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TRANSCRIPT
Prebiotics strengthen
the immune system
Paul Vos, PhD
Danone Research -
Centre for Specialised Nutrition
Wageningen
19-6-2009
Outline
Immune system
Immune regulation
Immune disorders
Natural imbalance
Oligosaccharides: immuno-nutrition breakthrough
In vitro results
In vivo preclinical results
Results from clinical trials
3
Major tasks of the immune system
Defence against pathogens: bacteria, viruses, parasites, fungi
Assistance in anti-cancer responses
Removal of foreign / non-self compounds
Inhibition self-reactive / auto-reactive responsiveness
Inhibition “over-activity/allergy”
YY
Granulocyte
Macrophage / Dendritic cell
Monocyte
T Lymphocyte
(Th1, Th2, Th3, Tr, ...)
B Lymphocyte / Antibodies
Natural Killer Cell
Epithelial cell
Bone marrow
Lymph nodes
Spleen
Thymus
Organs Cells
60 – 70 % of immune cells in the gastro- intestinal tract !!
Immune system
Non-specific first line defence: skin, lungs, intestine,
urinary system (physical/chemical barrier, low pH,
enzymes, mucus, ...)
Non-specific (innate) immunity:
(Macrophages, granulocytes, natural killer cells,..)
Adaptive immunity:
(T Cells, B Cells, memory, antibodies, vaccination)
Immune system
Humoral immunity:
• Antibodies
Cellular immunity:
• Immune-regulation
• Cytotoxicity
Adaptive immunity
T
TT
TT
T
IL-2
T
T
ExpansionActivation
Virus/bacteria/allergen
DC
B
Antibody Production
YYYYYYYY
The adaptive immune response
Th0Th1/Th17 Th2
APC
Immune regulationTh1/Th2 paradigm
Allergy
Antibody-mediated autoimmunity
Resistance to intracellular
infections
Anti-cancer
Delayed type
hypersensitivity reactions
Resistance to
extracellular infections
Tolerance
T-cell mediated autoimmunity
Graft rejection
Hallmark signaling molecules / antibody class:
Th1: IFN- , IL-12
Th2: IL-4, IL-13 / IgE antibodies
Tr/Th3: IL-10, TGF- 1 / IgG4 antibodies
Effector cellsActivation
Regulatory cellsInhibition, tolerance
Treg
Th3
Th1/Th17 Th2
APC / DC
Immune regulationThe extended Th1/Th2 paradigm
Hyper immune- responsiveness:
Allergy
Autoimmunity
Chronic inflammatory diseases
Hypo immune- responsiveness:
Infections
Tumors/metastasis
Immune disorders (1)
Th1 , Th2 , Th1/Th2
Th1
Th2 (type I allergy)
Th2
Th2
Th1
Th1 ?
Th1
Th1
Th1 , Th2
Immune disorders (2)
HIV
COPD
Allergies
Asthma
Atopic eczema
Coeliac disease
Cystic Fibroses
Cancer
Elderly
Infants
Natural imbalance
Pregnancy and neonates
Th2-directed immune system: cytokines downregulate cellular
immunity (Th1) no rejection of fetus and or mother tissue
After birth
Development of their own “healthy” Th1/Th2 balanced immune
system
Th 2
Normal
PregnanciesFailure
CD 69+
IL 2
TNF α
TNF δ
CD 94+
IL 4
IL 5
IL 10
Th 2Th 1
Normal
Pregnancies
• Genes
• Hygiene
• Drugs
• Diets
• Stress
• Hormones
years0 1 2 5 10 40 50 60 70 80 90
Th 1 Th 2
Th1 and Th2 activity as a function of age
Allergy
Natural imbalance
years0 1 2 5 10 40 50 60 70 80 90
Th 1 Th 2
Stimulate development of Th1/Th2 balanced
immune system during the first year of life
Field et al J Nutr 2005; 135: 1-4
Compounds with immunological properties in human milk Anti-microbial compounds Immune development Immunoglobiuines: sIgA, SIgG, compounds SIgM Macrophages Lactoferrin, lactoferrin B and H Neutrophils Lysozyme Lymphocytes Lactoperoxidase Cytokines Nucleotide-hydrolizing Growth factors Antibodies Hormones к-casein and α-lactalbumin Milk peptides Haptocorrin Long-chain polyunsaturated Mucins fatty acids Lactadherin Nucleotides Free secretory component Adhesion molecules Oligosaccharides and pre-
biotics Anti-inflammatory compounds Fatty acids Cytokines: Il-10 and TGFβ Maternal leukocytes and Il-1 receptor anatagonist Cytokines TNFα and Il-6 receptors sCD14 sCD14 Complement and complement Adhesion molecules receptors Long-chain polyunsaturated β-defensin-1 fatty acids Toll-like receptors Hormones and growth factors Bifidus factor Osteoprotegerin Tolerance/priming compounds Long-chain polyunsaturated Cytokines: Il10 and TGFβ fatty acids Anti-idiotypic antibodies Hormones and growth factors
Compounds with immunological
properties in human milk
Composition of Human Milk
53-61 g/l
30-50 g/l
8-10 g/l
Prebiotics
10-12 g/l
Made
of:
Amino Acids Mono Saccharides Fatty acids
Human milk oligosaccharides are not
commercially available
But:
mixture of specific prebiotic oligosaccharides has
been developed
GOS e.g. DP3 FOS e.g. DP10
[Gal( 1-4)Gal( 1-4)Glc [Frc( 2-]8 1)Frc( -1)Glc
90 % GOS: short-chain -Galacto-OligoSaccharides from lactose
10 % FOS: long-chain -Fructo-OligoSaccharides from chicory
Our oligosaccharides concept
Human milk (g/L) Cow's milk (g/L)
Lactose 55-70 40-50
Total oligosaccharides 3.0-15.0 Traces
Facts:
15-25% of total carbohydrates
a very heterogeneous fraction > 100 different structures
variability: genetic control, time of lactation
soluble, non-digestible, fermentable
Hypotheses:
Induce a healthy gut microbiota in the baby
Modulate the developing immune system
Human milk oligosaccharides
Clinical trials
in humans
In vivo
animal
models
In vitro
animal cells
In vitro
human cells
In vitro mechanistic
Translational Research Approach(as recommended by WHO, FDA)
From invited review on oligosaccharides: critical reviews immunology: Paul Vos et al., 2007
Potential mechanisms of immune modulation by orally
applied nog-digestible carbohydrates
Beneficial
bacterial
SCFA, pH
Immune systemPathogen
reduction
Barrier integrity /
Mucoprotection
Bacterial
fermentation
Boehm et al Nutrafoods 2005
Potential mechanisms of immune
modulation by HMOS
HMOS
Prebiotics must stimulate one or a limited number of beneficial bacteria, like bifidobacteria or lactobacilli, within the colonic
microbiota
Non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the
colon that can improve host health
Prebiotics must “survive” acidic conditions
Prebiotics must be selectively fermented in
the colon
Prebiotics must evade digestion in the small
intestine
Fuller & Gibson, 1997, Scand J Gastroenterol Suppl (222):32-40
Prebiotic concept
0.8g/100ml
(n=27)
0g/100ml
(n=33)
0.4g/100ml
(n=30)
log
10 o
f C
FU
/g w
et
fae
ce
s (
me
dia
n, IQ
R)
log
10 o
f C
FU
/g w
et
fae
ce
s (
me
dia
n, IQ
R)
0.8g/100ml
(n=27)
0g/100ml
(n=33)
0.4g/100ml
(n=30)
2
3
4
5
6
7
8
9
10
11
12
Reference range (IQR) of breastfed infants (n=15)
10
0
1
2
3
4
5
6
7
8
9
Reference range
(IQR) of breastfed
infants (n=15)
LactobacillusBifidus
Group difference
according to Mann-
Whitney U-test: *
p<0.05 vs. 0.0, # vs. 0.4
**#
* *
Moro et al .J Pediatr Gastroenterol Nutr 2002; 34:291-295
Group difference
according to Mann-
Whitney U-test: *
p<0.05 vs. 0.0, # vs. 0.8
Oligosaccharides promote growth of
healthy bacteria
Beneficial
bacterial
SCFA, pH
Immune systemPathogen
reduction
Barrier integrity /
Mucoprotection
Bacterial
fermentation
Boehm et al Nutrafoods 2005
Potential mechanisms of immune
modulation by HMOS
HMOS
Improve gut barrier/immunomodulation
Inflammation
induced tissue
destruction
Intestinal
Immune Cell activity
Epithelial
Function and
Regeneration
Permeability
Mesenchym
Mucus
SCFA, SIgA
Short chain fatty acids stimulate mucin-2
production by intestinal epithelial cells
Willemsen et al Gut 2003; 52: 1442-1447
Beneficial
bacterial
SCFA, pH
Immune systemPathogen
reduction
Barrier integrity /
Mucoprotection
Bacterial
fermentation
Boehm et al Nutrafoods 2005
Potential mechanisms of immune
modulation by HMOS
HMOS
Vaccination model
Blood sample
Primary influenza
immunization
Blood sample
Booster influenza
immunization
Study parameters:
• DTH
• Influenza specific antibody titers
• Ex-vivo lymphocyte restimulation
0 7 14 302821-20
Start dietary
intervention
DTH
End
scGOS/lcFOS stimulate Th1-
dependent vaccination response
Vos et al. International Immunopharmacology 2006; 6: 1277-1286
Conclusions so far
GOS/lcFOS can affect systemic immunity (Th1/Th2)
Not all oligosaccharides affect the immune system
What about improved resistance to infections?
What about reduced risk for IgE mediated allergy?
The increase in the prevalence of
asthma, eczema and hay fever
Devereux et al. Nat. Rev. Immunol. 2006;6: 869-874
Sensitisation
Allergen avoidance
Allergen Allergen Provocation
B cellT h 2 cellAntigen-
Presenting (DC) cell
Clinicaleffects
IgE binding
inflammatory cell
IL-4
IL-5
IL-13IgE
Mediators
Mechanism of allergy development
OVA-induced model for experimental
allergic asthma
0 7 35 41
i.p. OVA
Study parameters:
• Airway hyperresponsiveness
• Airway inflammation
• Allergen specific antibody titers
• Ex-vivo lymphocyte restimulation
-14
Start dietary
intervention
38 42
OVA aerosol
challenge
end
Oligosaccharides decrease the manifestation
of allergic asthma
Inflammatory cells in BALAirway hyperresponsiveness
Vos et al., International Immunopharmacology 2007; 7(12):1582-1587
Moro study
Double blind, placebo controlled, randomised prospective study
206 infants with at least a single family history of allergy
gestational age between 37 and 42 weeks (birth weight appropriate for
gestational age)
Feeding regime:
formula feeding within the first 2 weeks of life until the age of 6 months
mixed feeding accepted until the 6th week of life
exclusive formula feeding until the end of 4 months of life
Formulas:
Aptamil HA plus 0.8g GOS/FOS per 100ml (n=104)
Aptamil HA plus 0.8g maltodextrine per 100ml (control; n=102)
Outcome parameters:
primary outcome: atopic dermatitis
Microbiota, IgE and IgG4 in a subpopulation of the cohort
GOS/lcFOS
(n=10/102)Placebo
(n=24/104)
Cu
mu
lati
ve
in
cid
en
ce
of
ato
pic
de
rma
titi
s
0
5
10
15
20
25
30
p<0.03 Fisher‘s exact
Reduction of Atopic Dermatitis and increase in
bifidobacteria in high risk infants at 6 months
Placebo
(n=44)
Bif
ido
ba
cte
ria
co
un
t (l
og
cfu
/g s
too
l)
0
3
6
9
12
15
18
P<0.001 Mann Withney U-test
GOS/lcFOS
(n=50)
10.3
8.7
9.8%
23.1%
Moro et al. 2006, Arch. Dis. Child 91: 814-819
scGOS/lcFOS decrease serum IgE
in High-Risk Infants at 6 Months
**p=0.008
Placebo Gos/lcFos
1
10
100
1000
IgE
(kU
/l)
Hoffen et al. 2009, Allergy 64: 484-487
scGOS/lcFOS decrease CMP IgG1
in High-Risk Infants at 6 Months
P<0.0105
Placebo GOS/lcFOS
0.1
1
10
100
1000
IgG
1 (
AU
/ml)
P<0.0105
Hoffen et al. 2009, Allergy 64: 484-487
Vaccination response is not affected by
scGOS/lcFOS
Placebo Gos/lcFos10
100
1000
10000Ig
G1 (
AU
/ml)
DTP IgG1
Hoffen et al. 2009, Allergy 64: 484-487
Protective effect on infectious episodes
in High-Risk infants at 6 months
Arslanoglu et al, 2007, J .Nutr. 137: 2420-2424
0
10
20
30
40
50
All Infections URTI
infe
cti
ou
s e
pid
so
des
placebo
GOS/lcFOS
p=0.01
trend,
p=0.07
Early dietary intervention with scGOS/lcFOS reduce the
incidence of allergic manifestations during the first 2 years
of life
Arslanoglu et al., 2008, J. of Nutr. 138:1091-1095
Early dietary intervention with a mixture of prebiotic
oligosaccharides reduce the incidence infections during
the first 2 years of life
Arslanoglu et al., 2008, J. of Nutr. 138:1091-1095
Scholtens study
Double blind, placebo controlled, randomized prospective study
215 healthy infants without a family history of atopy
gestational age between 37 and 42 weeks (birth weight appropriate for
gestational age)
Feeding regime:
mixed feeding accepted
Formulas:
Non-hydrolyzed formula plus 0,6g/100 mL scGOS/lcFOS (n=58)
Non-hydrolyzed formula (n=64)
Reference group: full breastfeeding (n=20)
Outcome parameters:
primary outcome: fecal sIgA
scGOS/lcFOS increases faecal sIgA
level during the first 6 months of life
P. Scholtens et al., J Nutr 2008; 138: 1141-1147
sIgA increment:
-better first line defence
-less infections
-less allergies
-tolerance induction
Placebo scGOS/lcFOS Breastfed0
250
500
750
22
24
31
Faecal
sIg
A
(g
/g)
P 0.001
Bruzzese study
Open, placebo-controlled, randomised, prospective study 324 healthy infants
gestational age between 37 and 42 weeks (birth weight appropriate for
gestational age)
Feeding regime formula feeding for 12 months
Formulas Standard formula (n =173)
Standard formula plus 0.4 g/100 ml scGOS/lcFOS (n=169)
Outcome parameters Primary outcome: incidence of acute diarrhoea, upper- and lower respiratory
tract infection, use of antibiotics, number of hospital admissions
Secondary outcome: nutritional adequacy
scGOS/lcFOS reduce incidence of acute
diarrhea and antibiotic courses in infants
Bruzzese et al., 2009, Clinical Nutrition, in press
Reduction of mean number of
episodes of gastroenteritis
Trend for reduced recurrence
of upper respiratory tract
infections (URTI)
Reduction of antibiotics
courses used
at least 1 URTI 3 URTI0
10
20
30
40
50
6065 Placebo
scGOS/lcFOS
65/10960/94
29/65
17/60
% o
f ch
ild
ren
wit
h U
RT
I
0
25
50
75
100 Placebo
scGOS/lcFOS
At least 1 antibiotic
course 2 antibiotic
course
% o
f ch
ild
ren
wit
h a
nti
bio
tics
58/65
46/60
43/65
24/60
P=0.06 p<0.02
Placebo scGOS/lcFOS0.0
0.1
0.2
0.3
0.4
0.5
Nu
mb
er
of
ep
iso
des/c
hil
d/1
2 m
on
ths
p<0.01
Pathogens
Viruses
Parasites
Fungi
Proteins
mechanical defencee.g. skin, mucosa, mucus layer
chemical defencee.g. SCFA, acidic pH
biological defencehealthy microbiota prevents growth
of pathogens
immunological defencee.g. antibodies, cytokines,
white blood cells,
macrophages
Conclusion
Pathogens
Viruses
Parasites
Fungi
Proteins
mechanical defencee.g. skin, mucosa, mucus layer
chemical defencee.g. SCFA, acidic pH
biological defencehealthy microbiota prevents growth
of pathogens
immunological defencee.g. antibodies, cytokines,
white blood cells,
macrophages
Unique mixture of
Prebiotics
Conclusion