Breakthrough of Human Milk Oligosaccharides

Breakthrough of Human MilkBreakthrough of Human Milk
OligosaccharidesOligosaccharides
18-10-201818-10-2018
Khaled Saad, MDKhaled Saad, MD
Associate Professor of PediatricsAssociate Professor of Pediatrics
University of Assiut School of MedicineUniversity of Assiut School of Medicine
Board member of European Council for Nutritional andBoard member of European Council for Nutritional and
Environmental Medicine (CONEMEnvironmental Medicine (CONEM))

18-10-201818-10-2018 Breakthrough of HMO, K.SaadBreakthrough of HMO, K.Saad

ObjectivesObjectives
 To highlight the importance of HumanTo highlight the importance of Human
Milk Oligosaccharides (HMOs) onMilk Oligosaccharides (HMOs) on
Growth, Immunity and Morbidity.Growth, Immunity and Morbidity.

AgendaAgenda
““It’s all about Effects of Human MilkIt’s all about Effects of Human Milk
Oligosaccharides on Growth,Oligosaccharides on Growth,
Immunity and MorbidityImmunity and Morbidity””

IntroductionIntroduction
The transition from the protectedThe transition from the protected
intrauterine environment to aintrauterine environment to a
world of microbes presents aworld of microbes presents a
newborn with a set ofnewborn with a set of
immunological demands for self-immunological demands for self-
protection against pathogens.protection against pathogens.

 Because the adaptive immuneBecause the adaptive immune
system is impaired due to minimalsystem is impaired due to minimal
pre-exposure to microbes in uteropre-exposure to microbes in utero
and immature effector B- and T-and immature effector B- and T-
cells, the innate immune system iscells, the innate immune system is
the first-line of the newborn'sthe first-line of the newborn's
defense.defense.

 It consists of the physical barriersIt consists of the physical barriers
and cellular and humoral immunity,and cellular and humoral immunity,
which provide rapid protectionwhich provide rapid protection
against pathogens withoutagainst pathogens without
generating immunologic memory. Ingenerating immunologic memory. In
the first three months of life, thethe first three months of life, the
cellular immune system undergoescellular immune system undergoes
maturation with multiple factorsmaturation with multiple factors
involved.involved.

 Granulocytes (mostly neutrophils),Granulocytes (mostly neutrophils),
monocytes, macrophages, dendriticmonocytes, macrophages, dendritic
cells, natural killer (NK) cellscells, natural killer (NK) cells
together with the complementtogether with the complement
system are directed to combat asystem are directed to combat a
broad spectrum of invadingbroad spectrum of invading
pathogens. Notably, at birth the T-pathogens. Notably, at birth the T-
helper (Th) cell population is stillhelper (Th) cell population is still
biased towards the Th2 phenotype.biased towards the Th2 phenotype.

 This driving type-2 pathwayThis driving type-2 pathway
(“humoral immunity”) critical for(“humoral immunity”) critical for
the protection against extracellularthe protection against extracellular
pathogens and regulation of allergicpathogens and regulation of allergic
responses as opposed to theresponses as opposed to the
Th1/Th17 phenotype, driving type-1Th1/Th17 phenotype, driving type-1
pathway (“cellular immunity”) forpathway (“cellular immunity”) for
fighting intracellular pathogens.fighting intracellular pathogens.

 This peculiarity is a result of an
adaptation to avoid an alloimmune
reaction between mother and fetus
during the intrauterine period,
which leads to a newborn's
particular susceptibility to
pathogens and allergy.

 The gut epithelium constitutes a
functional barrier which delays
pathogenic invasion of the host
by gut microbiota and provides
active elements of the lymphoid
cell line, thus modulating
immune responses.

 The immature neonate's intestine
is characterized by overexpression
of inflammatory genes and
insufficient expression of genes
responsible for the feed-back
regulation of innate signaling,
which could lead to amplification
of immune responses resulting in
excessive inflammation.

What are HMOs?What are HMOs?
 HMOs comprise a family of freeHMOs comprise a family of free
oligosaccharides with high structuraloligosaccharides with high structural
diversity and represent the thirddiversity and represent the third
largest group of bioactive moleculeslargest group of bioactive molecules
in human milk. Colostrum containsin human milk. Colostrum contains
20-25 g/L of HMOs, however, further20-25 g/L of HMOs, however, further
milk production maturation ismilk production maturation is
accompanied by the decrease ofaccompanied by the decrease of
HMOs content to 5-20 g/L.HMOs content to 5-20 g/L.

 Over 200 unique HMOs have been
discovered and more than 100 have
been structurally solved and
characterized.
 The dominant oligosaccharide in
80% of all women is 2'-fucosyllactose
(2′-FL), a trisaccharide consisting of
glucose, galactose, and fucose.

 To date, there have been two large,
international analyses of HMOs in
human milk [Erney et al. J.
Pediatr. Gastroenterol. Nutr. 2000,
30, 181–192, and McGuire et al.
Am. J. Clin. Nutr. 2017, 105, 1086–
1100 ].

 The first comprehensive analysis
of HMOs from human milk in
approximately 400 lactating
women from 10 countries found
that 85% of human milk samples
had detectable 2′-FL at
concentrations of 0.06–4.65 g 2′-
FL/L.

 The second study found similar
results from 410 lactating women
from 11 international cohorts with
65–98% of human milk samples
having 2′-FL with mean
concentrations ranging from
0.702–3.440 g 2′-FL/L.

 The majority of HMOs contain a
lactose, polylactosamine or lacto-
N-biose core. Further modification
by the addition of differently
linked fucose or sialic acids
increases their structural diversity
and, in addition, provides
resistance against enzymatic
digestion.

 Human milk oligosaccharidesHuman milk oligosaccharides
(HMOs) are believed to stimulate(HMOs) are believed to stimulate
the growth of bifidobacteria in thethe growth of bifidobacteria in the
gastrointestinal tract (GIT) whilegastrointestinal tract (GIT) while
protecting against entericprotecting against enteric
pathogens.pathogens.

 These OSs contain lactose at theThese OSs contain lactose at the
reducing end and typically a fucosereducing end and typically a fucose
or a sialic acid at the nonreducingor a sialic acid at the nonreducing
end, the 2’- fucosyllactose (2’- FL)end, the 2’- fucosyllactose (2’- FL)
and lacto-N-fucopentaose- I (LNF-and lacto-N-fucopentaose- I (LNF-
I), both of which contain an a 1,2-I), both of which contain an a 1,2-
linked fucose, being the mostlinked fucose, being the most
common.common.

 HMOs are believed to have manyHMOs are believed to have many
roles in a developing infant, inroles in a developing infant, in
addition to putative prebioticaddition to putative prebiotic
functions, and may possess anti-functions, and may possess anti-
adhesive effects that reduce theadhesive effects that reduce the
binding of pathogenic bacteria tobinding of pathogenic bacteria to
colonocytes.colonocytes.

 They also have modulating effectsThey also have modulating effects
on immunological processes at theon immunological processes at the
level of gut-associated lymphoidlevel of gut-associated lymphoid
tissue plus may also decreasetissue plus may also decrease
intestinal permeability in pretermintestinal permeability in preterm
infants in a dose-related manner ininfants in a dose-related manner in
the first post-natal month.the first post-natal month.

HMO metabolismHMO metabolism
 HMOs resist digestion in the upper
GIT. Evidence suggests that the
majority of HMOs reach the large
intestine intact. A small portion of
ingested HMOs are absorbed intact
into the circulation and are excreted
in urine, which may explain the
systemic benefits of HMOs.

 For example, a key window of
immune development occurs
during the first few weeks of life
when innate immune cells peak in
the circulation. Furthermore,
because ~70% of immune cells
reside in the digestive tract, they
may interact directly with HMOs
that are consumed by infants .

 HMOs have been studied from
human milk for their immune
benefits. In a study of 93 infant, the
mother’s breast milk was analyzed
for HMOs between one and five
weeks postpartum and infant
feeding and diarrhea data were
collected for two years.

 Infants whose mother’s breast milk
had low concentrations of 2′-FL
had significantly higher rates
of Campylobacter diarrhea and
calicivirus diarrhea, respectively,
than infants who were fed with
their mother’s breast milk with
higher levels of these HMOs.

HMOs in immunomodulationHMOs in immunomodulation
and inflammationand inflammation

Immunomodulation functions of HMOsImmunomodulation functions of HMOs
 The molecules modulateThe molecules modulate
immune responses indirectly,immune responses indirectly,
by changing the infant gutby changing the infant gut
microbiota composition, andmicrobiota composition, and
directly, by affecting immunedirectly, by affecting immune
cells on a systemic level, aftercells on a systemic level, after
entering the bloodentering the blood
circulation. The absorptioncirculation. The absorption
rate of HMOs from the GIrate of HMOs from the GI
tract has been reported to betract has been reported to be
about 1%.about 1%.

1. Fucose-containing HMOs1. Fucose-containing HMOs
(fHMOs)(fHMOs)
 About 70% of human milkAbout 70% of human milk
oligosaccharides areoligosaccharides are
fucosylated. Fucose can befucosylated. Fucose can be
attached to galactoseattached to galactose
through an a1,2-linkage,through an a1,2-linkage,
and to glucose or N-and to glucose or N-
acetylglucosamine throughacetylglucosamine through
a1,3- or a1,4-linkagea1,3- or a1,4-linkage
 (Fig. 2)(Fig. 2)

 The production and distribution of
distinct fucosylated oligosaccharide
species depends on the maternal
blood group type and the stage of
lactation. A 1,2-Fucosylated
oligosaccharides were found to be
the most abundantly represented
HMOs in human milk of secretor
mothers.

 They are the first to appear, the
longest-lasting and the most active
in combating various pathogens
causing diarrhea.

 These oligosaccharides block the
microbial and viral pathogenicity
by inhibition of adhesion of
Campylobacter jejuni and E. coli
toxin to intestinal mucosa
receptors through a competitive
mechanism.

 For instance, 2'-fucosyllactose lower
the levels of mRNA and the
membrane-bound form of CD14,
which is a co-receptor for bacterial
lipopolysaccharides (LPS). This led to
weakening of LPS-induced
inflammation during infection by
different E. coli species which cause
diarrhea and urologic diseases.

2. Sialo-containing HMOs (sHMOs)
 The proportion of sialylated HMOs
(acidic HMOs, aHMOs) in human
breast milk has been reported to be
about 12-30%. Sialic acid residues
can be attached by a2,3- or a2,6-
linkages to terminal galactose or
subterminal N-acetylglucosamine
(GlcNAc) (Fig. 3).

 Similarly, to fucosyl oligo-
saccharides, sHMOs have been
demonstrated to block binding of
pathogenic bacteria to intestinal
epithelium. For example, an
inhibitory effect by 3'-SL on
Helicobacter pylori and
enteropathogenic E. coli adhesion
has been shown.

 Also they play a positive role in
the prevention of necrotizing
enterocolitis (NEC) in breastfed
newborns.

3. Galactosyllactoses3. Galactosyllactoses
 Of special interest regarding their
role in the neonate's protection are
galactosyllactoses (GL), molecules
with b1,3-, b1,4- and b1,6-galactosyl
residues linked to the lactose core,
which are typically present in
human colostrum rather than
mature milk (Fig. 3).

 A closely related class to GL is
comprised of microbiologically
derived galacto-oligo-saccharides
(GOS), the products of
transglycosylation.

Translating knowledge onTranslating knowledge on
the role of HMOs into thethe role of HMOs into the
treatment of diseases intreatment of diseases in
infantsinfants

Necrotizing enterocolitisNecrotizing enterocolitis
 Necrotizing enterocolitis (NEC) is one
of the main disorders of the GI tract in
premature infants which is
characterized by excessive
inflammation and resultant necrosis of
intestinal epithelium. The feasibility of
HMOs application as a medication
against NEC has been supported by
recent studies.

 HMOs exert a protective role
against NEC. HMOs may become
valuable supplement for the
prevention and treatment of this
devastating disorder in infants
which do not receive human milk.

Thrombo-inflammatory developmentThrombo-inflammatory development
 Several studies showed an important
interplay between platelets and
human milk oligosaccharides. HMOs
have been demonstrated to inhibit
microvascular inflammation
associated with the formation of
platelet-neutrophil complexes.

 HMOs species could be used as an
infant formula supplement or
potential therapeutics for the
prevention and treatment of
thrombo-inflammatory disorders.

 An interesting study was reported
on the possible role of milk
oligosaccharides in treating multiple
sclerosis (MS), a chronic
inflammatory autoimmune disorder
accompanied by demyelinisation
and destruction of nervous cells.
Multiple sclerosisMultiple sclerosis

 Given the anti-inflammatory
effects of HMOs, studies reported
immune-modulatory function if
HMOs by downregulating
monocyte-macrophages with a
subsequent reduction in deleterious
cytokine production

Viral and bacterial diseasesViral and bacterial diseases
A protective role of HMOs against
respiratory and urinary infections has been
reported. In experiments with respiratory
syncytial virus (RCV) and influenza virus,
milk oligosaccharides were shown to be able
to enhance innate antiviral response in
addition to the decrease of the viral infection
and/or inflammation in human airway
epithelial cells.

 In another study, HMOs, particularly
sialo-containing fractions, have been
demonstrated to protect bladder
epithelial cells from detrimental
cytotoxic and proinflammatory effects
of urinary infection caused by
uropathogenic E. coli. HMOs reduced
bacterial invasion and protected host
cells from bacteria-induced cell
damage.

2′-fucosyllactose (2′-FL)2′-fucosyllactose (2′-FL)

 Because 2′-FL is the most abundant HMO
that is present in the milk of most lactating
women, it has been the most studied HMO in
regard to its potential systemic effects.
Preclinical research shows that 2′-FL has
multiple functions including: acting as a
prebiotic, protecting against infections and
inflammation, modulating the immune
system, supporting brain development, and
reducing the risk of necrotizing enterocolitis.

 Synthesized 2′-FL is structurally identical
to the 2′-FL in human milk. The recent
availability of synthesized 2′-FL is
important because HMOs were previously
only found at significant levels in human
milk, however they are now available in
infant formulas.

 Further, several 2′-FL ingredients have been
the subject of Generally Recognized As Safe
(GRAS) notifications and the US Food and
Drug Administration has no questions on the
proposed addition to infant formula.
Internationally, the European Union has
approved the use of 2′-FL in infant formula.
Likewise, countries that refer to the
European authorization will also adopt the
safe addition of 2′-FL to infant formulas.

Growth and Tolerance of Infants Fed
Milk-Based Formula with 2′-FL
 The first clinical study [Marriage, et al.
J. Pediatr. Gastroenterol.
Nutr. 2015, 61, 649–58] to investigate
2′-FL in infant formula was a
prospective, randomized, controlled,
growth, and tolerance study that was
conducted in healthy term infants.

 Infants were enrolled by 5 days of life
(n = 420). There were four groups,
including three randomized formula
groups and a breastfed (BF) reference
group. The three study formulas
included a control formula (CF) without
added HMO and two study formulas
differing in the amount of 2′-FL: 0.2 g 2′-
FL/L versus 1.0 g 2′-FL/L (Abbott
Nutrition, Columbus, OH, USA).

 The amount of 2′-FL was chosen to be within
the range of 2′-FL in human milk. The
primary outcome was weight gain from 14–
119 days and this was not significantly
different among the formula groups or the
BF reference group. Similarly, tolerance
measures (including stool frequency, stool
consistency, incidence of spit-up, and vomit
associated with feedings) did not differ
among the formula groups.

 Overall, the 2′-FL containing
formulas were effective at
maintaining appropriate growth
and tolerance and had similar 2′-
FL uptake. Also, no safety
concerns were observed. Marriage
et al. was the first published
clinical study of an infant formula
with 2′-FL.

Inflammatory Cytokines of Infants Fed
Milk-Based Formula with 2′-FL
 Goehring et al. [J. Nutr. 2016, 146,Goehring et al. [J. Nutr. 2016, 146,
2559–2566] utilized blood samples that2559–2566] utilized blood samples that
were obtained from a subset of thewere obtained from a subset of the
infants from the Marriage et al. studyinfants from the Marriage et al. study
described before (n = 201). At six weeksdescribed before (n = 201). At six weeks
of age, blood samples were drawn forof age, blood samples were drawn for
markers of immune function.markers of immune function.

 10 plasma inflammatory cytokines
were measured and five were
significantly higher in the CF group
than both the BF and the 2′-FL
containing formula groups: interleukin
(IL)-1ra, IL-1α, IL-1β, IL-6, and tumor
necrosis factor (TNF)-α.

 There were no differences in the plasma
concentrations of any of the 10 plasma
inflammatory cytokines between the BF
infants and the infants that were fed
formulas with 2′-FL, demonstrating that
the addition of 2′-FL resulted in lower levels
of multiple cytokines, similar to the levels in
the BF infants.

 Overall, the circulating plasma
inflammatory cytokine profiles and
RSV-induced cytokine profiles of the
infants that were fed either formula
with 2′-FL were similar to those of the
BF group. For the first time, the effect
of 2′-FL on markers of immune
function was investigated in formula
fed infants.

Gastrointestinal Tolerance of Infants
Fed Milk-Based Formula with 2′-FL
 A prospective, randomized, multi-
center, double-blinded, controlled,
tolerance trial was conducted in
healthy term infants (Kajzer et al.
FASEB J. 2016, 30, 671).

 The study assessed GI tolerance of
infants who were fed formula that
was supplemented with 0.2 g 2′-
FL/L and 2.0 g scFOS/L (Abbott
Nutrition, Columbus, OH, USA),
compared to a CF without
oligosaccharides and a BF
reference group.

 A total of 131 infants were enrolled into
the clinical trial and 119 infants
completed the study duration, with 41 in
the formula with 2′-FL group, 36 infants
in the CF group, and 42 infants in the BF
group. There were no statistically
significant differences in the
demographic characteristics among the
three groups at enrollment.

 This 2′-FL clinical study evaluated the
GI tolerance of infants that were fed
formulas with and without 2′-FL,
compared to a BF reference group. No
clinically significant differences were
found among the three groups from
enrollment to 35 days of age for stool
consistency, formula intake,
anthropometric measures, and percent
feedings with spit-up/vomit associated
with feeding.

 Formula with 2′-FL was safe and well
tolerated in infants, as evidenced by
stool consistency, formula intake,
percent feedings with spit-up/vomit,
and reported AEs like those of the
infants who were fed formula without
oligosaccharides or those of the BF
infants.

Clinical Feeding Experience of Infants
Fed a Partially Hydrolyzed Whey-
Based Formula with 2′-FL
 A clinical feeding experience study was
recently conducted to assess the effects of
switching to a partially hydrolyzed whey-
based formula (PHF) supplemented with
2′-FL on symptoms of formula
intolerance.

 The study design was a
prospective, multi-center, single-
arm study investigating infants fed
a PHF with 0.2 g 2′-FL/L and 1.8 g
scFOS/L (Abbott Nutrition,
Columbus, OH, USA).

 A total of 59 infants were enrolled in
the clinical feeding experience study.
Forty-seven were evaluable on day 1
and 32 were evaluable at day 28. The
median reduction in the severity of
fussiness was statistically significant
after one day (p < 0.0001). The severity
of fussiness continued to decrease
throughout the duration of the study.

 Overall, the formula was safeOverall, the formula was safe
and was well tolerated by theand was well tolerated by the
infants, similar to the results thatinfants, similar to the results that
were reported by Marriage et al.were reported by Marriage et al.
and Kajzer et al.and Kajzer et al.

Final messageFinal message
 Several clinical studies reporting results of
infants who were fed formula that was
supplemented with 2′-FL. These clinical
experiences found that the supplementation
of infant formula with 2′-FL is safe and
well-tolerated, and that 2′-FL is absorbed
and excreted with similar efficiency
compared to 2′-FL in human milk.

 In addition, infants who were fed
formula with 2′-FL had immune
benefits like the BF reference group,
had fewer parent-reported infections,
specifically respiratory infections,
and had improved symptoms of
formula intolerance in fussy infants.

 Therefore, adding 0.2 g 2′-FL/L to
infant formula not only brings it
closer compositionally to human milk,
but also functionally. Additional
clinical research may reveal other
beneficial effects of 2′-FL in infant
formulas, including other study
populations such as preterm infants

Breakthrough of Human Milk Oligosaccharides

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