300
200 200
100 100
0 00 1 2 0 1 2
LSTb
[nm
ol/m
L]
α2-6
β1-4
β1-3
β1-3
P<0.0001 P<0.0001
LSTb
LSTc
[nm
ol/m
L]
α2-6β1-3
β1-4
β1-4LSTc
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GWAS reveals genes involved inHMO biosynthesis
FUT2_rs601338 (“Caucasian SNP”)
wt het homo
HMO sum
P<0.000130,000
20,000
10,000
00 1 2
HMO-bound FucP<0.0001
SUM
[nm
ol/m
L]
0 00 1 2 0 1 2
25,000 6,000
20,0004,000
15,000
10,0002,000
5,000
HM
O-b
ound
Fuc
[nm
ol/m
L]
HM
O-b
ound
Sia
[nm
ol/m
L]
HMO-bound SiaP<0.0001
What’s normal?Oligosaccharide concentrat ions and prof i les in milk
produced by healthy women vary geographical ly
Secretor [%]
McGuire M et al. Am J Clin Nutr 2017;105:1086–100
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Canadian CHILD cohort: (n=427)
Asian: 60% SecretorsCaucasian: 74% Secretors First Nation: 76% Secretors
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Maternal Drivers
Genetics• Secretor/Lewis• other?genomics, transcriptomics, HMO-mics• in silico pathway modeling• validation through in vitro enzyme
characterization and manipulation
Environment• diet
Diet and exercise alter mouse milk oligosaccharide concentration (3’-sialyllactose, 3’SL)
2,500
2,000
1,500
500
0
1,000
3'SL
[ug/
mL]
n=8
** ***
less sialylated HMO on high fat diet
In collaboration with Dr. Kristin Stanford, Ohio State University
Maternal Drivers of HMO Composition
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0
5000
10000
15000
20000
0
1000
2000
3000
4000
Conc
entr
atio
n(n
mol
/mL)
Conc
entr
atio
n(n
mol
/mL)
Sialylated HMOs Fucosylated HMOs
Maternal Drivers of HMO Composition
Maternal diet alters HMO composition
less sialylated HMO on high fat dietIn collaboration with Dr. Morey Haymond, Dr. Mahmoud Mohammad,
and Dr. Kjersti Aagaard, Baylor College of Medicine
Maternal Drivers of HMO CompositionMaternal use of probiotics during late pregnancy is associated with variation in HMO composition
In collaboration with Dr. Antti Seppo and Dr. Kirsi Järvinen-Seppo, University of Rochester
Seppo A et al. JAMA Pediatrics (In Press)
n.s. p = 0.0*
3
carb high fat glucose galactose
p = 0.02*
n.s.
carb high fat glucose galactose
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Maternal Drivers of HMO CompositionMaternal use of probiotics during late pregnancy is associated with variation in HMO composition
In collaboration with Dr. Antti Seppo and Dr. Kirsi Järvinen-Seppo, University of Rochester
Seppo A et al. JAMA Pediatrics (In Press)
Maternal Drivers
Genetics• Secretor/Lewis• other?genomics, transcriptomics, HMO-mics• in silico pathway modeling• validation through in vitro enzyme
characterization and manipulation
Environment• diet • supplements••
lifestyle (e.g. physical activity) exposures (e.g. smoking, drugs, etc.)
Time• lactation stage
Health / Disease
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Maternal Drivers of HMO Composition
Azad MB et al. J Nutr 2018;148:1–10
Maternal Drivers
Genetics• Secretor/Lewis• other?genomics, transcriptomics, HMO-mics• in silico pathway modeling• validation through in vitro enzyme
characterization and manipulation
Environment• diet • supplements••
lifestyle (e.g. physical activity) exposures (e.g. smoking, drugs, etc.)
Time• lactation stage• parityHealth / Disease
• season
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Maternal Drivers of HMO Composition
Azad MB et al. J Nutr 2018;148:1–10
Maternal Drivers
Genetics• Secretor/Lewis• other?genomics, transcriptomics, HMO-mics• in silico pathway modeling• validation through in vitro enzyme
characterization and manipulation
Environment• diet • supplements• lifestyle (e.g. physical activity)• exposures (e.g. smoking, drugs, etc.)
Time
Health / Disease• lactation stage• parity• age
• season
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Maternal Drivers and Infant Outcomes
gut microbiome necrotizing enterocolitis
diarrhea
HIV transmission
respiratory infections
gut-brain axisviral and bacterial infections asthma
brain development
cognitionIQ
food allergies
body composition
obesitycancer
lung developmentchronic inflammatory diseases
metabolic diseases
sepsis
What areHuman Milk Oligosaccharides
(HMOs) doing?
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HMO at the Interface of Host-Microbe Interactions
prebiotics
prebiotics
Shaping microbial community development
• structure-specific effects of HMO ex vivo (infant feces)
Dr. Karsten ZenglerUCSD
β1-4
β1-3 α1-2
β1-3
Lacto-N-tetraose (LNT) 2’-fucosyllactose(type 1 chain) (2’FL)
β1-4 β1-4
α2-3
3’-sialyllactose (3’SL)
!! !
#
!
#$% !$%
HMO as Prebiotics
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HMO as Prebiotics
prebiotics
• prebiotic effects of HMOs are structure-specific !
• different HMOs have different effects on microbial communities
• mixture of HMOs vs individual HMOs?
• short- and long-term consequences?(DOHaD: Developmental Origins of Health and Disease)
prebiotics bacteriostatic, bactericidal
antimicrobials
HMO at the Interface of Host-Microbe Interactions
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prebiotics
Dr. Victor Nizet UCSD
bacteriostatic, bactericidal
antimicrobials
HMO Exhibit a Unique Antimicrobial Property Against Group B Streptococcus
• identified structure-specific HMO(LNT)
GBS+HMO
GBS
Lin A et al. (2017) J Biol Chem 292(27):11243-11249
• identified potential GBS target (glycosyltransferase)
• suggest HMO as cell wall substrate that inhibits growth
• other bacteria? / pathogens?
HMO as Antimicrobials
prebiotics
Dr. Victor Nizet UCSD
bacteriostatic, bactericidal
antimicrobials
HMO Exhibit a Unique Antimicrobial Property Against Group B Streptococcus
Vancomycin IC50 = 0.25 µg/mLVancomycin IC50 (+HMO) = 0.00602 µg/mL
Ciprofloxacin IC50 = 1.37 µg/mLCiprofloxacin IC50 (+HMO) = 0.0021 µg/mL
I =0.30I =0.29
Log Ciprofloxacin (µg/ml)
Gro
wth
at 4
h (C
FU)
Log Vancomycin (µg/ml)
Lin A et al. (2017) J Biol Chem 292(27):11243-11249
HMO as Antimicrobials
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prebiotics bacteriostatic, bactericidal
antimicrobials
antiadhesives
HMO at the Interface of Host-Microbe Interactions
HMO as Antiadhesives
bacterial lectin
“glycocalyx”
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prebioticsDr. Lars Eckmann UCSD
bacteriostatic, bactericidal
antimicrobials
antiadhesivesHMO Protect Against EnteropathogenicE. coli (EPEC) Attachment in vitro andColonization in Suckling Mice
attachment in vitro
HMO as Antiadhesives
prebioticsDr. Lars Eckmann UCSD
bacteriostatic, bactericidal
antimicrobials
antiadhesivesHMO Protect Against EnteropathogenicE. coli (EPEC) Attachment in vitro andColonization in Suckling Mice
attachment in vitro
Control pHMO treated0.0
0.5
1.0
Control pHMO treated
A/E
lesi
onco
unt/c
ell
A/E lesions (phalloidin staining) *
HMO as Antiadhesives
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prebiotics bacteriostatic, bactericidal
antimicrobials
antiadhesivesepithelial cell modulators
HMO at the Interface of Host-Microbe Interactions
prebiotics bacteriostatic, bactericidal
antimicrobials
antiadhesives
epithelial cell modulators
HMO Protect Bladder Epithelial Cells Against Uropathogenic Escherichia coliInvasion and Cytotoxicity
Lin A et al. (2014) J Infect Dis 209:389-98• other epithelial cell responses?
HMO as Epithelial Cell Modulators
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prebiotics bacteriostatic, bactericidal
antimicrobials
antiadhesivesepithelial cell modulators
immune cell modulators
HMO at the Interface of Host-Microbe Interactions
prebiotics bacteriostatic, bactericidal
antimicrobials
• identified specific HMOsthat block macrophage inflammation
epithelial cell modulators
Dr. Philip GordtsUCSD
antiadhesives
immune cell modulators
HMO as Immune Cell Modulators
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SummaryWhat are the potential benefits of HMOs?
• indirect effects on the infant (through effects on the microbiome)
• prebiotic• anti-microbial• anti-adhesive
• direct effects on the infant• epithelial cell response modulators• immune cell response modulators
• multiple potential other mechanisms
• health implications throughout the life cycle
HMO health implicat ions throughout the life cycle
effects at every stage of the life cycle (“synthetic” HMOs)
Jantscher-Krenn E et al.AJP EndoMetabolism effects on pregnant and Nov 14, 2018 breastfeeding women
immediate immediate short-term short-term
effects effects
Wise A et al. Front. Pediatr.Oct 2, 2018
lasting, long-term effects (DOHaD)
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Necrotizing Enterocolitis (NEC) in the Preterm Infant
immediate short-term
effects
inci
denc
e [%
]
Necrotizing Enterocoli t isN Engl J Med 2011
• one of the most common and fatal GI disorders in preterm infants• ~5% of all VLBW infants (<1,500g)• high mortality (>25%)
• ~6- to 10-fold higher risk in formula-fed infants (FF) compared to breast-fed infants (BF)
HMOs?which one?
Lucas A and Cole TJ (1990) Lancet 336: 1519-1523
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HMO isolation from humanmilk
In vivo efficacy testing in preclinical models
Necrotizing Enterocoli t is
Jantscher-Krenn E et al. (2012) GUT 61:1417-25
A 100
1.0
0.0
4.0
3.0
2.0
DF, Normal (score: 0) FF, Complete Destruction (score: 4)
*** ***
Villus Edema Epithelial Sloughing Misaligned Nuclei
C D
DF
Pat
holo
gyS
core
FF FF+HMO +GOS
FF
90
80FF+GOS76.5% (13/17)
FF70 73.1% (19/26)
0 24 48 72 96time post-partum [h]
Sur
viva
l[%
]
DF100% (18/18)
FF+HMO95% (19/20)
HMO isolation from humanmilk
In vivo efficacy testing in preclinical models
Yes No
Identification of most effective oligosaccharides
Necrotizing Enterocoli t is
Jantscher-Krenn E et al. (2012) GUT 61:1417-25
0.0
1.0
2.0
3.0
4.0
A
FF+HMO-2
Pat
holo
gyS
core
***
***
DF
FF 0 -
3-4
-1
0.0
1.0
2.0
3.0
4.0
Pat
holo
gyS
core
E *****
*
FFFF
+HMO 2 HMO 3+4
DF
FF+
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HMO isolation from humanmilk
In vivo efficacy testing in preclinical models
Yes No
Identification of most effective oligosaccharides
β1-4α2-6
β1-3
β1-3
α2-3
DSLNT
Necrotizing Enterocoli t is
Jantscher-Krenn E et al. (2012) GUT 61:1417-25Yu H et al. (2014) Angew Chem Int Ed Engl. 53(26):6687-91 Autran CA et al. (2016) Br J Nutr 116(2):294-9Yu H et al. (2017) J Org Chem. 82(24):13152-13160
HMO isolation from humanmilk
In vivo efficacy testing in preclinical models
Yes No
Identification of most effective oligosaccharides
Clinical intervention study
Mother-infantpair cohorts
HMO composition Identification of associationsanalysis between HMO and clinical metadata
Match!
Mechanism?
inform
α2-6
β1-4
β1-3
β1-3
α2-3
DSLNT
Van Nierkerk E et al. (2014) J Nutr 144(8):1227-33 Autran CA et al. (2018) GUT 67:1064–1070
Jantscher-Krenn E et al. (2012) GUT 61:1417-25Yu H et al. (2014) Angew Chem Int Ed Engl. 53(26):6687-91 Autran CA et al. (2016) Br J Nutr 116(2):294-9Yu H et al. (2017) J Org Chem. 82(24):13152-13160 Etzold S et al. (in preparation)
Necrotizing Enterocoli t is
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Necrotizing Enterocoli t isHMO effect is highly structure-specific !
1.0
0.0
4.0
3.0
2.0
Path
olog
y Sc
ore
***
FF FF+ FF+ FF+ DSLNT DSLNT DSLNT
-α2-3/6 -α2-3
β3 β3 β3β3 β3 β3
α3
Jantscher-Krenn E et al. (2012) GUT 61:1417-25
β4α
6
β4
β4α
6
Autran CA et al. (2018) GUT 67:1064–1070
Food Sensitization and Allergy
lasting long-term effects (DOHaD)
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Miliku K et al. (2018) Allergy
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Miliku K et al. (2018)
Individual HMOs or HMO profiles?What we’ve learned so far…
• sometimes specific individual HMOs are effective
• effects are highly structure-specific
• dose-response effect
• mechanisms likely receptor-mediated (host and/or microbe)
• sometimes specific mixtures (composition profiles) are effective
• relative abundance of HMOs, ratio of HMOs
• mechanisms likely indirect through shaping microbial communities, microbial metabolites, and/or direct on host (immune system and other)
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What is the ’optimal’Human Milk Oligosaccharide
(HMO) composition?
Is there an ’optimal’Human Milk Oligosaccharide
(HMO) composition?
Infant(genetics)
Time
EnvironmentGeographic Location
Personalized, Context-dependent Composition
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Conclusion
• HMOs are the third most abundant component of human milk
• maternal (genetic and environmental) factors driving variation in complex HMO composition not fully understood
• combination of preclinical, cohort and clinical studies required to fully assess effects, functions and potential claims
• sometimes individual HMOs are effective; sometimes complex mixtures of HMOs in specific relative abundancies are required (personalized?)
• HMOs benefit the breastfed infant and potentially the fetus
• HMOs may have potential long-term effects (DOHaD)
• HMOs may benefit maternal health
• HMOs serve as template for novel therapeutics way beyond the first 1,000 days
www.bodelab.com www.milk.ucsd.edu
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