Download Can fish oil in pregnancy and lactation alter maternal and

Linköping University Medical Dissertations
No. 1206
Can fish oil in pregnancy and lactation alter maternal and
infant immunological responses and prevent
allergy in the offspring?
Catrin Furuhjelm
Division of Pediatrics
Department of Clinical and Experimental Medicine
Faculty of Health Sciences, Linköping University, Linköping, Sweden
Linköping 2010
©Catrin Furuhjelm 2010
Cover Design: Familjen Furuhjelm
ISBN: 978-91-7393-314-8
ISSN: 0345-0082
Published papers are reprinted with permission of the publishers
Printed by LiU-Tryck, Linköping, Sweden 2010
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To my family
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ABSTRACT
Background: A connection has been proposed between the increase of allergic
disease and the altered composition of fatty acids in the diet in the westernised
world. Less oily fish and more vegetable oil are consumed today compared to
50-100 years ago. Programming of the immune responses takes place very early
in life and environmental factors, such as fish in the diet, have been suggested to
protect from infant allergy.
Aim: The general aim of this thesis was to assess the effects of maternal dietary
supplementation with ω-3 long chain polyunsaturated fatty acids (LCPUFA),
i.e. fish oil, in pregnancy and lactation on the development of allergic symptoms
and sensitisation in the infants as well as some immunological markers in
mothers and infants.
Subjects: This thesis is based on the results from a prospective double-blind
placebo-controlled multi-centre trial comprising 145 families.
Methods: Pregnant women, at risk of having an allergic infant, were recruited at
the antenatal clinics in Linköping and Jönköping and randomised to daily
supplementation with 1.6 g eicosapentaenoic acid (EPA, C20:5ω-3) and 1.1 g
docosahexaenoic acid (DHA, C22:6ω-3) or placebo, starting in the 25th
gestational week and continuing through 3.5 months of breastfeeding.
Phospholipid fatty acids in maternal and infant serum/plasma were analysed
before, during and after the intervention to assess compliance. Prostaglandin E2
(PGE2), leukotriene B4 (LTB4) and infant vaccine induced responses were
analysed with ELISA technique. Maternal cytokines and infant chemokines
were analysed with Luminex technique. Clinical outcomes were allergic disease
and positive skin prick test/detectable circulating IgE antibodies to common
allergens.
Results: Phospholipid proportions of ω-3 LCPUFA increased significantly in the ω-3
supplemented women and their infants. Lipopolysaccharide-induced PGE2 secretion
from whole blood culture supernatants decreased in a majority of the ω-3supplemented mothers (p<0.01). The decrease in PGE2 production was more
pronounced among non-atopic than atopic mothers. No difference in the prevalence of
allergic symptoms was found between the intervention groups. The cumulative
incidence of IgE associated eczema and IgE mediated food allergy was though
reduced in the ω-3 group during the first years (OR= 0.3 and 0.1 compared to placebo,
p<0.05 for both) and up to two years (OR= 0.2 and 0.3 compared to placebo, p<0.05
for both). The cumulative incidence of any IgE associated disease during the first two
years of life was 13% in the ω-3 supplemented group compared to 30% in the placebo
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group (p=0.01, OR 0.3, p<0.05). This effect was most evident in infants of nonallergic mothers. Higher maternal and infant proportions of DHA and EPA were
associated with lower prevalence of IgE associated disease (p=0.01-0.05), in a dose
dependent manner. In addition, no allergic symptoms as compared to multiple allergic
symptoms, in the infants, regardless of sensitisation, were related to higher maternal
and infant ω-3 LCPUFA status (p<0.05).
High infant Th2-associated CC-chemokine ligand 17 (CCL17) levels were associated
with infant allergic disease (p<0.05). In infants with non-allergic mothers the ω-3
supplementation was related to lower CCL17/ CXC-chemokine ligand 11 (CXCL11)
(Th2/Th1) ratios (p<0.05). This was not seen in infants whose mothers had allergic
disease. Furthermore in non-allergic, but not in allergic infants, ω-3 supplementation
was linked with higher Th1-associated CXCL11 levels (p<0.05), as well as increased
IgG titres to diphtheria (p=0.01) and tetanus (p=0.05) toxins.
Conclusions: A decreased cumulative incidence of IgE associated disease in the
infants was found after maternal ω-3 LCPUFA supplementation in pregnancy
and lactation. This result was supported by a reverse dose response relationship
between maternal ω-3 LCPUFA status and infant IgE associated disease.
Higher proportions of DHA and EPA in maternal and infant serum/plasma were
also associated to less severe allergic disease. A tendency towards strengthened
Th1 associated response after maternal ω-3 LCPUFA supplementation was
indicated in the analysis of maternal and infant immunological markers. These
effects, as well as the clinical outcomes, were more pronounced in non-allergic
individuals, suggesting gene-by-environment interactions.
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SAMMANFATTNING
Bakgrund: I takt med att sammansättningen av fettsyror i vår kost förändrats har
också allergifrekvensen ökat i västvärlden. Vi äter mindre fisk och mer
vegetabiliska oljor idag än för 50-100 år sedan. Immunförsvaret programmeras
tidigt i livet och epidemiologiska studier talar för att ökat fiskintag under
graviditeten och barnets första år kan förebygga allergi hos barnet.
Syfte: Avhandlingens syfte är att ta reda på om tillskott av fiskolja under
graviditet och amning kan påverka några av de immunologiska markörer hos
mor och barn som har betydelse för allergisjukdom, samt förekomsten av
allergiska symptom och sensibilisering hos barnen upp till två års ålder.
Metod: Blivande mödrar i Jönköping och Linköping med allergisjukdom hos
dem själva eller i närmaste familjen, rekryterades via mödravårdscentraler och
slumpfördelades till att äta 2.7 g fiskolja dagligen eller icke aktiva kapslar från
graviditetsvecka 25 till och med 3.5 månaders amning.
Andelen fiskolja i blodets fospholipider mättes upprepade gånger hos mödrar
och barn. Luminex- och ELISA- tekniker användes för att analysera
immunfaktorer som prostaglandiner, leukotriener och cytokiner i mödrarnas
blod samt kemokiner i barnets blod och barnens immunsvar efter vaccination.
Barnen undersöktes upprepade gånger och genomgick pricktest för vanliga
allergiframkallande ämnen. Även cirkulerande IgE-antikroppar mot dessa
ämnen mättes.
Resultat: Nivåerna av ω-3-fettsyror ökade i den ω-3-behandlade gruppen hos
både mödrar och barn och var högre än i placebogruppen upp till ett år efter
barnets födelse. De stimulerade cellkulturerna från mammor som fått omega-3fettsyror visade mindre produktion av prostaglandin E2 (PGE2), som visat sig ha
betydelse för allergisk sensibilisering. Produktionen av cytokiner hos mödrarna
samt kemokiner hos barnen skiljde sig inte mellan omega-3-gruppen och
placebogruppen.
Under sina första två levnadsår hade barnen till mammor som fått
fiskoljetillskott mindre ofta positiv pricktest mot födoämnen, speciellt ägg.
Däremot var det ingen skillnad mellan grupperna med avseende på förekomst av
allergiska symptom, så som eksem, astma, födoämnesallergi eller hösnuva. De
barn som hade både allergiska symptom, oftast i form av eksem eller
födoämnesreaktioner och sensibilisering (positiv pricktest/detekterbara IgEantikroppar i blod), s.k. IgE associerad allergisk sjukdom var dock vanligare i
placebo gruppen än i den ω-3-behandlade gruppen (30 % mot 13 %, p=0.01).
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Det visade sig också att högre andel omega-3-fettsyror både i mammans och i
barnets blod gav lägre sannolikhet för barnet att utveckla IgE-associerad
allergisk sjukdom under sina första två år. Dessutom hade barn med mer än ett
allergiskt symptom, d v s en allvarligare form av allergisk sjukdom, lägre nivåer
av omega-3-fettsyror i blodet än barn utan allergiska symptom.
Effekten av fiskoljetillskottet var tydligast i den grupp där mammorna själva inte
hade allergisjukdom, både gällande PGE2 hos mamman och IgE associerad
allergisk sjukdom samt positiv allergitest hos barnet. Hos barn till icke allergiska
mammor fanns dessutom lägre nivåer av kemokiner aktiva i allergisk
inflammation i omega-3-gruppen jämfört med kontrollgruppen (p<0.05). Icke
allergiska barn byggde också upp ett bättre skydd mot stelkramp och difteri efter
vaccination om deras mammor fått ω-3-tillskott (p<0.05 respektive 0.01).
Slutsats: En skyddande effekt sågs av fiskoljetillskott mot IgE-associerad
allergisk sjukdom hos barnen upp till två års ålder. Det verkar som höga nivåer
av fiskfetter i barnets och moderns blod var associerat till en minskad
omfattning av den allergiska sjukdomen hos barnen, möjligen pga. mindre
allergisk sensibilisering. Effekterna av fiskoljetillskottet, både på
immunologiska markörer och på klinisk sjukdom, var tydligast hos barn till
mödrar som själva inte hade allergisk sjukdom vilket kan tyda på ett samspel
mellan gener och kostfaktorer.
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CONTENTS
ORIGINAL PUBLICATIONS ............................................................ 11
ABBREVIATIONS ............................................................................. 13
INTRODUCTION ............................................................................... 15
REVIEW OF THE LITERATURE ..................................................... 17
Introduction to allergic disease ........................................................................... 17
The allergic march ................................................................................................. 17
Allergic sensitisation - atopy ................................................................................. 18
Food allergy ......................................................................................................... 19
Sensitisation and tolerance in the gut .................................................................... 21
Eczema ................................................................................................................. 22
Asthma and rhinoconjunctivitis............................................................................ 23
Introduction to the immune system ...................................................................... 24
Immune components involved in allergic inflammation ...................................... 25
T-cells and cytokines ............................................................................................. 25
Chemokines ........................................................................................................... 28
B cells and immunoglobulins ................................................................................ 30
Eosinophils ............................................................................................................ 31
Mast cells ............................................................................................................... 31
Intrauterine sensitisation and foetal immune responses ...................................... 32
Maternal immune responses during pregnancy ..................................................... 32
The foetal immune system .................................................................................... 34
Early T cell responses to allergens ........................................................................ 34
Early specific IgE .................................................................................................. 35
Total IgE in cord blood ......................................................................................... 36
Prevention of allergic disease .............................................................................. 38
Environmental factors ........................................................................................... 39
Early allergen exposure ......................................................................................... 39
Smoking and air pollution ..................................................................................... 40
Psychological factors ............................................................................................. 41
Microbial components - the hygiene hypothesis ................................................... 41
Infections - Respiratory Syncytial Virus (RSV). ................................................... 42
Antibiotics ............................................................................................................. 43
Protective factors in the diet of mother and child ................................................. 43
Long chain polyunsaturated fatty acids ............................................................... 45
Fish oil intake early in life and infant allergic disease........................................ 48
Clinical outcomes .................................................................................................. 49
Immunological outcomes ...................................................................................... 50
Breast milk ............................................................................................................ 51
Immune modulating mechanisms. ........................................................................ 52
Gene level .............................................................................................................. 52
Membrane fluidity ................................................................................................. 52
Lipid mediators...................................................................................................... 53
Gene-by-environment interaction ........................................................................ 56
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AIMS AND HYPOTHESIS ................................................................ 57
MATERIALS AND METHODS ........................................................ 59
Inclusion of participating families ....................................................................... 59
Intervention .......................................................................................................... 61
The content of the capsules ................................................................................... 61
Compliance ............................................................................................................ 64
Blinding ................................................................................................................. 65
Safety ..................................................................................................................... 66
Study subjects ....................................................................................................... 67
Clinical methodology ........................................................................................... 68
Diagnostic criteria ................................................................................................. 69
Laboratory methodology ...................................................................................... 71
Statistics ............................................................................................................... 72
Ethical considerations .......................................................................................... 73
RESULTS AND DISCUSSION.......................................................... 75
Maternal diet and fatty acid status. ..................................................................... 75
Pregnancies and deliveries .................................................................................. 79
Allergic heredity and environmental factors ....................................................... 82
Maternal eicosanoid secretion ............................................................................. 84
Infant fatty acid status .......................................................................................... 85
Clinical outcomes ................................................................................................. 85
Intention-to-treat analysis ...................................................................................... 88
Infant chemokines and vaccine induced immune responses ................................ 89
Previous ω-3 supplementation trials - design and outcomes .............................. 90
Phospholipid fatty acids in relation to allergic disease ....................................... 92
Possible gene-by-environment interaction ........................................................... 94
CONCLUSIONS ................................................................................. 97
FUTURE PERSPECTIVES ................................................................ 99
ACKNOWLEDGEMENT ................................................................. 101
REFERENCES .................................................................................. 103
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ORIGINAL PUBLICATIONS
I. The effects of omega-3 fatty acid supplementation in pregnancy on maternal
eicosanoid, cytokine and chemokine secretion.
Kristina Warstedt, Catrin Furuhjelm, Karel Duchén, Karin Fälth-Magnusson,
Malin Fagerås Böttcher
Pediatr Res 2009; 66(2): 212-7.
II. Fish oil supplementation in pregnancy and lactation may decrease the risk of
infant allergy.
Catrin Furuhjelm, Kristina Warstedt, Johanna Larsson, Mats Fredriksson,
Malin Fagerås Böttcher, Karin Fälth-Magnusson, Karel Duchén
Acta Pediatr 2009; 98(9): 1461-7.
III. Allergic disease in infants up to two years of age in relation to plasma
omega-3 fatty acids and maternal fish oil supplementation in pregnancy and
lactation.
Catrin Furuhjelm, Kristina Warstedt, Malin Fagerås, Karin Fälth-Magnusson,
Johanna Larsson, Mats Fredriksson, Karel Duchén
Pediatr Allergy Immunol. In press.
IV. Th1 and Th2 chemokines, vaccine induced immunity and allergic disease in
infants after maternal ω-3 fatty acid supplementation during pregnancy and
lactation.
Catrin Furuhjelm, Maria C. Jenmalm, Karin Fälth-Magnusson, Karel Duchén
Pediatr Res. Accepted.
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ABBREVIATIONS
AA
arachidonic acid
APC
antigen presenting cell
ARC
allergic rhinoconjunctivitis
CAPS
the Childhood Asthma Prevention Study
CBMC
cord blood mononuclear cells
CCL
CC-chemokine ligand
CD
cluster of differentiation
COX
cyclooxygenase
CV
coefficient of variance
CXCL
CXC-chemokine ligand
d
day
DBPCFC
double-blind placebo controlled food challenge
DC
dendritic cell
Der P
Dermatophagoides pteronyssinus
DHA
doxohexaenoic acid
dns
data not shown
EPA
eicosapentaenoic acid
ER
endoplasmatic reticulum
FABPpm
plasma membrane fatty acid binding protein
FADS
fatty acid desaturase encoding gene
FсεRI
high affinity IgE receptor
FLG
filaggrin gene
GST
glutation s-transferase
gw
gestational week
HDM
house dust mite
HLA
human leukocyte antigen
IFN
interferon
Ig
immunoglobulin
IL
interleukin
ISAAC
the International Study of Asthma and Allergies in Childhood
LA
linoleic acid
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LCPUFA
long chain polyunsaturated fatty acids
LNA
alfa-linolenic acid
LOX
lipoxygenase
LPS
lipopolysaccharide
LT
leukotriene
MHC
major histocompatibility complex
NFκβ
nuclear factor kappa beta
NK
natural killer
PAF
platelet aggregating factor
PBMC
peripheral blood mononuclear cells
PCB
polychlorinated biphenyles
PGE
prostaglandin E
PHA
phytohemagglutinin
PKC
protein kinase C
PL
phospholipid
PLA
phospholipase A
PPAR
peroxisome proliferator-activating receptor
PRR
pathogen recognition receptors
PUFA
polyunsaturated fatty acids
RDI
recommended daily intake
RSV
human respiratory syncytial virus
SIgA
secretory IgA
SNP
single nucleotide polymorphism
SPT
skin prick test
TCR
T cell receptor
Th
T helper
TLR
toll like receptor
TNF
tumour necrosis factor
Treg
regulatory T-cell
VCAM
vascular cell adhesion molecule
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INTRODUCTION
Until recently, a common allergy preventing advice for mothers of infants at risk
of developing allergic disease was to avoid fish in the infant diet until the end of
first year (1). Now the former risk factor “early fish intake” is becoming a
potentially beneficial element in the prevention of allergic disease. Some of the
ideas, theories and facts behind this change are discussed in this thesis.
The prevalence of allergic disease varies widely between different countries.
This indicates that there are many factors, including genetic and environmental
aspects, accounting for the occurrence of allergies (2). The reason for the
increased prevalence of allergic disease seen in affluent countries during the last
decades is not clear (2) but among other environmental factors, the quality of
dietary fat might be of importance (3, 4). The International Study of Asthma
and Allergies in Childhood (ISAAC) indicates that countries with a low
frequency of allergic disease, such as Asian countries (including India and China
but excluding Hong Kong and Japan), Eastern European countries (including
Russia and Poland), and some Southern European countries (including Greece),
share a relative low dietary intake of oils containing the omega (ω)-6 fatty acid
linoleic acid, i.e. various vegetable oils. Instead their diets contain olive oil (ω-9)
and/or fish oil (ω-3). Thus, the ω-6 (vegetable oil)/ω-3 (fish oil) ratio is low (4).
Furthermore, the prevalence of sensitisation to inhalant allergens in
schoolchildren from Greenland, who supposedly consume considerable amounts
of fish, is low compared to European children (5). Still, the prevalence of
allergic disease has increased in the Arctic, just like in the Western countries (6).
Efforts to treat already established asthma in children and adults with ω-3 fatty
acids, thus lowering the ω-6/ω-3 ratio, have not been successful (7).
On the other hand, attempts to influence early foetal immune responses with
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maternal ω-3 fatty acid supplementation in pregnancy have been more fortunate
(8, 9). In this thesis we have investigated the effects of ω-3 fatty acid
supplementation in pregnancy and lactation on infant allergic sensitisation and
disease in addition to maternal and infant immunological markers.
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REVIEW OF THE LITERATURE
Introduction to allergic disease
Allergic disease is caused by a hypersensitivity reaction initiated by specific
immunologic mechanisms. An allergen is an antigen causing allergic disease
and allergy can be antibody- or cell-mediated. The reaction is IgE mediated
(type I hypersensitivity) in most patients with allergic symptoms from the skin
and mucosal membranes in the airways and gastrointestinal tract. In non-IgE
mediated allergy, the inflammation can be brought about by antibodies of the
IgG isotype (type II or III, involving antibodies directed against cell
surface/matrix associated antigens or soluble antigens, respectively) or by
allergen specific T-lymphocytes (type IV). However, in the more chronic stages
of the IgE mediated allergy, the inflammation causing the symptoms is
dominated by allergen specific T lymphocytes and eosinophils as well (10).
Primarily, IgE mediated allergy in the first years of life will be discussed in this
thesis.
The allergic march
The term “allergic march” refers to the natural history of sensitisation to
allergens and allergic symptoms. It starts in infancy as IgE-related eczema,
sometimes accompanied by allergic gastrointestinal disease, and then progresses
to respiratory allergy. The initial IgE reactivity is directed to food allergens and
reactivity to inhaled allergens develops later (11). The term “allergic march” has
been questioned lately from a clinical perspective. It is quite clear that there are
many different phenotypes in the temporal sequence of asthma and allergic
diseases (12). For example, the majority of children with eczema and asthmatic
symptoms at age 7 already wheezed in early childhood, indicating an early co- 17 -
manifestation of these allergic phenotypes rather than an allergic march (13).
The IgE mediated allergic reaction varies between different tissues. For instance,
asthma is dominated by the long lasting reactions mediated by leukotrienes and
cytokines while histamine effects dominate in urticaria. Individual sensitivity in
mast cells in different organs regulates where the symptoms appear (14).
Allergic sensitisation - atopy
Individuals who are sensitised (i.e. atopic (10)) synthesise IgE antibodies
towards food or inhalant proteins. These IgE antibodies can be demonstrated
through a skin prick test where an allergen is injected in the skin. Mast cells with
IgE towards the allergen bound to high affinity IgE receptors (FсεRI) on their
surface subsequently degranulate and release histamine. The skin reaction, a
wheal appearing where the allergen was injected, can be measured with a ruler.
Circulating IgE antibodies to allergens can also be detected in serum (14).
Eczema, food reactions, asthma and rhinoconjunctivitis are allergic diseases
where IgE mediated mechanisms can be involved. The IgE associated disease is
also entitled extrinsic (15, 16). There are children who have symptoms without
measurable IgE sensitisation (i.e. intrinsic), as well as sensitised children who
are asymptomatic (10). For example, sensitisation to foods in young children
without food related symptoms seems to be a common phenomenon (17).
About 40% of chronic allergic disorders was attributable to sensitisation
(extrinsic) and 60 % to organ based and other factors (intrinsic) in a study of
four year old children in the United Kingdom (18).
Nevertheless, sensitisation can be a predictive marker of future allergic
manifestations. In the Danish DARC birth cohort study, IgE sensitisation to at
least one food allergen between 3 and 18 months was significantly associated
with eczema and asthma at the age of 6 years. Around 50% of children suffering
from eczema, rhinoconjunctivitis or asthma at 6 years were sensitised to food at
6 months (19). In agreement with this, children with IgE associated eczema at 2
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years had a greater risk of developing asthma at 6 years than non-sensitised
children in a recent study (20). Hence, recognition of IgE sensitisation can
provide useful information on the risk of childhood asthma and allergic
rhinoconjunctivitis. However, early IgE responses to allergens are often
transient in infants who remain non allergic whereas they reach higher and more
persistent levels in those who develop allergy (21). In a German report, children
persistently sensitised to food had a 3.4 fold higher risk of developing allergic
rhinitis and a 5.5 fold higher risk of developing asthma than infants who were
only transiently food sensitised. Persistent food sensitisation in combination
with a positive atopic family history was a strong predictor for the development
of allergic rhinitis and asthma at five years of age (22).
In conclusion, although sensitisation to food allergens during the first years of
life is not compulsory in allergic disease, it represents a predictive factor for
future allergic manifestations, especially if the early sensitisation persists
through the first two years.
Food allergy
Any food can cause a reaction, but there are few foods responsible for the large
majority of the symptoms: milk, eggs, wheat, peanuts, nuts, fish and shellfish.
Of these, cow‟s milk and egg allergy is frequently presented in infants (23). The
major food allergens involved in children‟s allergies are heat, acid, and protease
stable water-soluble glycoproteins 10 to 70 kD in size. Heating or preparing
foods might reduce (egg) or enhance (roasted peanut) allergenicity by modifying
conformational epitopes (24).
The term food allergy can be further split into IgE and non-IgE mediated
reactions (Fig. 1). IgE mediated food allergy begins in the first 1-2 years of life
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with the process of sensitisation. For an allergic reaction to occur, re-exposure is
needed with binding of the allergen to allergen-specific mast cell- or basophilbound IgE antibodies (23). The symptoms of IgE mediated food allergy include
angioedema, urticaria, rashes, flushing, anaphylaxis, bronchospasm,
rhinoconjunctivitis and immediate gastrointestinal symptoms. The non-IgE
mediated immune reactions that can arise in the gastro-intestinal tract are not so
well defined and more difficult to recognise. Eosinophil eosophagitis and
gastroenteritis as well as eczema are considered mixed IgE and cell mediated
while more delayed gastrointestinal symptoms, contact dermatitis, dermatitis
herpetiformis and pulmonary hemosiderosis are considered cell mediated (25).
Indeed, it has been well established that approximately 30% of children
suffering from moderate to severe eczema present an associated food allergy
that worsens their eczema. Double blind placebo controlled food challenge
(DBPCFC) is considered the “gold standard” for diagnosing any kind of food
allergy (26) but IgE testing and epicutaneous tests can be helpful to diagnose
IgE mediated and non-IgE mediated food allergy respectively (23).
Food reactions
Food allergy
Non allergic
food reactions
IgE-mediated
food allergy
Non-IgE-mediated
food allergy
Figure 1. Classification of food hypersensitivity reactions. Modified from (10).
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A diagnosis of food allergy is very often suspected in early childhood with at
least 25% of parents reporting one or more adverse food reactions. IgE mediated
food allergy can be confirmed in 5-10 % of young children in a normal
population and the peak prevalence is at approximately 1 year of age (27). Most
patients with egg allergy and egg IgE level less than 50 kU/L are likely to
develop egg tolerance by late childhood (28). In a population based study, 76%
of children with IgE mediated milk allergy and 100% of those with non IgE
mediated milk allergy were tolerant by the age of three years (29). In addition,
adverse reactions to fruits, vegetables and other cereal grains are typically very
short-lived (27). Children who present with one food allergy, especially if it is
IgE mediated, have a very high risk of developing additional food and inhalant
allergies (reviewed in (27)). Interestingly, reported food hypersensitivity at
young ages, even though transient, increased the risk for other allergic diseases
at 8 years in one study (30).
Sensitisation and tolerance in the gut
In the gastrointestinal tract, a single-cell layer of columnar intestinal epihelial
cells separates the internal sterile environment from the external world (31). An
intricate „„gastrointestinal mucosal barrier‟‟ has evolved consisting of
physiologic and immunologic components to process food to a form that can be
ingested but also prevent the penetration of harmful pathogens into the body.
However, the components of this mucosal barrier are immature in infants (31)
and this may contribute to the increased prevalence of both gastrointestinal tract
infections and food allergies seen in the first years of life. Factors that influence
the outcome of an immune response to oral antigens include antigen availability,
the immune environment and actions by immune cells with concomitant
cytokine secretion. However, how the mucosal immune system “decides”
whether and when to induce tolerance or sensitisation when exposed to fed
antigens remains largely unclear (32).
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In conclusion, different forms of food hypersensitivity are common during the
first two years of life, possibly due to the immaturity of the gut immune system.
Although most of the children become tolerant after a few years, the risk of
future allergic disorders is increased if the food allergy is IgE mediated.
Eczema
Eczema is the most common inflammatory skin disease in childhood with a
course marked by exacerbations and remissions. In a Swedish cohort of high
risk infants 35% developed eczema before 2 years of age and 28% had IgE
associated eczema (33). It usually clears in about one third of the patients after 2
years, and in another third after 5 years of age (13, 34). Eczema often coincides
with food allergy (35).
The definition of eczema is complicated due to variability of the distribution of
symptoms and morphology, the inconsistency of the time course of disease and
the lack of a diagnostic test. The diagnostic criteria, defined by Hanifin and
Rajka (36), to identify children with eczema has been a matter of debate,
especially with respect to the significance of some minor features in younger
children. Seymour (37) and Oranje (35) modified the criteria for infants. After
comparing five different sets of diagnostic criteria for children up to 18 months
of age, Johnke et al. found the agreement acceptable but the importance of
repeated examinations was underlined (38).
Major determinants of the prognosis of early eczema are severity of disease and
early allergic sensitisation (13). The mechanism of sensitisation may differ
between individuals. In some children with eczema, an intrinsic defect of the
skin barrier facilitates sensitisation due to damaged skin, while sensitisation in
the gastrointestinal tract precedes the development of eczema in other children
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(39). Filaggrin is a key protein for the skin barrier. It consolidates the keratin
filaments into dense bundles, thus being crucial for development of the cornified
cell envelope, maintaining the barrier function of the uppermost layer of the skin
(40). Two independent loss-of-function genetic variants (R510X and 2282del4)
in the gene encoding filaggrin (FLG) are very strong predisposing factors for
eczema (39). The FLG gene is located on the human chromosome 1q21, within
the epidermal differentiation complex, composed of more than 30 genes, all
involved in terminal differentiation of the epidermis (41). Weidinger et al (42)
evaluated an association of FLG null alleles with eczema, allergic sensitisation
and asthma in a German population of 476 families. Strong associations were
found between the FLG variants and eczema in combination with allergic
sensitisation. In agreement with this, eczema has been proposed to be a predictor
for subsequent development of sensitisation in non-sensitised children (43, 44).
Consequently, restoring skin barrier function in filaggrin deficient infants may
help prevent the development of sensitisation and halt the development and
progression of allergic disease (45). Filaggrin gene mutations also increased the
risk of asthma in people with atopic eczema.
In summary, eczema is the most common symptom of allergic disease in early
childhood, appearing with or without sensitisation. The skin barrier defect
associated with eczema seems to facilitate sensitisation but sensitisation can
also precede the onset of eczema. The combination of eczema and allergic
sensitisation is a strong predictor of future allergic disease in the airways.
Asthma and rhinoconjunctivitis
Asthma is a chronic inflammatory disease in the lower respiratory tract where
mast cells, eosinophils and T lymphocytes take part. This inflammation causes
repeated attacks of wheeze and cough, especially at night. The attacks may be
- 23 -
induced by allergen or viral infections, primarily infections, in early childhood
(46). About 10% of children in a high risk cohort in Sweden developed IgE
mediated asthma (33), also called allergic asthma (10). Without concomitant
sensitisation or asthma in the family, infants with non-allergic asthma do not
have an increased risk of asthma at 7 years of age according to the German
Multicentre Allergy study (47). Rhinoconjunctivitis mediated by allergens is
uncommon in the first two years of life but the incidence increases with age and
is about 20 % in the teen-age years (48).
In summary, allergic asthma and rhinoconjunctivitis are not as common as
eczema during the first two years of life. Wheeze is a frequent symptom in early
childhood but it is commonly triggered by infections and does not always
predispose for future asthma.
Introduction to the immune system
The immune system consists of innate and acquired immune defences. The
actions of macrophages and neutrophils, the phagocytes of the innate immune
system, do not depend on prior exposure to a particular antigen. They engulf and
degrade the microbe by phagocytosis. Complement, natural killer (NK) cells,
mast cells, basophiles and eosinophils also participate in the inflammatory
processes destined to destroy microbes (14, 49). Innate immune cells express
pathogen recognition receptors (PRRs), e.g. Toll like receptors (TLR),
recognising microbial associated molecular patterns, for instance
lipopolysaccharide from Gram- bacteria and lipoteichoic acid from Gram+
bacteria (50). TLR ligation induces secretion of cytokines including tumour
necrosis factor (TNF), IL-1β, IL-6, IL-10 and IL-12 and up regulation of cell
surface proteins (50).
- 24 -
The cells in the acquired immune system are T- and B-lymphocytes and they
respond to specific antigens and provide an immunologic memory. There are
two types of T lymphocytes: CD4+ (helper/regulatory) and CD8+cells
(cytotoxic) (14). B-lymphocytes that mature into plasma cells produce
antibodies; soluble molecules that can neutralise microbes and toxins, activate
complement, enhance phagocytosis and trigger cell degranulation.
Communication between the innate and the acquired immune systems is brought
about by cell-to-cell contact involving adhesion molecules and by the production
of chemical messengers (14). The immune responses vary between individuals
due to age, sex, smoking habits, alcohol consumption, menstrual cycle, stress
and infections, vaccination and early environment (49).
Immune components involved in allergic inflammation
The allergens are ingested, internalised and expressed as peptides bound to
major histocompatibility (MHC) molecules at the surface of antigen presenting
cells (APC). The APC present the antigen to T-lymphocytes which may promote
the transformation of B-lymphocytes to plasma cells that secrete IgE (23). Once
formed and released into the circulation, IgE binds to high affinity receptors on
mast cells, ready for future interaction with allergen (Fig. 2) (23).
T-cells and cytokines
The APCs carrying out the antigen presentation to naïve CD4+T cells are
dendritic cells (DC), principal regulators of T-cell memory function (51). The
antigen presentation takes place in the lymph nodes where the naïve T-cells
circulate. DC´s process protein antigens and express peptide fragments of the
antigen at the membrane with MHC class II molecules. To fully activate a naïve
T-cell, a second signal is needed; B7 on the APC that has been up regulated
binds to the CD28 on the T cell (Fig. 2) (52). T cell activation also requires
- 25 -
cytokines, a heterogeneous group of proteins that mediate intercellular signalling
and induce proliferation and differentiation. Cytokines are secreted from
different cells, such as activated antigen presenting cells, monocytes,
macrophages, mast cells and lymphocytes (53). Conventional CD4+ Th cells
control the adaptive immunity by activating, in an antigen-specific fashion, other
effector cells such as CD8+ cytotoxic T cells, B cells and macrophages (54).
Thus far, four CD4+ Th cell lineages are documented based on their cytokine
profiles and regulatory properties: namely Th2, Th1, Treg and Th17 cells (55).
Allergens that are presented to the naïve T cell makes it mature into a IL-4, IL-5,
IL-9, and IL-13 producing Th 2 lymphocyte (Fig. 2) (56). IL-4 and induces
production of IgE from plasma cells (53).This cytokine also contributes to the
differentiation of naïve Th lymphocytes toward a Th2 phenotype and prevent
apoptosis of Th2 lymphocytes (57). Another important activity of IL-4 in
allergic inflammation is its ability to induce expression of vascular cell adhesion
molecule 1 (VCAM-1). This produces enhanced adhesiveness of endothelium
for T cells, eosinophils, basophiles, and monocytes, but not neutrophils, as is
characteristic of Th2-mediated allergic reactions (58). IL-13 shares much of IL4‟s biologic activities on mononuclear phagocytic cells, endothelial cells,
epithelial cells, and B cells but it also induces mucus production and airway
hyper responsiveness (53). IL-5 is the most important cytokine for recruiting
and enhancing the survival of eosinophils and their progenitors and primes these
cells for activation and chemo taxis. Mast cells are recruited by IL-9 (56).
Antigens like bacteria and viruses induce IL-12 production from the APC and
the naïve T cell, which then stimulates CD4+ Th cells to a Th1 immune
response. This is characterised by production of interferon gamma (INFγ) and
TNF, with activation of macrophages and cytotoxic T- cells that eliminate
intracellular microbes (14).
- 26 -
IL-17
IL-21
IL-22
IL-10
TGFβ
Treg
IL-12
TGFβ
TGFβ
+ IL-6
Th17
CD 28
naive
Th cell
TCR
B
Th2
IL-4,
IL-13
Allergen
reexposure
IFNγ
TNF
Th1
IL-4
Allergen
peptide
CD4
Lipid
mediators
Mast
cell
eosinophils
Histamine
IL-4, IL-9,
IL-13, IL-5
basophils
VCAM
IgE
Plasma
cell
IL-5, IL-9,
IL-4, IL13
Figure 2: Schematic overview of the differentiation of Th cells with emphasis on allergic sensitisation. The allergen is
presented to the naïve T cell by the antigen-presenting cell (APC) and the T-cell receptor (TCR) binds to the Major
Histocompatibility Complex (MHC) class II and the peptide; CD4 stabilises this interaction and the local cytokine milieu is
essential for the differentiation. It is dominated by IL-4 in this example of Th2 differentiation (solid arrows) that causes IgE
27
production by plasma cells. Upon re-exposure of the allergen the mast cell is activated. Modified from (52, 53).
B7
APC
MHC II
There is a regulatory element in immune responses to allergens in healthy
individuals (14). It consists of regulatory T cells (Treg) cells that can suppress
Th2 responses to allergen, airway eosinophilia, mucous hyper secretion, and
airway hyper responsiveness, partly through the cytokines IL-10 and TGFβ.
Suppression by these cells may be decreased in allergic individuals (59).
Th17 cells produce many cytokines including IL-17A, IL-17F, IL-22 and IL-21.
In addition to their involvement in autoimmune diseases, Th17 cells also play
critical roles during immune responses against fungi and extra cellular bacteria,
e.g. via neutrophil activation (60).
Chemokines
Chemokines, i.e. small chemotactic proteins attracting immune cells, are
produced by macrophages, DCs, keratinocytes, bronchial epithelial cells and
fibroblasts (61). They are characterised by the presence of 3 to 4 conserved
cysteine residues and can be subdivided into 4 families based on the positioning
of the N-terminal cysteine residues: CC, CXC, C and CX3C (Fig. 3) (53).
C
C
C C
CX C
C
C
CC chemokine
CXC chemokine
Figure 3: Chemokines of the CC and CXC families. Black line: peptide chain.
Grey line: disulphide bond C: cystein residue, X: amino acid
- 28 -
The differential display of chemokine receptors by Th1 and Th2 subsets of T
lymphocytes after polarisation allows the cells to selectively respond to multiple
chemokines (62). The CC chemokine family has been extensively studied in
allergic diseases because of some of its members‟ ability to recruit eosinophils,
T-lymphocytes, and monocytes to regions of inflammation. The CXC
chemokines, on the other hand, target neutrophils (53). For example, the CCchemokine receptor (CCR) 3 and CCR4 are frequent on cells involved in the
allergic inflammation (63).
The chemokines analysed in this thesis are CXCL10, CXCL11, CCL17 and
CCL22 (Table 1). CCL17 and CCL22 bind to the CCR4 receptor on the Th2
cells (14). Up regulation of CCL17 and CCL22 has been associated with
presence and severity of eczema in children and adults (64, 65). Both IL-4 and
IL-13 promote CCL17 expression, amplifying Th2 responses (66). In addition,
nasal epithelial cells express CCL17, and expression of this chemokine and its
receptor, CCR4, was higher in patients with allergic rhinitis compared with nonallergic control subjects in one study (66). Furthermore, CCL17 and CCL22
levels clearly differentiate asthmatic children from non-atopic children with
chronic cough (67). On the other hand, the chemokines CXCL10 and CXCL11
are induced by IFNγ from Th1 cells and NK cells and act chemotactically on
Th1 cells by binding to their specific receptor CXCR3 (68).They are linked to
Th1-like diseases like Crohn´s disease (68). As CXCL10 also may be produced
in airway epithelial cells during the early phases after allergen exposure (69),
possibly induced by contact with the allergen (53), its role in allergic disease is
not completely clear. Nevertheless, a Th2 like deviation, i.e. increased plasma
levels of CCL22 and CCL22/CXCL10 ratios, in cord blood (63) and during the
first year (33) was associated to development of allergic disease in recent
studies.
- 29 -
Table 1. The chemokines analysed in this thesis, their corresponding receptors
and cells.
Receptor
Ligand
CCR4
CCL17
CCL22
CXCR3
CXCL10
CXCL11
Cells
Th2 lymphocyte
Dendritic cell
Natural killer cell
Mast cell
Induced by
Th1 lymphocyte
Mast cell
IFNγ
IL-4
IL-13
B cells and immunoglobulins
The immunoglobulins are produced by plasma cells derived from B cells. IgE,
IgA and IgG subtypes are linked to the allergic inflammation. IgE activates mast
cells and it is produced by plasma cells that have encountered a specific allergen
and have been stimulated by IL-4 and IL-13 from activated CD4+T helper cells
(Fig. 2) (70). The primary physiologic function of IgE might be to protect
against parasites as high levels of IgE are found in worm-infected subjects (14).
IgE is very biologically active but has a short half-life (<1d) when not bound to
mast cells and it is present in exceptionally low concentrations in the circulation.
However, IgE responses over long time can be attributed to the long life of IgE
producing B cells and stability of mast cells in the skin (70).
IgG responses to a range of purified house dust mite (HDM) allergens showed
that both IgG1 and IgG4 antibodies are present in sensitised individuals (71).
IgG1 activates complement and stimulates phagocytosis (70). IgG4 production
has been associated to high exposure to cat (70) and food antigens (72). It is
dependent of IL-4, a feature of Th2 responses (70), but is considered a
physiological response of the immune system (72) that has been associated to
tolerance (73, 74). The regulatory cytokine IL-10 can stimulate production of
IgG4 from B-cells while IgE production is decreased. In fact, data from
experimental animal studies have shown that high-dose allergen exposure
- 30 -
independent of the route of administration favours immune tolerance, while lowdose allergen exposure favours immune responsiveness. Observational
epidemiological studies in humans suggest that exposure to pets decreases the
risk of pet allergy, possibly involving this mechanism, reviewed in (75).
IgA exists in two forms: the monomeric form in blood and secretory IgA on
mucosal surfaces. The secretory IgA is dimeric and very resistant to acid and
enzymes of the gastrointestinal tract. It represents the primary line of protection
against incoming pathogens (e.g. allergen), preventing attachment to the
underlying epithelium (76). The very high concentration of secretory IgA in
human colostrum and milk strongly suggests that it must play an important role
in the immune protection of the newborn (76).
Eosinophils
Eosinophils are recruited to the allergic inflammation site and activated mainly
by the cytokine IL-5 and the chemokine CCL11 (eotaxin) (77). They adhere to
VCAM1 on endothelial cells, reach the tissues, degranulate and synthesise the
lipid metabolites LTC4 and platelet-activating factor (PAF). Eosinophilia in the
blood is present in severe allergic reactions (53). Eosinophils are important in
the chronic allergic inflammation and in the airways they can contribute to a
reconstruction of lung tissue, which may cause progressing hyper reactivity.
Attempts to reduce eosinophilia in humans by anti-IL-5 antibody therapy has
resulted in decreased eosinophilia and improved asthma control (77). This is,
however, not in clinical use.
Mast cells
The mast cells are situated around the vessels in the airways, the intestinal
mucosa, in connective tissue and the epidermis. They have receptors for the Fc
part of IgE on the surface, i.e. FcεR, as well as receptors for C3a from
- 31 -
complement activation and substance P from nerves. The mast cell is activated
through binding of any of these receptors or by heat, cold or pressure (14).
When the mast cell becomes activated, histamine is immediately released (Fig.
2) and makes plasma pass through the vessel walls and into the tissues. The
endothelial cells produce nitric oxide that dilates capillaries and venules and the
axon reflex, which involves substance P, also generates dilatation of the vessels.
The swelling increases over several hours. Eosinophilic granulocytes and T cells
are recruited from the bloodstream by chemokines and the cytokines IL-4, IL-5,
IL-9 and IL-13 are secreted by the mast cell (70). Phospholipase A2 is mobilised
when the mast cell is activated and it releases the arachidonic acid (AA) from
the intracellular membrane phospholipids. Lipoxygenase (LOX) and
cycloxygenase (COX) convert AA into eicosanoids; lipid mediators that
maintain allergic inflammation (78). These mediators are released to the tissues
several days after the activation of the mast cell and they contribute to the late
symptoms in the allergic reaction (14).
In summary, several cells and immune mediators are active in the allergic
inflammation. The cytokine, chemokine and eicosanoid responses associated
with allergic inflammation are considered Th2 deviated, as opposed to the Th1
deviated responses that take part in the defence against intracellular microbes.
The T regulatory cells are thought to balance the Th2/Th1 responses.
Intrauterine sensitisation and foetal immune responses
Maternal immune responses during pregnancy
As foetal tissue is regarded as semi-allogeneic, expressing human leukocyte
antigen (HLA) from the father, a successful pregnancy depends upon tolerance
of a genetically incompatible foetus by the maternal immune system (79). The
innate branch of the immune system is activated during pregnancy in healthy
- 32 -
women, possibly to compensate for the increased immunoregulation of the
adaptive immune system (80). The presence of maternal Th2/Treg cytokines,
such as IL-4, IL-10, and IL-13, during pregnancy is thought to suppress Th1mediated immunity, usually associated with transplant rejections, and to
promote acceptance of the foetus in the womb by the mother (81). In contrast,
the Th1 cytokine, IFN-γ, is an abortificant, whose effect may be mediated
through the promotion of cytotoxic lymphocyte and NK cell development (82,
83). Consequently, cytokine responses to phytohemagglutinin (PHA), allergen
extracts and lipopolysaccaride (LPS) are influenced by pregnancy regardless if
the mother is allergic or not. Further, there are findings that support the view of
hormones, especially progesterone, as critical regulators of the Treg populations
that influence the Th2/Th1 balance in pregnancy (84). Hence, lower IgE levels 2
years after pregnancy than during pregnancy have been detected in unselected
women (85). However, the levels of total IgE in early pregnancy were higher in
sensitised women with allergic symptoms than in healthy women in a recent
study, indicating that the Th2 deviation is augmented by allergic disease (86).
Combined information from old and new studies indicates that interaction
between Th1 and Th2 type cytokines is important for reproductive success (87).
The maternal CD4+ T-cell is assumed to play a central role in the interface
between maternal and foetal immune systems. T-cells release regulatory
cytokines (IL-4, IL-13, IL-10) that may cross the placenta and interact with the
foetus (82). Maternal cells themselves may also cross the placenta and cells
within the placenta may be stimulated to release various cytokines. The transfer
of maternal CD4+ Th2 skewed T cells across the placenta could potentially
skew foetal immune development toward a Th2 bias (88). The exact nature and
mechanism of this maternal influence and how it might be associated with the
development of allergic sensitisation and asthma in the child is not clear (82).
- 33 -
The foetal immune system
Stem cells are present in the human yolk sac at 21 days of gestation and the first
lymphocytes appear in the thymus at the end of the ninth week of gestation.
From 14 weeks, the lymphocytes can be seen in a range of organs, including the
lungs and gut, but the maturation of these cells occur in the third trimester of
pregnancy. By 19–20 weeks, circulating B-cells have detectable surface
immunoglobulin M (81). At delivery, the immune system is quite complete
although some cells, like phagocytes and dendritic cells, are not yet adequate in
number and function. The microbial flora colonising the gut facilitates the
expansion of the lymphoid population (89). If the neonatal immune system is
unable to down-regulate the pre-existing Th2 dominance effectively, then an
allergic phenotype may develop (79).
Early T cell responses to allergens
IgE and allergens might be amniotically transferred and ingested by the foetus
through the skin, respiratory tract, and gastrointestinal tract (90). The second
route of allergen exposure is by direct transfer across the placenta, possibly
mostly in complexed form with immunoglobulin G in the third semester of
pregnancy (91). Jones et al found that infants of mothers who were exposed to
birch pollen at 22 weeks of gestation or beyond have significantly higher cord
blood mononuclear cell (CBMC)-proliferative responses to birch pollen at the
time of birth than infants of mothers who had either not been exposed at all
during their pregnancy or whose exposure had been prior to 22 weeks. These
findings suggest that inhaled allergens might have crossed the placenta during a
critical period during foetal development and primed the infant T cells (92).
Allergen specific CD45 RA+ CD3 T cells have been found in cord blood (93,
94) but recent evidence suggests that they might be immature and cross reactive.
They are recent thymic emigrants that respond to antigens/allergens not
- 34 -
previously encountered and express altered antigen receptors that lack the fine
specificity of conventional T cells. These cells rapidly apoptose after allergen
triggered proliferation but have a greater chance of survival if IL-2 is present.
There is no correlation between cytokine pattern from recent thymic emigrants
in cord blood and future atopy (93, 95).
Generally the T cells from infants at high risk of developing atopy seem to
secrete less Th1 and Th2 cytokines than T cells from low risk individuals, the
reduction being more profound in Th1 cytokines (96), for example reduced IFNγ
production was associated to the development of eczema in a recent study (97).
This reduced capacity to generate Th1 polarising mediators, such as IL-12p70
from dendritic cells and macrophages (98) during infancy may compromise or
delay the transition to a Th1-competent immune response to allergens
(reviewed in (82)). Except for the influences of the maternal Th2 skewed
environment another possible explanation for this is that the neonatal T
lymphocytes have a reduced ability to activate mitogen-activated protein
kinases, which are crucial for cytokine production. This is associated with
reduced expression of several protein kinase C (PKC) isozymes. Variations in
PKC isozyme expression in CD4+Th cells lead to corresponding differences in
maturation characteristics, patterns of cytokine response, and disease
susceptibility. Lower levels of PKCzeta have been associated to allergic disease
(99).
Early specific IgE
The issue of intrauterine sensitisation is controversial. The foetal gut, with its
Peyers patches built up of T cells and other immune cells, is the principle route
by which sensitisation might occur through allergen in the amniotic fluid (100).
Intrauterine sensitisation may have evolved to facilitate enhanced neonatal host
response to maternal helminths. Indeed, infants born to helminth infected
- 35 -
mothers have Th2 biased immune responses to helminth antigen and IgE
antibodies to these antigens (81). Contrarily, recent studies indicate that allergen
specific IgE in cord blood does not reflect intrauterine sensitisation. For
example, allergen specific IgE was found in 14% of cord blood samples from
mothers with asthma and specific IgE in cord blood completely matched specific
IgE in maternal blood with respect to allergen specificity and level of specific
IgE. The IgE was no longer detectable at 6 months (n=411) and small placental
bleedings during late pregnancy or delivery may be a plausible mechanism
(101). These findings are supported by Bertino et al (102) but not by Nambu et
al (103).
However, epidemiological studies show that prenatal exposure to stables or farm
life is protective from inhalant allergies (104, 105). The nature of this response
may be regulated by the maternal environment, particularly the mother's
IgE/IgG ratio (106). For instance, protective specific IgG4 antibodies may cross
the placenta after maternal allergen exposure (107). After birth, sensitisation to
foods in infants without food related symptoms seem to be a common
phenomenon (17). Children who remain non-allergic produce detectable but low
levels of specific IgE up to 12 months of age before returning to baseline (95),
whereas the IgE levels reach higher and more persistent levels in those who
develop allergy (21).
Total IgE in cord blood
Some infants destined to have allergic disease have raised total IgE in cord
blood. This has proved to be a specific but very insensitive marker of later
disease (108, 109). In average, 23 % of cord blood samples in a group of women
from Taiwan (n=334) had levels above 0.35 IU/ml. This was associated with
maternal total serum IgE > 100 IU/l one month before delivery, maternal allergy
to dog dander and atopy in maternal grand parents (110). Maternal history of
- 36 -
asthma was shown to be the most important determinant for high cord blood
total IgE in two different studies. There was no relationship with paternal
asthma, demonstrating the impact of the maternal environment (111, 112).
Maternal atopic history, elevated total IgE levels and allergic sensitisation were
associated with infants with elevated CB IgE levels and infantile eczema (113).
A predictive value has been shown for cord blood IgE for urticaria due to food
allergy at 12 months (111), atopy at 18 months (114) and asthma at 11 years
(109). Other studies have shown no associations between cord blood total IgE
and future sensitisation and allergic symptoms in the child (108). The question
remains if cord blood IgE levels merely reflect the maternal atopic status or if
the immune responses in the child are influenced by the IgE antibodies in cord
blood (81).
The mechanisms are not completely clear and some results are contradictory on
the subject of intrauterine sensitisation and immune responses of the foetus.
There is epidemiological evidence that pre- and perinatal events like low
maternal vitamin E (115) and fish (116, 117) intake in pregnancy as well as
smoking in pregnancy (118) and delivery by caesarean section (119, 120) all
increase the risk of sensitisation and allergic disease in childhood. This indicates
that future sensitisation and allergic disease is associated with some kind of preand perinatal immune programming.
In summary, all infants are born with a somewhat Th2 skewed immune response
but infants who develop allergic disease seem to have less Th1 immune
responses (IFNγ and IL-12) at birth and more sustained and elevated production
of IgE antibodies (Th2) than healthy infants. This immune deviation might be
influenced by early environmental factors through mechanisms that need further
characterisation.
- 37 -
Prevention of allergic disease
As discussed earlier, the intrauterine environment seems to have an impact on
the risk of developing allergic disease. Interestingly though, studies on adopted
children who move to industrialised countries before 2 years of age indicate that
they develop allergies to the same extent as children born in the country as
opposed to children who move here later in life (14), which indicates
environmental effects during the first years of life. The hypothesis of an
environmentally based deviation of the immune response towards a Th2 and
away from a Th1 response in allergic disease is questionable from an
epidemiological point of view. The occurrence of Th1 diseases such as type 1
diabetes is positively rather than negatively associated with asthma (121) and
both Th1 and Th2 diseases have increased under the same environmental
influences (122). A lack of Treg cells might be the factor of importance as they
may suppress Th2 and Th1 immune responses through IL-10 (123).
On the other hand allergic disease is a hereditary disorder. When both parents
have the same expression of atopic disease, their child has a 70% risk of
developing the same manifestations. If both parents are atopic there is a 30-50%
risk for the child to develop atopic disease (124-126). Ten percent of children
without atopic heredity and 30% with a single heredity develop disease. As
many as around 30% of newborns have at least one parent/ or older sibling with
previous or current atopic disease (124, 126).
Apart from the genetic predisposition for allergic disease, there are mechanisms
for interaction between genes and environment, i.e. epigenetic mechanisms and
gene-by-environment interaction that have an impact on the individual risk of
developing allergies. The term epigenetics is defined as environmentally caused
changes in gene expression, inherited in the absence of mutations in the DNA
- 38 -
sequence as well as the event that initiated the change. DNA methylation
(binding of a methylene group to a DNA CpG dinucleotide), and methylation,
acetylation or phosphorylation of histones and chromatin are examples of
epigenetic modifications. Factors that may have an impact on the epigenetic
mechanisms are nutrition, toxic chemicals, radiation, exposure to air pollution
and tobacco smoke (127). Gene-by-environment interaction, on the other hand,
describes that different genotypes imply different susceptibility to environmental
factors and their ability to protect from or increase the risk of disease (128, 129).
In conclusion: when working with prevention of allergic disease, knowledge of
genetic and environmental influences, as well as their complex interaction, is
essential.
Environmental factors
There are several environmental factors that have been considered regarding
their impact on the development of allergic disease. The most studied ones are
early allergen exposure, smoking and air pollution, psychological factors,
microbial exposure, respiratory infections, use of antibiotics and maternal and
infant dietary content including micronutrients. They are each discussed
separately below and for some of them gene-by-environment interaction
mechanisms have been reported.
Early allergen exposure
Inhalants
Early-life exposure to pets or lifestyle factors associated with exposure to pets
seem to reduce the risk of developing atopy-related diseases in early childhood.
However, these findings might also be explained by selection for keeping pets
(130). There is a dose dependent relationship between early exposure to
aeroallergens, e.g. HDM, and sensitisation and asthma in many but not all
- 39 -
studies (126). Exposure to HDM antigen together with dampness at home was a
significant risk factor for the persistence of bronchial hyper reactivity and
respiratory symptoms in children with asthma in one study (131). In another
report, the level of prenatal exposure to Dermatophagoides pteronyssinus (Der
p) 1 influenced the immune profile of cord blood T lymphocytes and the
prevalence of eczema in early life (132). Yet, prevention studies addressing the
environmental issue with drastic avoidance measures show disappointing results
(133).
Foods
Repeated intervention studies have not shown a protective effect of an allergenpoor diet on the part of the mother during pregnancy and lactation (134-136). On
the contaty, recent findings indicate that early introduction to food allergen, at 4
months rather than at 6 months, can be protective of allergic disease (discussed
in (137)).Høst et al state that the most effective dietary regimen for the child at
high risk of developing allergic disease is exclusive breastfeeding for at least 4-6
months or, in absence of breast milk, formulas with documented reduced
allergenicity for at least the first 4 months, combined with avoidance of solid
food and cow's milk for the first 4 months (138). This, however, is questioned in
a recent update where the authors declare that hypoallergenic formula does not
prevent the allergic symptoms, it merely delays them (139). Consequently, the
Swedish Paediatric Society recommends extensively hydrolyzed formulas for
the first 4 months in a very limited number of cases; only for infants in families
with at least two family members with documented severe and long lasting
allergic disease and where breast-feeding is not sufficient (144).
Smoking and air pollution
Smoking during pregnancy can increase recurrent wheezing during infancy
(reviewed in (126)) but the possible effect on allergic sensitisation is
- 40 -
contradictory (118, 140). Thus, if passive smoking early in life has any impact
on allergies, it is likely to increase the severity of disease in those who have
additional factors promoting the development of allergic sensitisation (140). The
Glutation S-transferase (GST) –enzymes (GSTM1, GSTP1 and GSTT1) protect
cells from toxic substances and oxidative stress. In some genotypes, these
proteins do not function properly and there is an increased risk of damage to the
airway epithelium after exposure of air pollution and second hand smoke (141).
There are indications that children with the GSTP1-genotype (valin 105) have
an increased risk of allergic disease in areas with high air pollution (142) and
children with a GSTM1-deletion have a susceptibility to develop asthma from
maternal smoking during pregnancy (143).
Psychological factors
Psychological stress can influence the balance between Th1 and Th2 cytokines
which might strengthen the allergic inflammation especially in allergic
individuals (144). A meta analysis revealed a robust relationship between
psychosocial factors and atopic disorders. In addition to conventional physical
and pharmacological interventions, psychological intervention might be
beneficial for successful prevention and management of atopic disorders (144).
Microbial components - the hygiene hypothesis
Epidemiological studies suggest that microbial components in the environment
early in life are mediators of allergy-preventing effects (145). For example, the
International Study of Asthma and Allergies in Childhood (ISAAC),
including more than 200 000 children, found that an increase in the tuberculosis
notification rate was associated with an absolute decrease in wheeze.
Mycobacterial notification rate served here as a surrogate marker for
mycobacterial exposure (146). Increased levels of microbial exposures
- 41 -
recognized by innate immune cells may affect adaptive immune responses
resulting in decreased risk of atopic sensitisation and asthma (104).
Poverty, multiple older siblings and growing up on a farm with animals reduce
the risk of developing allergy with good evidence (14, 104), maybe due to
increased microbial exposure. A number of gene-by-environment interactions
have been observed with polymorphisms in genes of innate immunity receptors
and exposure to farm surroundings. One example involves the CD14-gene,
coding for a receptor that influence immune responses towards endotoxin that is
frequent in farming environment. The protective effect on the development of
asthma and allergies from drinking farm milk seems to be influenced by
variations of this gene (147). Some studies (33, 148), but not all (149) find a
protective effect from day-care on different allergic manifestations and there are
indications of genotype specific responses (150).
It was suggested that changes in the microbial gut flora may mediate changes in
the prevalence of atopy (151). Delivery by caesarean section has been found to
be associated with a moderately increased risk of allergic rhinitis, asthma,
hospitalisation for asthma, and perhaps food allergy (119), but not with eczema
(120). These findings demonstrate that the mode of delivery may have
significant effects on immunological functions in the infant, possibly via gut
micro biota development. Trials using supplementation with different strains of
probiotics early in life have been performed in order to enrich the gut flora and
prevent allergic disease and the results have been slightly favourable (152, 153).
Infections - Respiratory Syncytial Virus (RSV).
Whether the RSV bronchiolitis is a risk factor for allergic sensitisation and
asthma is a matter of debate (154, 155). RSV infection that is severe enough to
require hospitalisation does not cause asthma but is an indicator of the genetic
- 42 -
predisposition to asthma, is the conclusion of a recently published twin study
(156). A deficient cytokine response in relation to infection with RSV has been
proposed as an indicator of host susceptibility to severe disease. Several
polymorphisms in cytokine genes have been proposed. In particular,
polymorphisms in the IL-4, IL-10, IL-13, CCR5, TGFβ, and TLR-4 genes have
been related to RSV severity (157).
Antibiotics
Early life exposure to broad-spectrum antibiotics may have a causative role in
sensitisation in combination with expression of wheeze (158). In the KOALA
birth cohort in the Netherlands, early antibiotic use preceded the manifestation
of wheeze but not eczema or allergic sensitisation during the first 2 years of life
(159). The effect of antibiotics on respiratory disease may though be due to
confounding by chest infections at an early age when asthma may be
indistinguishable from infection (160).
Protective factors in the diet of mother and child
Breast feeding
In a recent meta analysis of 21 studies there was no strong evidence of a
protective effect of exclusive breastfeeding for at least 3 months against eczema,
even among children with a positive family history (161). In another meta
analysis the conclusion was that breast-feeding fully for 3 months and partly for
3-4 months reduces the risk of eczema in children with atopic heredity during
the first 4 years of life (162). This is supported by observational studies from a
Swedish group where at least 4 months of breast feeding offered protection from
eczema up to 4 years (163), as well as asthma and sensitisation up to 8 years of
age (164). However, there are also opposing results (165, 166). A possible
preventive effect of breastfeeding on the development of allergic disease might
be due to either a protective effect of human milk or avoidance of high dose cow
- 43 -
milk protein, or a combination of these factors. There is also a risk of reverse
caution in some studies: mothers in families with a high risk of allergy
breastfeed longer (126). The diverse results from the studies could be caused by
the varying composition of breast milk and individual susceptibility to the
protective factors in the milk. In line with this, the protective effect of
breastfeeding on eczema in children was found in non-allergic mothers but not
in allergic mothers in the Dutch KOALA Birth Cohort Study (167).
Maternal diet
There might be components of the maternal diet in pregnancy that protect from
development of allergies. For example adequate intake of anti-oxidants (168),
apples (117) and fish (116, 117) have been associated with lower risk of allergic
disease in the child. Vitamin E is an antioxidant which has been reported to
possibly decrease the risk for asthma, eczema (168) and sensitisation (169) in
childhood. Murine studies indicate that dietary folate intake in pregnancy can
alter the allergic predisposition through epigenetic mechanisms (170). The
possible preventive effects of fish are discussed further on in this thesis.
In summary: so far no special diet in pregnancy or lactation is recommended in
Sweden, in order to prevent allergic disease. Very high-risk children, who have
two family members with severe allergic disease, may benefit from
hypoallergenic formula before 4 months of age in absence of breastfeeding
(primary prevention). Children with established allergic disease should avoid
tobacco smoke and exposure to relevant allergens (secondary prevention)
(171). For the general health of the children, the Swedish primary childcare
advice all families to avoid tobacco smoke during pregnancy and lactation and
to breastfeed exclusively for 4-6 months followed by the introduction of solid
foods. Cows milk formula is the supplement of choice for most infants not fully
breast-fed.
- 44 -
Long chain polyunsaturated fatty acids
As early as in the 1940´s a relationship between atopic eczema and abnormal
serum essential fatty acids was found (172). Fatty acids are long carbon chains
with a methylene at one side and a carboxyl at the other side. The number of
carbons varies and there are one or multiple double bonds between the carbons.
Polyunsaturated fatty acids (PUFAs) contain multiple double bonds and the two
families of essential PUFAs; ω-6 and ω-3 are named according to the position of
the terminal double bond at the methylene end of the chain. Each fatty acid also
has the number of carbons in the chain and the number of double bonds in its
denomination.
COOH
5
2
4
3
1
CH3
eicosapentaenoic acid (EPA)
Figure 4: C20: 5 ω-3 - 20 carbons, 5 double bonds, and the terminal double
bond is situated at the 3rd carbon counted from the methylene end.
The essential fatty acids, which have to be provided from the diet, are linoleic
acid (LA, 18:2ω-6) and α-linolenic acid (LNA, 18:3ω-3). By insertion of
additional double bonds into, and elongation of, the acyl chain, long chain
PUFA (LCPUFA) can be produced from these fatty acids (173). The metabolic
steps take place in the liver cells, mainly in the endoplasmatic reticulum, except
for the last step that requires β oxidation in the peroxisomes (174) (Fig.5).
- 45 -
PUFA comprise 15% of dietary fat in the European diets and 5-6% of the total
energy intake (175, 176). LA comprises 85-90 % of the PUFA intake, and LNA
about 10 % while AA, DHA and EPA contribute to less than a few percent of
this according to data from the diets of women in Australia and Belgium (176,
177). All vegetable oils are rich in LA while rapeseed oil and linseed oil also
contain significant amounts of LNA. Additionally, walnuts and green plants are
important sources of LNA. Meat and eggs provide high quantities of AA while
DHA and EPA are abundant in seafood and fatty fish like herring, mackerel,
salmon and tuna.
ω-6 PUFA
ω-3 PUFA
enzymes
18:3 ω-3 LNA
18:2 ω-6 LA
Δ -6-desaturase
18:4 ω-3
18:3 ω-6
elongase
20:4 ω-3
20:3 ω-6
Δ-5-desaturase
20:4 ω-6 AA
20:5 ω-3 EPA
ER
elongase
22:5 ω-3
22:4 ω-6
elongase
24:5 ω-3
24:4 ω-6
Δ -6-desaturase
24:6 ω-3
24:5 ω-6
peroxisome
ß-oxidation
22:5 ω-6
22:6 ω-3 DHA
Figure 5. Conversion of LA and LNA into long chain polyunsaturated fatty
acids (LCPUFA). LA: linoleic acid, AA: arachidonic acid, LNA: α-linolenic
acid, EPA: eicosapentaenoic acid, DHA: docosahexaenoic acid, ER:
endoplasmatic reticulum
- 46 -
The ratio of ω-6 to ω-3 has increased in the northern European diet during the
last half of the 20th century, mainly because of increased intake of vegetable oils
and decreased intake of fish oil (178). The ratio used to be close to 1:1 (ω-6/ω-3)
(178) but is currently between 5:1 and 20:1 (179). An additional explanation to
this increase is that meat and dairy products from ruminant animals contain less
ω-3 fatty acids (i.e. LNA) nowadays because of the practice of feeding them
concentrated cereal grain-based feed rather than green grass (178).
On the other hand, the conversion of LNA into EPA and DHA in the human
body is quite inefficient as less than 10 % of LNA is converted to DHA in adults
(180). In women of reproductive age and in neonates, particularly prematures,
this process may be a little more proficient (181, 182). In adults DHA is most
abundant in retina, sperm and the cerebral cortex while AA can be found mostly
in red blood cells, spleen and liver (180). The elongation and desaturation of
PUFA seems very important. In a patient, with reduced Δ-6-desaturase activity,
there were skin abnormalities, growth failure, corneal ulcerations and feeding
intolerance shortly after birth and this was corrected with LCPUFA
supplementation (183).
In pregnancy, fatty acids are released from maternal triglycerides by lipoprotein
lipase on the maternal surface of the placenta and bound to plasma membrane
fatty acid binding protein (p- FABPpm) and cytoplasmatic transport proteins. In
order to meet the foetal demand, the placental transfer is selective and results in
higher levels of both DHA and AA in cord blood compared to maternal blood
(184). Omega-3 LCPUFA are incorporated into phospholipids and sphingolipids
in cell membranes of primarily the liver and the brain during the last trimester of
pregnancy (180) and they are important for growth of the foetus and
development of the CNS and the retina. LCPUFA status in the child is related to
gestational age at birth, maternal diet and maternal LCPUFA status in
- 47 -
phospholipids (185). There is evidence that the maternal requirements of DHA
in pregnancy and lactation are not always met by our western diet and the
maternal stores are not easily replenished after pregnancy (186, 187).
Consequently, women who are pregnant with their second or third child have
less available ω-3 fatty acids, especially DHA (186, 188).
In summary, the ω-3 LCPUFAs DHA and EPA are abundant in oily fish and
especially DHA is important for foetal development. There is a selective
transport of DHA across the placenta to the foetus.
Fish oil intake early in life and infant allergic disease
Observational studies indicate that fish consumption 2-3 times a week during
pregnancy reduces the risk of atopic eczema and sensitisation in the children at
one year (116) and atopic eczema at five years of age (117). Fish consumption
during the first year of life may also reduce the risk of allergic rhinitis (189) and
any allergic disease at 4 years of age (190). There are however a few studies that
show negative effects or no effect at all from fish consumption on allergic
disease in childhood (reviewed in (191). Low levels of EPA and DHA and a
high ratio of AA /EPA in maternal milk were reported to relate to increased risk
of allergic disease during the first 18 months of life (192).
These findings have encouraged the performance of supplementation studies.
Due to the inefficient conversion of LNA into LCPUFA, the most proficient
way of substituting EPA and DHA is by giving the pure substance, not a
precursor (193). Trials with EPA and DHA supplementation in pregnancy have
thus resulted in increased levels of these LCPUFAs in both maternal and infant
plasma phospholipids (194, 195) as well as in cord blood erythrocytes (196).
- 48 -
Supplementation with EPA and DHA to adults led to a decreased ratio of
AA/EPA acid in the membrane phospholipids of mononuclear cells (197).
Clinical outcomes
Dunstan et al reported beneficial effects on the severity of infant eczema
(SCORAD≥25, p=0.045) and SPT to egg (p=0.055) at one year of age after
supplementing 98 atopic mothers with 3.3 g EPA and DHA during the last half
of pregnancy. The positive effect was noted even though the study was not
designed to analyse clinical outcomes (9).
The Danish study of Olsen et al reported that fish oil supplementation in late
pregnancy is associated with a marked reduction in allergic asthma in the
offspring at age 16 years of age compared to placebo (olive oil), suggesting a
long-term effect of any immunologic change that occurred in pregnancy and
early life of those children. Unexpectedly, the group who did not take any
supplementation had a risk of asthma similar to the ω-3 supplemented group.
The study was originally designed to investigate pregnancy related effects of the
supplementation (198).
In the Australian Childhood Asthma Prevention Study (CAPS), cooking oils and
spreads with low ω-6 content starting in the last month of pregnancy and tuna
fish oil supplementation (500 mg/d starting at weaning or at 6 m of age)
decreased the prevalence of wheeze at 18 months of age. High plasma ω-3
PUFA levels were associated with less bronchodilator use, irrespective of the
supplementation group but there was no effect on sensitisation (199, 200).
Follow-up at 3 years showed that the atopic children in the fish oil group had a
reduced prevalence of cough, but not wheeze (201). However, no effect of fish
oil was seen on the other endpoints: eczema, serum IgE concentration or doctor
diagnosis of asthma. At 5 years of age, there was no significant effect of fish oil
- 49 -
on any of the clinical outcomes relating to lung function, allergy, or asthma
(202). At 5 years of age the children stopped taking the supplementation but the
children were examined at 8 eight years of age and no significant effects on
atopic disorders sere reported (203). The possible reasons for the lack of
beneficial effects of long-chain ω-3 PUFAs after 3 years of age may be related
to suboptimal adherence to and/or implementation of the intervention (50% and
56% compliance in the intervention and control group, respectively), as well as
to the low dose of fish oil used. The intervention started in late pregnancy and
this may have been too late to have an impact on the development of the foetal
immune system.
Immunological outcomes
High intakes of ω-3 LCPUFA suppress a wide range of immune variables such
as lymph proliferation (204-206), CD4+ cells antigen presentation (207, 208),
adhesion molecule expression (209) as well as proinflammatory cytokine (206)
and eicosanoid production (205, 210) in humans and rodents. A few human
studies of these effects early in life were performed:
An observational study reported that increased levels of cord blood EPA and AA
in cell membranes attenuated cord blood lymphocyte proliferation and decreased
allergen stimulated IFNγ secretion. A lower AA/EPA ratio was also associated
to reduced IFNγ secretion (211), indicating that the balance between ω-6 and ω3 fatty acids is important.
In a ω-3 supplementation study of pregnant women, lower neonatal IL-13 (Th2
related cytokine) levels were noted in the ω-3 group (8) and there was a trend
towards lower IL-5, IL-13, IL-10 and IFNγ responses to all allergens in the fish
oil group (9). Further, fish oil supplementation modulated expression of infant T
cell PKC zeta, one of the mitogen-activated protein kinases, which are crucial
- 50 -
for cytokine production, in the neonatal period. Allergic disease is associated
with a decreased expression of PKC zeta (99).
The Danish study of maternal fish oil supplementation during lactation (212)
investigated immune outcomes in the offspring. Infants of lactating mothers who
received fish oil supplementation had a higher IFNγ production at 2.5 years of
age compared to children whose mothers had received olive oil as a placebo, an
observation that may reflect faster maturation of the immune system.
The “early-in-life” ω-3 LCPUFA supplementation trials reporting
immunological outcomes in maternal and infant blood and clinical outcomes in
the infants are summarised in Table 9.
Breast milk
Maternal ω-3 LCPUFA supplementation increases the content of these lipids in
breast milk (213, 214) but a low dose did not alter milk cytokine content (214).
After supplementation with 3.3 g ω-3 LCPUFA daily during pregnancy, the
levels of S-IgA and soluble CD14 in breast milk were increased (213), which
may have allergy preventing effects by influencing immunological responses in
the intestine of the infant (215). Furthermore, addition of small amounts of AA
and DHA (close to the content in human milk) to formula for preterm babies
may influence the concentration, proportion, maturation and production of
peripheral blood lymphocytes in the children (216).
In summary: Fish in the diet during pregnancy and lactation has been
associated to decreased risk of allergic disease in the early years. The fish oil
supplementation studies performed so far indicate a slight protective effect but
the studies have either not been designed to investigate the clinical outcomes or
- 51 -
used very small doses of fish oil. Nevertheless, they point out that ω-3 LCPUFA
in pregnancy and lactation have effects on neonatal immune responses.
Immune modulating mechanisms.
The immune modulating actions of long chain ω-3 PUFAs are executed at the
gene level, in the cell membranes, and in the metabolism of lipid mediators
(217) and it is not clear which mechanism is the most important. The previously
mentioned effects on cytokines, cell functions and clinical symptoms may be
executed through these different mechanisms.
Gene level
Omega-3 LCPUFAs have been reported to decrease the activity of inflammatory
genes such as COX 2 and VCAM-1 through modification of nuclear factor κβ
(NFκβ) activity, and thereby direct alteration of gene expression (218, 219). The
ω-3 LCPUFA are also natural ligands of nuclear receptors such as peroxisome
proliferators activated receptors (PPAR) α and γ that are transcription factors in
inflammatory cells (218). These implications on gene expression may contribute
to possible epigenetic changes by ω-3 LCPUFA.
Membrane fluidity
AA, and to a lesser extent LA, are major fatty acid components of lymphocyte
membrane phospholipids. For example, CD4+ T cells, which play a central and
critical role in immune regulation, have a high proportion of membrane AA
(25%), but very little DHA (4%) (210). Recent data indicate that the
organisation and trafficking of LCPUFAs play an important role in the
stabilisation of cell membrane lipid rafts and the acylation of membrane proteins
involved in cell regulation. Lipid rafts are small domains of the plasma
- 52 -
membrane that are platforms for cell activation and signalling between cells
(220). Th1 cell activation, opposite to Th2 cell activation, is proposed to be
dependent on lipid rafts. Increased ω-3 LCPUFA proportions in cell membranes,
which can be obtained by a fish oil rich diet, alter membrane fluidity and
modulate lipid raft composition and hereby may influence T cell responses (197,
217, 219), supposedly by down regulation of Th1 responses (221). Additionally,
increased content of ω-3 LCPUFA in the APC cell membranes may reduce the
capacity to present antigens by inhibiting the up-regulation of MHC class II
receptors, cytokines and co stimulatory molecules in murine cells (207, 208).
This has been difficult to prove in neonatal APCs probably due to their
immaturity and reduced antigen presenting capacity (222).
Lipid mediators
When an inflammatory reaction takes place, AA in the cell membranes is
released by the enzyme phospholipase A2. Further on, eicosanoids are
synthesised through the enzymes COX, that induces production of
prostaglandins and tromboxanes, and LOX that generates leukotrienes. COX is
present in all types of cells but LOX only in inflammatory cells like monocytes,
macrophages, mast cells and granulocytes (14). Lipid mediators like
prostaglandins and leukotrienes are also formed from EPA in the cell
membranes. However, the membrane fatty acid composition influences the
production of lipid mediators in the cells (223).
Omega-6 lipid mediators
Eicosanoids formed from AA are the two-series of prostaglandins (e.g. PGD2
and PGE2), and the four-series of leukotrienes (e.g. LTB4). Lipoxines are also
formed from AA through the 5-LOX pathway and they are active in the late
phase of inflammation resolution (224).
- 53 -
Activated endothelial cells and macrophages release PGE2, that can inhibit T
cell proliferation (225), regulate APC function, inhibit IL-1, IL-2, IL-6 and IFNγ
and possibly enhance the production of IL-4 and IL-5 (226). PGE2 also
stimulates the production of IgG1 and IgE antibodies in LPS and IL4 stimulated
mature B cells (225). In line with this, it has been reported that PGE2 enhances
antigen-specific IgE responses in murine lymphocytes (227). On the other hand
PGE2 is suggested to be protective against airway inflammation (228). It is
possible that PGE2 promotes sensitisation via its effect on T- and B-cells but is
protective against the subsequent manifestations of inflammation (191).
Membrane phospholipids
Phospholipase A2
arachidonic acid
AA
COX1/
COX 2
5-LOX
eicosapentaenoic acid docosahexaenoic acid
EPA
DHA
COX1/
COX 2 COX 2
5-LOX
COX 2
5-LOX
Lipoxines
LTB5
LTB4
LTC4
Protectines
E Resolvins
D Resolvins
PGE2
PGD2
TXA2
PGE3
PGI3
TXA3
Figure 6. AA, EPA and DHA are released from cell membranes and form
substrates for cyclooxygenases (COX) and 5- lipoxygenase (5-LOX). PGE:
prostaglandin E, TXA: Thromboxane, LT: Leukotriene. Solid lines: pro
- 54line:
inflammatory mediators, striped
less inflammatory mediators, dotted line:
anti-inflammatory mediators. Modified from (14, 174).
PGD2 is released from mast cells and causes bronchoconstriction, dilatation of
vessels and chemotaxis for inflammatory cells. It also suppresses activation of
neutrophil granulocytes (14).LTB4, released from macrophages and neutrophils,
and LTC4 from eosinophils and mast cells, increase vascular blood
permeability, enhance local blood flow and stimulate mucus secretion and
constriction of the airways (14). They are also chemotactic for leukocytes and
promote cytokine production, such as TNFα, IL1, IL6, IL2, IFNγ (229).
Omega-3 lipid mediators
Eicosanoids, such as PGE3 and LTB5, are formed from EPA in the cell
membranes. It has been assumed that these mediators have lower biological
potency than those formed from AA (230). Yet, more recent results indicate that
PGE3 and PGE2 may have equivalent inhibitory effects upon production of
TNF and IL-1 and consequently, EPA and AA derived eicosanoids do not
always have different potencies (223). However, there are recently revealed
mediators formed from DHA and EPA through COX2 and 5-LOX that have
anti-inflammatory properties, i.e. resolvins and protectines (Fig. 6) (231). They
block production of inflammatory mediators and stop infiltration of
polymorphonuclear cells and thereby help resolve inflammation (224). This
indicates that DHA might have similar potency as EPA as an anti-inflammatory
fatty acid (232).
In summary, when the availability of DHA and EPA is increased, i.e. by a diet
rich in fish oil this might influence gene expression, modify immune cell
responses through altering the composition of the cell membranes and decrease
the eicosanoid production from AA in favour of lipid mediators from EPA and
DHA. These mechanisms together or separately might prevent the development
of allergic sensitisation and hamper the allergic inflammation.
- 55 -
Gene-by-environment interaction
The fatty acid desaturases genes (FADS) 1 and 2, code for the rate limiting
PUFA desaturases Δ-5 desaturase and Δ-6 desaturase respectively (Fig. 5) (233,
234). Genetic variants of FADS1/FADS2 influence breast milk essential fatty
acid composition and plasma phospholipid content during pregnancy (235). It
has been shown that the biological effects of LCPUFAs differ between
individuals or population subgroups and this may partly depend on different
polymorphisms in the FADS genes (174, 236, 237). For example, Caspi et al
studied the association between breastfeeding and IQ in children with different
polymorphisms of the FADS1/FADS2 genes. Children carrying the C allele
inrs174575 polymorphism had marked advantage in IQ due to breastfeeding
(and supposedly its content of LCPUFAs) while children without this allele had
no advantage from breastfeeding (237).
Similar mechanisms might contribute to the diverse data on the protective effect
from breast milk on allergic disease. Allergic disease and sensitisation have been
associated with a disturbed fatty acid metabolism in maternal blood (238) and
low ω-3 LCPUFA in mature breast milk (192). It has also been linked to the
same region in chromosome 11 as the FADS1/FADS2 genes (233). Moreover, a
lower prevalence of allergic rhinitis and eczema was associated to several single
nucleotide polymorphisms in the FADS genes. However, these associations
were not significant after correction for multiple testing (234).
In summary: The biological effects of ω-3 LCPUFA might differ between
subgroups of individuals due to gene-by-environment interaction. In future
studies of the effects of ω-3 LCPUFA, gene analyses would be useful.
- 56 -
AIMS AND HYPOTHESIS
The general aim of this thesis was to assess the effect of maternal dietary
supplementation with ω-3 LCPUFA, i.e. fish oil, in pregnancy and lactation on
the development of allergic symptoms and sensitisation in the infants and to
explore the associations between some maternal and infant immunological
markers and the ω-3 LCPUFA supplementation.
Specific aim and hypothesis of each paper:
I. To evaluate the effect of ω-3 LCPUFA supplementation in pregnancy on
maternal eicosanoid and cytokine production. We hypothesise that PGE2 and
LTB4 as well as the proinflammatory cytokines IL-1, IL-6 and TNF will
decrease during ω-3 LCPUFA supplementation.
II. To assess the effect of ω-3 LCPUFA supplementation during pregnancy and
lactation on the risk of allergic sensitisation, food reactions and eczema during
the first year of life. We hypothesise that the ω-3 LCPUFA supplementation will
decrease the incidence of these clinical outcomes.
III. First, to investigate the effect of ω-3 LCPUFA supplementation during
pregnancy and lactation on the risk of allergic sensitisation and disease during
the first two years of life. Second, to explore the relationship between the
proportions of DHA, EPA and the AA/EPA ratios in maternal and infant
serum/plasma phospholipids and the frequency of IgE associated disease as well
as the severity of eczema and number of allergic symptoms in the infants. We
hypothesise that the ω-3 LCPUFA supplementation will decrease the incidence
of the clinical outcomes. We also believe that high maternal and infant
proportion of ω-3 LCPUFA will be associated with low prevalence of allergic
- 57 -
symptoms and sensitisation, lower SCORAD index in eczematous children and
a reduced number of allergic symptoms.
IV. To relate circulating Th2- and Th1-associated chemokines and IgG antibody
responses to tetanus and diphtheria vaccines to the ω-3 fatty acid
supplementation and allergic disease in mother and child. We hypothesise that
the infant Th2 chemokines will be suppressed by the ω-3 LCPUFA
supplementation in favour of Th1 chemokines, especially in infants of nonallergic mothers. We also believe that the vaccine-induced responses will be
strengthened by the ω-3 LCPUFA supplementation.
- 58 -
MATERIALS AND METHODS
Inclusion of participating families
All papers in this thesis are based on a prospective double blind placebo
controlled randomised trial conducted at the Department of Paediatrics at the
University hospital in Linköping and in the county hospital of Jönköping, all in
South Eastern Sweden. 185 families were willing to participate when asked at
the antenatal clinic between March 2003 and May 2005. The inclusion criteria
were allergic disease in at least one family member (mother, father and/or
sibling of the future child) and an intention to breastfeed the child for at least
three months. The mothers had to be included before gestational week 25. A
structured interview by one of three experienced allergy nurses covered the
family history of eczema, asthma, allergic urticaria, gastrointestinal allergy or
allergic rhinoconjunctivits before inclusion of the family.
Women with allergy to soy or fish, those under treatment with anticoagulants
and those who were unwilling to stop taking their own fish oil supplements were
not included in the study. In all, 145 families were enrolled. Two mothers gave
birth to twins but only the first born in each couple was included. The reasons
for non-inclusion and for discontinuing the intervention are displayed in Fig. 7.
Some of the families missed planned visits due to loss of interest or relocation
but they were encouraged to a phone call or visit at 24 months. Finally, data on
allergic symptoms in the infant during the first two years was obtained from
143/145 families.
- 59 -
24 months visit (n=53)
12 months visit (n=52)
6 months visit (n=52)
Lost interest (n=1)
3 months visit (n=53)
24 months visit (n=63)
Assessment
at 24 months (n=143)
- 60 -
Missed 6 months
visit (n=7)
24 months visit (n=19 )
Lost interest (n=3)
6 months visit (n=15)
Lost interest (n=2)
24 months telephone contact and allergy questionnaire (n=8)
moved
abroad
(n = 1)
12 months visit (n=63)
Lost interest (n=1)
6 months visit (n=65)
3 months visit (n=65)
Moved (n=1)
Intervention not completed (n=25)***
lost to follow up (n = 1)
Excluded (n=40):
Not meeting inclusion criteria (n=31)*
Declined participation (n=7)
Other reasons (n=2)**
Placebo group (n=75)
Discontinued intervention (n=9)
Moved (n=1)
Moved (n=1)
ω-3 group (n=70)
Discontinued intervention (n=16)
Randomized (n=145)
Assessed for eligibility (n=185)
Figure 7: Inclusion and clinical follow up.
*: Treated with anticoagulants (n=2), had passed 25th gw (n=12), allergy to soy
(n=2), wished to continue their own fish oil supplements (n=1), no allergies in
the family (n=4), planned birth date after end of study (n=10).
** : moved (n=1), miscarried (n=1).
*** : Reasons for discontinuing intervention: inability to swallow capsules
(n=9), nausea (n=6), abdominal pain (n=3), ignorance (n=3) and
miscellaneous (n=4). The content of the capsules did not have an impact on
reasons for withdrawal.
Intervention
The content of the capsules
Block randomisation was performed by the producer of the supplements
(Pharma Nord, Vejle, Denmark) and the material was double blinded until the
end of the two-year follow-up of the children. The women in the ω-3 group
(n=70) were supplemented with Bio Marin capsules (Pharma Nord, Vejle,
Denmark), containing a minimum of 35% EPA and 25% DHA. The
recommended daily dose of nine 500-mg capsules contained in average 1.6 g
EPA, 1.1 g DHA and this corresponds to a meal of approximately 100 g salmon
daily. It also contained 23 mg α-tocopherol as an antioxidant, a necessary
ingredient, according to the standard procedure of the manufacturer, to assure
the durability of the oil.
The mothers in the placebo group (n=75) received nine soy oil capsules a day,
containing mainly the ω-6 PUFA LA (58%, 2.5 g/day), a small amount of the ω3 PUFA LNA (6%, 0.28 g/day) and 36 mg α – tocopherol. The fatty acid content
of the capsules is presented in detail in
Table 2.
- 61 -
Table 2. The content of the capsules.
Analyses performed at professor Birgitta Strandviks laboratory in Gothenburg,
resulting in slightly higher proportions of EPA and DHA than reported as a
minimum content by the manufacturer. This is due to the variation in EPA and
DHA content of the fish used in the production. The placebo capsules do not
contain EPA or DHA and the analysis of the placebo content agreed well with
the data from the manufacturer.
12:0
14:0
14:1ω-5 *
16:0
16:1ω-7
18:0
18:1ω-9
18:2ω-6 (LA)
18:3ω-6
20:0
18:3ω-3 (LNA)
20:2ω-6
20:3ω-9
20:3ω-6
22:0
20:4ω-6 (AA)
20:5ω-3 (EPA)
24:0
24:1ω-9
22:6ω-3 (DHA)
ω-3
mol %
0.0
0.5
0.0
1.1
0.9
0.9
2.8
0.3
0.1
0.6
0.3
0.2
0.2
0.3
0.1
2.6
53.5
0.0
0.4
35.4
Placebo
mol %
0.0
0.1
0.0
11.7
0.1
3.0
19.7
58.5
0.0
0.3
6.2
0.0
0.0
0.0
0.3
0.0
0.0
0.1
0.0
0.0
The choice of soy oil as a placebo was based on the ability of the producer to
manufacture the capsules. Estimated intakes of LA in women according to a
nation wide study in Sweden in 1989 was 7.8 g daily (239), which means that
the placebo capsule would add about one third of the daily intake to the
mothers´ diets. The daily intake of LNA (1.2 g) was increased by about 25 %.
However, there are indications that the ω-6/ω-3 ratio has an impact on
inflammatory responses (211, 240) and in that respect the placebo capsule, with
- 62 -
a ratio of ω-6/ω-3 (LA/LNA) of about 9 was close to the ratio LA/LNA of 7,
registered in the diet of Swedish women (239). The ratio LA/LNA in the ω-3
capsule was 1 and the total ω-6/ω-3 was <1.
The metabolism of LNA into DHA and EPA is inefficient so it can be presumed
that only insignificant amounts of DHA and EPA were added through the LNA
in the placebo capsule (181). On the other hand, the ω-3 capsule increased the
EPA and DHA intake 8-10 times (239) and the ratio of the active metabolites
AA/EPA (ω-6/ω-3) was more than ten times less than in the normal diet (239).
This supports the idea that the possible changes in LCPUFA composition of
blood and breast milk caused by the placebo capsule should be negligible,
particularly compared to those of the ω-3 LCPUFA oil.
The content of α-tocopherol (vitamin E) in the capsules differed somewhat
between the two groups. Vitamin E is an antioxidant with a recommended daily
intake during pregnancy of 15-20 mg. It has been reported to possibly decrease
the risk of developing asthma, eczema (168) and sensitisation (169) in
childhood. However, the reduced risk of sensitisation has been seen at intakes of
vitamin E up to 7 mg/day, but higher doses did not give significant additional
effects (169). All mothers in this study received more than 25 mg/day.
Furthermore, 30 mg vitamin E daily did not modify the immunomodulatory
effects of fish oil in a study of healthy men (241). The content of
polychlorinated biphenyles (PCB) and dioxins in the capsules was 20 times less
than in a corresponding portion of oily fish according to the manufacturer
(Pharma Nord).
- 63 -
Compliance
Dietary supplementation started at the 25th (range 23.0-27.1) week of gestation,
which is around the time when the first immune responses to allergen have been
detected (92). The women, who completed the intervention, took the capsules
for an average time of 30.9 weeks, range: 15.1–42.1(-66.1), SD ±5.6 weeks,
(Table 4, Fig. 8).
The nurses called the mothers twice during pregnancy to remind them of the
capsules and compliance was assessed by analysis of phospholipid fatty acids in
maternal and infant serum /plasma. Each mother received enough capsules to
last through the 3-4 first months of breastfeeding but there were some mothers
who did not manage to take full dose and consequently the capsules lasted
longer. These mothers still fulfilled the study. Nine capsules daily were taken
once a day or, if there were swallowing difficulties 3 capsules 3 times a day
were recommended. There was one extreme outlier, as one woman in the
placebo group took a lower daily dose for 66 weeks (Fig. 8). Additionally, not
all women breastfed fully during the whole study period (Table 6) and therefore
the babies‟ daily dose and length of treatment varied. This emphasises the
importance of measuring LCPUFA proportions in phospholipids in order to
monitor compliance and detect associations to allergic disease but it makes this
study unable to determine the appropriate dose for maternal ω-3
supplementation in allergy prevention. When breastfeeding was not sufficient
extensively hydrolysed formula was recommended for infants with signs of
cow‟s milk allergy and cow‟s milk formula was suggested for everyone else.
- 64 -
Mothers
(n)
18
16
14
12
10
8
6
4
2
0
1
4
7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67
Intervention not
completed
Included in ”as treated analysis”
” analysis
Full weeks of supplementation
Figure 8. Duration of supplementation. Grey: placebo Black: ω-3
supplementation. Dotted line: Minimum 15 weeks of supplementation was
required for inclusion in the analysis of outcomes in the intervention groups
(”as treated analysis”).
Blinding
After about 4 weeks of supplementation, 11/80 (14%) mothers reported
belching. Ten of them were supplemented with ω-3 fatty acids (p= 0.001).
Belching has been reported previously when similar doses of fish oil have been
given in pregnancy (242, 243). Thus, the blinding to the subjects might have
been unmasked. This could be a problem as diagnosis of clinical symptoms is
dependent on the mother‟s description and expectations. However, positive
SPTs and detection of specific IgE antibodies in blood samples are objective
measures. Further, all staff members working with the intervention and followup were blinded throughout the whole study.
- 65 -
Safety
Increased consumption of fish oil in pregnancy could theoretically increase
bleeding tendency at delivery (244) and prolong gestation but also reduce the
risk of reoccurrence of preterm delivery (245). Replacement of AA by EPA in
the cell membranes induces vasodilatation and decreases platelet aggregation
(244). However, supplementation with the commonly used dose of 3 g
EPA+DHA per day, which is10-20 times the average intake of most pregnant
women in westernised countries, has not been associated with increased risk of
maternal bleeding complications (reviewed in (246)). That risk is clinically
significant only if the consumption of ω-3 fatty acids is 7-10 g per day (244).
Further, the mean length of infant hospital stay or the relative risks of admission
to neonatal care, congenital malformation, neonatal bleeding and non-bleeding
disorders have not been affected by 3g EPA and DHA daily during pregnancy
(reviewed in (245, 246)).
On the other hand, consumption beneath 150g ω-3 LCPUFA per day may
increase the risk of preterm delivery and low birth weight (247) and therefore a
minimum intake of 200-300 mg daily for pregnant women has been
recommended (217, 246). The saturation dose of DHA was 1.2 g / day when a
combination of DHA and EPA was provided to healthy adult volunteers (180,
248), which supports our choice of supplementing the mothers with 1.1 g DHA
daily.
- 66 -
Study subjects
Paper I. Fifty-nine mothers were included in this paper. Inclusion was based on
the availability of plasma samples for whole blood cultures. Twenty-eight of
these mothers were supplemented with ω-3 LCPUFA and 31 with placebo.
Paper II. 117 families, who completed the follow-up to one year after delivery,
were included in this paper. Fifty-two of the mothers were supplemented with
ω-3 LCPUFA and 65 with placebo. Infants were excluded from sub-analyses if
data were missing from any time point. This was the case for sensitisation and
IgE associated eczema at one year, since blood samples were not obtained from
all infants.
Paper III. 119 families (54 ω-3 and 65 placebo) were included in this paper and
the outcome for 24 of the 25 mothers who did not complete the intervention was
also discussed. Infants were excluded from sub-analyses, if data were missing
from any time point. This was the case for sensitisation and IgE associated
disease up to two years, since blood samples were not obtained from all infants.
Paper IV. The chemokine analyses in this paper were confined to infants from
Linköping, from whom blood was available from any time point during the first
two years of life (n=80). Chemokines were analysed in plasma from the
umbilical cord (n=72), 3 months (n=42), 12 months (n=61) and 24 months
(n=61). For analysis of vaccine induced immunity, samples from Jönköping and
Linköping were used and blood from 94 infants were available for analysis.
Number of subjects in each intervention group is given in Table 1, paper IV.
- 67 -
Clinical methodology
Table 3. The families were assessed according to the research protocol below.
MOTHER
gw25 partus
Heredity + clinical history
X
Questions maternal diet
X
Blood sample
X
Breast milk samples*
1m
3m
6m
12m 24m
X
X
colostrum
X
X
X
X
X
X
X
X
X
X
X
X
X
X
CHILD
Physical investigation (nurse)
Physical investigation (paediatrician)
X
Allergy questionnaire
SPT
Blood sample
cord blood
X
Saliva sample*
Faeces sample*
X
X
X
X
X
X
X
X
X
X
X
X
* Samples collected but not analysed in the papers included in this thesis.
The infants were examined by an experienced allergy nurse at 3, 6 and 12
months and in case of possibly allergic symptoms, a paediatrician, CF or JL, reexamined the child (Table 3). At 24 months of age all children were examined
by a paediatrician (CF or JL). Serum (obtained from families in Jönköping) and
plasma samples were immediately frozen and stored in –70 °C. An allergy
questionnaire used in prior studies (192), covering allergic symptoms, diet and
environmental factors, such as exposure to pets, tobacco and day-care were
filled out by the parents regularly. Records from primary care units, private
paediatricians and the paediatric clinics were scrutinized if the parents reported
that a paediatrician had examined the child.
Twenty-four-hour maternal food diaries were registered 3 days in a row at
gestational week 25 and at 6 months after delivery. A certified dietician assessed
the maternal intake of energy and fat according to routine clinical praxis using
- 68 -
the software „Dietist XP‟ (Kost- och näringsdata, Bromma, Sweden,
www.kostdata.se). The amount of each provision was estimated from the
registered weight, volume or number of portions (Table 4).
Diagnostic criteria
Symptoms of eczema, food reaction, asthma or rhinoconjunctivitis were
considered as clinical symptoms of allergic disease. Concomitant sensitisation,
i.e. at least one positive SPT and/or detectable circulating allergen specific IgE
antibodies against tested allergens, defined IgE associated allergic disease.
The diagnostic criteria for eczema used in this thesis are modified from the
criteria by Sampson (249) adapted by Seymour (37), for AEDS < 2 years.
The major features were:
 Evidence of chronic pruritic dermatitis
 Typical facial or extensor eczematous or lichenified or nummular
dermatitis
 Eczema-free skin of nose-mouth area and /or diaper area
The minor features were:
 Xerosis/ichtyosis/hyperlinear palms
 Peri-auricular fissures
 Chronic scalp scaling
 Perifollicular accentuation
For diagnosis of eczema 3 criteria were needed and pruritus was compulsory. At
least two of the criteria needed to be major. All children in the study had allergic
disease in the family, hence that criteria, originating from the definitions by
Samson (249), Oranje (35) and Seymour (37) was fulfilled for all children. If
detectable IgE antibodies or a positive SPT were present in combination with
eczema, it was defined as IgE associated eczema. Internal workshops were
- 69 -
arranged for the two examining paediatricians before and during the follow-up
of the children in order to achieve uniform assessments of eczema.
A food reaction was defined as gastrointestinal symptoms, hives, aggravated
eczema or wheezing following ingestion of a certain food with recovery after
food elimination and reoccurrence of symptoms after re-ingestion of the
particular food. If food specific positive SPT or serum IgE antibodies were
present, the food reaction was considered as IgE mediated food allergy (food
allergy in paper II).
Asthma was defined as doctor diagnosed wheezing at least three times during the
first two years. IgE associated asthma was defined as asthma with the presence
of any IgE antibodies or positive SPT.
Rhinoconjunctivitis was seasonal itching and running eyes and nose. It was
considered as IgE mediated rhinoconjunctivitis if there was a corresponding
positive SPT or detectable specific IgE antibodies.
Allergic mothers were mothers with current, or a history of, eczema, asthma,
food reactions or rhinoconjunctivitis according to interviews at inclusion. Atopic
mothers were mothers with any of the allergic symptoms and a positive
Phadiatop®, i.e. detection of IgE in a panel of inhalant allergen. Mothers with
positive Phadiatop® and no symptoms or vice versa were excluded from the
sub-analyses of atopic/non-atopic women (paper I).
- 70 -
Laboratory methodology
The laboratory methods used in the papers included in this thesis are listed
below. The methods are described in detail in each paper.
 ELISA
Analyses of PGE2 and LTB4 from whole blood cultures (paper I).
Analyses of tetanus IgG and diphtheria IgG (paper IV).
 Luminex
Analyses of cytokines from whole blood cultures (paper I).
Analyses of chemokines (paper IV).
 Gas chromatography:
Analyses of phospholipids (PL) fatty acids in serum or plasma (paper I-IV).
We have chosen to analyse LCPUFA in phospholipids of serum /plasma instead
of investigating the immune cell membranes, which might be the preferable
method when assessing immunological effects. Unfortunately, we did not obtain
enough cells to accomplish this. Phospholipids are transporters of fatty acids
that have not yet been incorporated into cellular membranes and they give the
best reflection of the overall PUFA status (188). The relative concentrations of
PUFA in plasma PL correlate to fatty acid content of erythrocytes in pregnancy
(250, 251) and both are reasonable markers for tissue LCPUFA content (180).
Ianalysis of PUFA in PL in serum and plasma provide analogous results.
 Skin prick testing (paper II-IV).
 UniCap®, Immunocap100 Phadiatop®
Analyses of total IgE, circulating IgE against allergens (paper I-IV).
- 71 -
Statistics
Power calculations for planning adequate sample size were performed based on
a previous study (252). In order to detect a 40% difference in the prevalence of
clinical symptoms of allergic disease, with 80 % power and a probability of
0.05, we needed at least 134 women to be included in this study. Preferably
there should be 67 in each intervention group. With a presumed drop out
frequency of 20 % around 160 mothers would have been desirable in this study.
We did not reach this goal due to the difficulties in recruiting mothers to this
kind of intervention and a limited durability of the capsules that did not allow us
to continue the recruitment of mothers for more than two years.
The categorical data of the two groups, outcome variables and background data,
were analysed by chi-2 tests (χ2). Fisher‟s exact test was used when the
expected frequency for any cell was less than five. The means of the continual
variables were analysed by Student‟s t-test. For the variables that were not
normally distributed, such as some of the fatty acids, the chemokines, PGE2,
LTB4, the cytokines and the vaccine induced IgG, Mann Whitney U-test was
used for unpaired analyses and Wilcoxon signed rank test for paired analyses
(paper I). Spearman‟s correlation was used for correlation of non-parametric
variables (paper I and IV). Samples with a concentration below the limit of
detection were assigned a value corresponding to half the cut-off value (paper I
and IV). Multiple logistic regression was employed to identify effect modifiers
among the possible confounders and calculate adjusted and unadjusted odds
ratios (paper II and III). Number needed to treat (NNT) was calculated with the
substitution method described by Daly (253) (paper I). χ2 and χ2 trend
(Extended Mantel-Haenszel chi-square test for linear trend) were used to assess
the differences in prevalence of IgE associated disease between the quartiles of
- 72 -
phospholipids fatty acid proportions in paper III. Friedman‟s test was used for
analysis of repeated measures of CCL17 in paper IV.
A difference with a p-value < 0.05 was considered to be statistically significant,
except for the analysis of cytokines and chemokines in paper I, where
differences were considered significant at a p-value of <0.01 as a consequence
of multiple comparisons. Statistic analyses were performed using Stat View for
Windows Version 5.9 (SAS Institute Inc., Cary, North Carolina, USA) in paper I
and SPSS software 15.0 for Windows (SPSS Inc, Chicago, Illinois, USA) in
paper II-IV.
Ethical considerations
The effect of maternal LCPUFA supplementation on the development of allergic
disease early in life was not documented before 2003, when this study was
planned, but there was enough data to assure the safety of this study design (see
paragraph on safety). Pain connected with blood sampling was minimised with
topical anaesthesia.
Pharma Nord had no impact on the study design, data collection, data
interpretation or publication of the results. An informed consent was obtained
from both parents before inclusion. In fact, the close surveillance and early
diagnosis of infant allergic disease might even have been an advantage for the
participating families. The Regional Ethics Committee for Human Research at
Linköping University approved the study.
- 73 -
- 74 -
RESULTS AND DISCUSSION
Maternal diet and fatty acid status.
The average registered dietary intake of DHA, EPA and LA at inclusion was 0.2
g/day, 0.1 g/day and 8 g/day respectively (Table 4), which corresponds well
with data from previous diet registrations in Sweden (239).
The proportions of ω-3 and ω-6 fatty acids in serum/plasma phospholipids of the
mothers were similar in the two groups at the time of inclusion, with a few
exceptions (Table 5). The placebo group had higher proportions of LA and the
ω-3 group had higher proportions of AA before the intervention (p<0.05 for
both). Dietary habits did not seem to explain these differences as both groups
showed similar diet registrations (Table 4). In the analysis of clinical outcomes
AA or LA levels at inclusion were adjusted for when they turned out to be
significant effect modifiers (paper II and III).
Higher relative levels of EPA and DHA in maternal serum/plasma phospholipids
and lower AA/EPA ratio were found in the ω-3 supplemented group compared
to the placebo group one week after delivery (p for all <0.001). Still 8 months
after ending the supplementation, i.e. 12 months after delivery AA/EPA ratios
remained lower (p=0.008) in the ω-3 supplemented group compared to the
placebo group indicating a long lasting effect of the ω-3 supplementation (Table
5). The proportions of both ω-6 PUFA, AA and LA, in the ω-3 supplemented
group were lower compared to the placebo group (p<0.001) one week after
delivery, an effect of the ω-3 supplementation seen in previous studies (180). At
12 months after delivery, however, ω-6 PUFA levels were similar in both
groups.
- 75 -
Table 4. Duration of supplementation and diet registrations
ω-3
Placebo
n
Mean(SD)
n
Mean(SD)
p
54
29.8(5.0)
66
31.4(6.3)
ns
Energy(kcal/d)
49
2168(366)
63
2172(386)
ns
% of RDI energy(kcal/d)
49
97(18)
63
96(17)
ns
Total fat(g/d)
49
77(18)
63
80(20)
ns
% of RDI fat(g/d)
49
102(24)
62
103(25)
ns
18:2ω-6 (LA, g/d)
47
7.7(2.5)
63
8.5(3.7)
ns
20:4ω-6 (AA, g/d)
48
0.7(0.06)
63
0.7(0.1)
ns
18:3ω-3 (LNA, g/d)
48
1.4(0.64)
63
1.6(0.8)
ns
20:5ω-3 (EPA, g/d)
48
0.1(0.1)
63
0.1(0.2)
ns
22:6ω-3 (DHA, g/d)
48
0.2(0.3)
63
0.2(0.4)
ns
Tot ω-6/tot ω -3
48
5.2(2.2)
63
5.0(1.6)
ns
Energy(kcal/d)
45
2089(429)
54
217(441)
ns
% of RDI energy(kcal/d)
45
84(17)
54
84(18)
ns
Total fat(g/d)
45
84(21)
54
83(23)
ns
% of RDI fat(g/d)
45
99(24)
54
96(27)
ns
18:2ω-6 (LA, g/d)
45
9(3)
54
11(10)
ns
20:4ω-6 (AA, g/d)
45
0.07(0.06)
54
0.1(0.05)
ns
18:3ω-3 (LNA, g/d)
45
1.8(1.2)
54
1.9(1.1)
ns
20:5ω-3 (EPA, g/d)
45
0.07(0.1)
54
0.1(0.1)
ns
22:6ω-3 (DHA, g/d)
45
0.15(0.2)
54
0.25(0.4)
ns
Tot ω-6/tot ω-3
45
4.7(4.3)
54
5.2(4.4)
ns
Daily supplementation (weeks)
Maternal diet 25th gestational week *
Maternal diet 6 months after delivery*
*The study capsules not included,
ns: not significant,
RDI: recommended daily intake.
DHA and EPA increased in the ω-3 supplemented group between week 25 of
gestation and one week after delivery (p<0.001). Eight months after the end of
the intervention the DHA proportions were the same as in pregnancy but the
EPA levels were still higher than the mid pregnancy levels (p<0.001, Table 5).
- 76 -
Children are provided ω-3 fatty acids from maternal stores, her diet and her
metabolism of LNA during gestation. LCPUFA are delivered through the
placenta and, after delivery, through breastfeeding (254). We found a decrease
in DHA proportions during pregnancy and lactation in our placebo group
(p<0.01) but the EPA levels increased after delivery (p<0.001). This most likely
reflects the utilization of DHA for breast milk as supported by the work of Otto
et al, 2001 (255). They also found an increase in the other ω-3 PUFAs after
parturition, represented by the increase in EPA in our placebo group, suggesting
that there is a selective transport of DHA into breast milk without any increased
conversion of EPA into DHA. In agreement with this, Jorgensen et al found a
decrease of DHA in maternal red blood cell-phosphatidylcholine during the first
4 months of lactation (256). Relative DHA levels increased in plasma
phospholipids during normal pregnancy until gestational week 18 and thereafter
the proportions decreased until >6 months after delivery in a Dutch study (257).
Current studies indicate that pregnant women today have difficulties meeting the
demands for ω-3 fatty acids from the foetus, i.e. women who are pregnant with
their second or third child have less available ω-3 fatty acids (186, 256). In our
study, there were weak but significant negative correlations between DHA
levels and parity in the whole group at inclusion (rho= -0.21. p=0.01) and in the
placebo group 12 months after delivery (rho= -0.27, p=0.03). No correlation was
found one week after delivery in the placebo group. The maternal relative levels
of DHA in mid pregnancy were generally a little higher than reported from the
Netherlands (186) which could explain, like in the study by Dunstan (196), that
the stores were not significantly compromised during pregnancy. Interestingly,
the levels of LNA decreased during the study period in both our intervention
groups.
.
- 77 -
The AA content in maternal serum/plasma phospholipids increased in the
placebo group (p<0.001) until delivery and remained at the higher level at 12
months after delivery. The LA proportions in the placebo group also increased
during the study period, but not until after delivery (p<0.001). Compared to the
Dutch studies of pregnant women our placebo population tended to have slightly
lower proportions of ω-6 PUFA and higher proportions of ω-3 PUFA in their
serum/plasma phospholipids in mid pregnancy (186, 187)
There were no differences in serum/plasma phospholipid fatty acid levels of
allergic and non-allergic mothers prior to supplementation. Other studies have
shown inconsistent associations between ω-6 and ω-3 PUFA levels in serum
phospholipids or cell membranes and allergic conditions (reviewed in (258)).
There are also studies reporting no associations between levels of ω-6 or ω-3
fatty acids in serum phospholipids and allergic disease in adults (n=740, 427
men) (259) and in cord blood vs. allergic disease in the infants ( n=1238) (260).
However, mothers who are allergic tend to have an abnormal balance of fatty
acids in their breast milk (254, 261). In our study the changes in serum/plasma
phospholipid fatty acid proportions due to ω-3 LCPUFA supplementation were
similar in the allergic and non-allergic mothers, data not shown (dns).
- 78 -
Table 5. Proportions of ω-6- and ω-3-PUFA in serum/plasma phospholipids
of the mothers before, during and after the intervention.
Gestational week 25
First week after delivery
12 months after delivery
ω-3
(n=52)
mean(SD)
Placebo
(n=64)
mean(SD)
t-test
p
ω-3
(n=51)
mean(SD)
Placebo
(n=61)
mean(SD)
t-test
p
ω-3
(n=53)
mean(SD)
Placebo
(n=62)
mean(SD)
t-test
p
18:2ω-6
(LA)
18.9(2.5)
19.9(2.1)
<0.05
15.2(2.3)
20.1(3.0)
<0.001
21.7(2.9)
22.2(2.4)
**
**
ns
20:4ω-6
(AA)
9.3(1.7)
8.6(1.5)
<0.05
8.1(1.6)
10.2(2.0)
10.6(1.7)
**
<0.001
10.3(1.9)
**
**
**
18:3ω-3
(LNA)
0.5(0.1)
0.5(0.1)
ns
0.4(0.1)
0.4(0.1)
0.3(0.1)
**
ns
0.3(0.1)
**
**
**
20:5ω-3
(EPA)
1.3(0.8)
1.2(0.6)
ns
7.6(2.5)
1.3(0.6)
<0.001
2.6(1.5)
2.2(1.2)
**
**
22:6ω-3
(DHA)
20:4ω6/20:5ω-3
AA/EPA
5.5(1.1)
5.4(1.2)
ns
7.9(1.5)
5.2(1.6)
<0.001
5.4(1.2)
4.9(1.4)
9.1 (4.2)
8.6(4.0)
ns
1.4(1.3)
8.9(3.6)
<0.001
4.8(1.9)
5.9(2.5)
**
**
Fatty acid
(mol%)
**
**
**
**
*
*:p<0.01 compared to gestational week 25 (paired t-test),
** p<0.001 compared to gestational week 25 (paired t-test)
ns : not significant.
Pregnancies and deliveries
The data on pregnancies and deliveries are presented in Table 6. The increase in
gestational length reported in several large ω-3 supplementation trials (reviewed
in (245)) was not seen in this study. This could be explained by two facts, the
relatively small size of the study but also by the significant increase of
pharmacological induction of deliveries in the ω-3 group (p=0.04, Table 6B).
Even though the inductions due to prolonged gestation were as common in the
two groups there were more inductions for various other reasons in the ω-3
group. This could conceal a significant difference in gestational length. The
need of emergency caesarean sections was similar in the two groups, though
planned sections were only performed in the placebo group. The motivations for
planned caesarean sections were humanitarian reasons, i.e. anxiety (n=5),
- 79 -
ns
ns
ns
0.05
<0.01
osteoporosis (n=1) and narrow pelvis (n=1). These results seem peculiar in such
a small study. However, in trials designed to investigate the psychological
effects of ω-3 supplementation by assessment of test anxiety (262), and other
psychological stress (263, 264), both biochemical and behavioural effects of
PUFA supplementation have been reported. An association between low seafood
consumption in pregnancy and maternal depression has been observed (265) and
ω-3 LCPUFAs have been considered in treatment of perinatal depression (266).
Table 6 A and B. The pregnancies, the deliveries and the babies.
A
n
ω-3
value(SD)
n
Maternal age at delivery (years)
53
31.1(4.0)
65
31.7(3.8)
ns
Parity, including study child
54
1.4(0.6)
66
1.5(0.7)
ns
Weight gw 8-10 (kg)
53
69.6(10.2)
65
68.1(14.7)
ns
Weight gain until delivery (kg)
45
13.1(3.8)
60
13.8(4.9)
ns
Height (cm)
53
167(5)
65
165(6)
ns
53
25(3.5)
65
24.8(3.6)
ns
BMI increase until delivery (kg/m )
45
4.7(1.4)
60
5(1.8)
ns
Gestational length (days)
54
282(9.8)
66
281(11.1)
ns
Bleeding at delivery (ml)
52
490(258)
64
492(279)
ns
APGAR 1 minute
54
8.5(1.1)
66
8.3(2.0)
ns
APGAR 5 minute
54
9.7(0.7)
66
9.6(1.0)
ns
APGAR 10 minute
54
10(0.2)
66
9.8(0.7)
ns
Head circumference baby (cm)
53
35(1.1)
65
35.2(1.5)
ns
Birth weight baby (g)
54
3511(499)
66
3567(554)
ns
Birth length baby (cm)
54
50(2.0)
66
50.5(2.6)
ns
Weight baby at 6 months (kg)
48
7.8(0.9)
57
7.8(1.0)
ns
Weight baby at 12 months (kg)
49
9.9(1.1)
62
10(1.1)
ns
Weight baby at 24 months (kg)
51
13(1.4)
61
13(1.2)
ns
Duration of breastfeeding (months)
53
9.6(5.0)
66
9.3(3.6)
ns
2
BMI gw 8-10 (kg/m )
2
§ = t-test, gw = gestational week, ns = not significant
- 80 -
Placebo
value(SD)
p§
n
ω-3
freq(%)
54
1(2)
66
2(3)
ns
Delivery after the end of 41 gw
54
20(37)
66
20(32)
ns
Induction
54
11(20)
66
5(8)
0.04
54
3(6)
66
3(4)
ns
“ “ preeclampsia
54
3(6)
66
0(0)
ns
“ “ anxiety
54
2(4)
66
1(2)
ns
“ “ various reasons*
54
3(6)
66
1(1.5)
ns
54
4(7)
66
14(21)
0.035
emergency
54
4(7)
66
7(11)
ns
planned
54
0(0)
66
7(11)
0.01
VE
54
6(11)
66
10(15)
ns
CPAP
54
4(7)
66
7(11)
ns
Neonatal ward
54
5(9)
66
3(4)
ns
Girls
54
30(56)
66
34(52)
ns
Infant colic pains
52
20(40)
65
24(40)
ns
VOC
54
2(4)
66
2(3)
ns
Exclusive breastfeeding until 3 m
54
46(85)
66
59(89)
ns
Exclusive breastfeeding until 6 m
53
18(34)
66
28(42)
ns
B
th
Delivery before end of 36 gw
st
Due to prolonged gestation
Section
Placebo
n
freq(%)
p#
#: Chi-2 test. When the expected values were less then 5 Fisher’s exact test was used.
*: Symphyseolysis, maternal kidney problem, poor growth, premature rupture of membranes,
twins. VE: Vacuum Extraction, CPAP: Continuous Positive Airway Pressure needed in the
resuscitation procedure, VOC: Vitium Organicum Cordis. ns: not significant
Apart from the belching in the ω-3 group mentioned before, maternal nausea
and abdominal pain occurred in both intervention groups (Fig. 7). Three to four
percent of the infants had any kind of heart defect, i.e. vitium organicum cordis
(VOC), which does not exceed the prevalence reported previously in a Swedish
population (267). Only one VOC led to surgery during the first two years in our
study.
- 81 -
Allergic heredity and environmental factors
There were no significant differences between the intervention groups in terms
of allergies in the family and environmental factors of possible importance for
allergic disease, except for caesarean sections (Table 7, data on parity,
breastfeeding and caesarean sections are presented in Table 6). Nevertheless,
variables that turned out to be effect modifiers were adjusted for in the odds
ratio analysis of the clinical results (paper II and III).
All families used cow‟s milk formula if the breast-feeding was insufficient and
there were no signs of cow‟s milk allergy in the children (Table 7). None of the
families gave formula containing ω-3 fatty acids to their infant. Solid foods were
introduced at 4.7 month of age (SD 0.7) in the placebo group and at 4.8 months
(SD 0.9) in the ω-3 group (ns). Five infants were introduced to solid foods
between 3 and 4 months of age (1 in the placebo group and 4 in the ω-3 group).
Fish was introduced in the diet of three infants in the placebo group and two
infants in the ω-3 group between 4 and 6 months of age, i.e. 4 %, while 16-17 %
of the families chose not to introduce fish to their child before the first birthday.
This reflects the impact of the previous recommendations given to families with
high-risk infants.
- 82 -
Table 7. Allergic history and environmental factors.
ω-3
Allergic history *
n
Placebo
freq(%)
n
freq (%) p#
mother 54
40(74)
66
42(64)
ns
father 54
36(67)
66
44(67)
ns
both parents 54
22(42)
66
21(32)
ns
sibling only 54
1(2)
66
1(2)
ns
Positive Phadiatop® mother
54
32(59)
66
35(53)
ns
Positive Phadiatop® and allergic
history mother¶
44
31(71)
59
35(59)
ns
Eczema in the family
54
24(44)
66
25(38)
ns
Food reactions in the family
54
10(19)
66
14(21)
ns
Exposure to furry pets (0-24m)§
54
35(65)
65
36(55)
ns
Exposure to smoking (0-24m) †
53
10(19)
62
12(19)
ns
Any AB treated infections (0-24m)
50
20(40)
58
33(57)
ns
More than 5 common colds (0-24m) 49
40(82)
57
43(75)
ns
Day-care
51
42(82)
59
44(75)
ns
Cow‟s milk formula before 4 m
52
13(25)
65
13(20)
ns
No fish in infant‟s diet during the
first year
51
8(16)
64
11(17)
ns
Environmental factors
*: Current or previous symptoms like eczema, asthma, allergic urticaria, gastrointestinal
allergy or allergic rhinoconjunctivits.
¶: atopic disease ( paper I)
§: Exposure to furry pets at least once a week.
†: Tobacco smoke in the home or where the child spends the day and/or smoking family
member.
AB: antibiotics
- 83 -
Maternal eicosanoid secretion
Eicosanoids, cytokines and chemokines were analysed in whole blood
supernatant from the mothers at inclusion and at delivery with and without LPS.
The results are presented in paper I. The mothers allocated to ω-3 fatty acids
supplementation responded with higher LPS-induced PGE2 secretion from
whole blood cell culture supernatants at inclusion compared with the mothers
who were later treated with placebo (p= 0.01). To overcome the influence of
this random difference already present at inclusion, we chose to analyse the
change in secretion during supplementation, rather than the absolute levels. The
LPS induced PGE2 secretion increased in the placebo treated group (p<0.001)
between gw 25 and partus but remained unaltered in the ω–3 LCPUFA treated
group. Furthermore, LPS-induced PGE2 secretion decreased during the
supplementation period in a majority of the ω-3 LCPUFA-supplemented women
but increased in the majority of the placebo-supplemented women (p=0.002,
Fig. 1A, paper I).
The changes in LPS-induced PGE2 production during intervention correlated
positively with changes in plasma phospholipid AA proportions and negatively
with changes in EPA (Fig. 2 AB, paper I). A positive correlation was also
observed between changes in LPS-induced PGE2 secretion and changes in
AA/EPA ratios (Fig. 2C, paper I). The effects of PGE2 in allergic inflammation
are complex, both facilitating and hampering inflammatory reactions (reviewed
in refs. (225, 229)). However, PGE2 seems to facilitate sensitisation as it inhibits
the production of Th1 cytokines, primes immature T cells to produce IL-4 and
IL-5, and facilitates B-lymphocyte immunoglobulin isotype switching to IgE
(225, 227, 268). Consequently a decreased PGE2 secretion may have a impeding
effect on allergic sensitisation and influence the predisposition to IgE associated
disease (191).
- 84 -
Infant fatty acid status
The infant serum/plasma EPA and DHA proportions were higher in the ω-3
supplemented group compared to the placebo group up to one year of age, about
nine months after the end of supplementation (Table 3, paper III). At two years
of age there were no differences between the groups. The DHA and AA
concentrations were considerably higher in cord blood compared to maternal
blood after delivery due to the preferential accretion of DHA and AA relative
the precursors EPA and LA by the placenta (184, 269).
Until 12 months of age the LA and AA proportions in the ω-3 supplemented
group were lower than in the placebo group, reflecting the response to the DHA
and EPA supplementation seen in previous studies (180). The LA and AA
proportions in the placebo group correspond with (188) or are lower (270) than
in previous studies which makes a significant contribution from the placebo
capsule less likely.
Clinical outcomes
Throughout the follow-up the number of children with eczema and food
reactions were lower in the ω-3 group compared to the placebo group but these
differences were not statistically significant (Table 1, paper III ). Our sample
size was calculated based on the cumulative incidence of allergic disease during
the first 18 months of life (252). However, for instance the cumulative incidence
of eczema in this study was 20% in the -3 and 31 % in the placebo group, and
thus less common than expected. In order to dismiss such a difference between
the two groups as a type II error, with an 80% power at a 0.05 significance level,
246 mothers would have been needed in each group. Consequently our study
- 85 -
cannot rule out a protective effect of ω-3 fatty acids on allergic symptoms per
se, due to the limited sample size.
On the other hand the cumulative incidence of positive skin prick tests up to
two years of age was 19 % in the ω-3 group and 36 % in the placebo group (p=
0.05), with the most obvious difference in SPT to food (15% vs 34%, p= 0.02),
especially egg (13% vs. 30%, p=0.04). This agrees with our hypothesis that
EPA outstrips AA and the production of PGE2 is reduced, leading to less IgE
antibodies (271). No significant differences in detectable specific IgE antibodies
between the groups were found (Table 1, paper III), possibly due to the small
number of samples.
IgE associated eczema (eczema and positive SPT/detectable specific IgE) during
the first two years of life was less common in the in the ω-3 group compared to
the placebo group (Fig. 9). It has been reported that sensitised infants with
eczema are at increased risk for later development of allergic asthma and
allergic rhinoconjunctivitis (ARC)(20, 272), which makes these results clinically
important. Twenty-two children had any food reaction during the first two years
and 17 of them were IgE mediated. The symptoms were hives: n = 14 (10 of
them were IgE mediated), gastointestinal symptoms: n =11 (7 IgE mediated),
aggravated eczema: n = 6 (3 IgE mediated), wheezing:n =1, which was IgE
mediated. Hence, some infants experienced more than one symptom. There were
no significant differences between the ω-3 and placebo groups regarding the
symptoms caused by the food reactions. Most of the food reactions and all of the
IgE mediated ones were caused by milk or egg. One child developed hives after
eating fish but no sensitisation to fish was found.
- 86 -
Figure 9 A. Cumulative incidence (0-24m) of eczema and IgE associated
eczema in the placebo and ω-3 groups.
Grey diamond: any eczema in the placebo group,
Grey circle: any eczema in the ω-3 group,
Black square: IgE associated eczema in the ω-3 group.
Black triangle: IgE associated eczema in the ω-3 group.
Data from the three months visit is presented at six months.
B. Cumulative incidence of allergic symptoms and IgE associated disease 0-24
months. Grey bars: allergic symptoms, i.e. asthma, rhinoconjunctivitis, eczema
and/or food reaction. Black bars: IgE associated disease: any allergic symptom
and allergic sensitisation. Three children with symptoms and negative SPTs
were excluded from the analysis of IgE associated disease because of missing
IgE data.
Chi-2 test was used for calculation of p in A and B.
Up to one year of age the risk of developing food allergy (IgE mediated food
reactions) was ten times higher in the placebo group compared to the ω-3 group
(Table 2, paper II). The odds ratios for developing IgE associated eczema, IgE
mediated food allergy or any IgE associated disease (e.g. atopic disease) (10) up
to two years of age indicate a 3-5 fould increased risk in the placebo group
compared to the ω-3 group (Table 2, paper III).
- 87 -
Intention-to-treat analysis
There were 25 women who did not complete the intervention but their infants
were followed up according to Figure 7. Their infants did not differ
significantly from the placebo group in terms of positive SPTs at 6 months or
allergic symptoms or IgE associated disease during the first two years of life.
However, 19/25 children of mothers who did not complete the intervention,
were skin prick tested at 24 months and they had a slightly higher incidence of
positive tests compared to the placebo group (Table 8). One explanation to that
could be a higher interest in the skin-prick testing by families with allergic
compared to healthy children. The families that did not attend the follow-up
visits were interviewed by phone regarding allergic symptoms (Fig. 7) and only
one of the mothers who did not complete the intervention was not reached
(n=24).
An intention-to-treat analysis was performed regarding the main clinical
outcomes and there was a trend toward lower cumulative incidence of IgE
associated disease in the group allocated to ω-3 supplementation compared to
the group receiving placebo capsules. However, most of the differences seen in
the “as treated” analysis vanished when including the children whose mothers
did not complete the intervention (Table 8). This indicates that compliance
needs to be increased by making the supplementation more tolerable and easy to
ingest in order to improve the results.
- 88 -
Table 8. The main clinical outcomes in “as treated” and “intention to treat”
analysis.
“As treated”
Any positive SPT
at 6m†
Any positive SPT
at 24 m‡
Allergic symptoms
up to 24m
IgE associated
disease up to 24 m
“Intention to treat”
ω-3
n (%)
Placebo
n (%)
Intervention
not completed
n (%)
ω-3
n (%)
Placebo
n (%)
6/52 (12)
16/65 (25)
3/15 (20)
8/62 (13)
17/70 (24)
6/52 (12)
12/62 (19)
8/19 (42)*¶
12/64 (19)
14/69 (20)
19/54 (35)
28/65 (43)*
15/24 (62)*
28/69 (41)
34/74 (46)
6/54 (11)
19/62 (31)**
7/18 (38)**
11/65 (17)
21/68 (31) #
*: p< 0.05 vs. ω-3 group.
**: p= 0.01 vs. ω-3 group.
¶: p<0.05 vs. placebo group,
#: p=0.06 vs. ω-3 group,
†: egg, milk, wheat and/or cat ,
‡: egg, milk, wheat, cat, birch, and/ or timothy
Infant chemokines and vaccine induced immune responses
High Th2-associated CCL17 levels and high CCL17/CCL11 (Th2/Th1) ratios
were associated with allergic disease in the child (p<0.05 for both), which is in
agreement with earlier research (64-67).
CCR4 mRNA levels, i.e. the receptor for Th2 associated chemokines CCL17
and CCL22, decreased in the fish oil intervention group compared with those
seen in the placebo group in a recent supplementation study with 0.5 g DHA and
0.15 g EPA from the 22nd gestational week until delivery (273). In our study,
including all children with available chemokine data, we found no significant
differences in CCL17, CCL22, CXCL10 or CXCL11 in the ω-3 compared to the
placebo group but we did not analyse the chemokine receptors.
- 89 -
Allergic children have been shown to be intrinsically hyporesponsive to
vaccines, possibly due to Th2 skewed immune responses, even though this
seems to be overcome by common vaccination regimens (274). In this study the
vaccine induced responses did not differ significantly between allergic and nonallergic children (dns). Further, ω-3 fatty acids have been shown to enhance the
Th1 responses through IL-2 and IFNγ production (219) and may therefore
enhance vaccine antibody responses. However, anti-tetanus and anti-diphtheria
IgG levels were similar in the placebo and the ω-3 groups (Table 2, paper IV).
Previous ω-3 supplementation trials - design and outcomes
Trials with ω-3 supplementation in pregnancy and/or lactation analysing clinical
and/or immunological outcomes in the offspring are summarised in Table 9.
There are though several methological differencies between those and our study.
They were either not originally designed for investigating clinical outcomes in
the children (9, 198, 212, 273), the dose of ω-3 LCPUFA was very low (200) or
the supplementation was given merely in pregnancy (9, 198, 273) or only in
infancy (200, 212). To the best of our knowledge, our study is the first originally
designed to analyse clinical outcomes in the children after ω-3 supplementation
during pregnancy and lactation. The intervention lasts for a 6 months period and
the dose is appropriate (248) and safe (245).
Interestingly, a transient protective effect from black currant seed oil
supplementation in pregnancy and childhood on eczema, but not sensitisation at
1 year was shown in an additional recent trial (275). The composition of black
currant seed oil corresponds to the recommended optimal dietary intake of ω-3
and ω-6 PUFAs; in other words, the oil has a ratio of ω-3/ω-6 from 1/3 to 1/4.
Besides, the black currant seed oil was well tolerated by the mothers (275).
- 90 -
Country
Australia
Australia
Norway
Denmark
Germany
Spain
Hungary
First authors
Dunstan
Prescott
Mihrshahi,
Peat
Marks
Olsen
Lauritzen
KraussEtschmann
Pregnant
women
Lactating
women
Pregnant
women
Infants
with
family
history of
asthma
Atopic
pregnant
women
Subjects
Milk
based
suppl.
n=50
Olive oil
n=28
Olive oil
=136
Sunola
oil
n=149
Olive oil
n=46
Placebo
- 91 -
Tuna fish oil
500mg
(37% ω-3
LCPUFA)
low ω-6
cooking oil
n=159
2.7 g ω-3
LCPUFA
n=263
1.5g ω-3
LCPUFA
n=37
0.5 g DHA,
0.15 g EPA
n=49
Intervention
daily dose
2.2 g EPA,
1.1 g DHA,
0.4 g
other ω-3
PUFA
n=52
In vitro LPS stimulated IFNγ,
IL-10 production in children 2
1/2 yrs
Maternal and cord blood
mRNA: CCR4, IL-13, IL-4,
CRTH2, CXCR3, IFNγ, IL-1,
TGFβ
22nd gw delivery
mRNA levels of CCR4 ▼,
IL-4▼ and IL-13▼ in cord
blood .
IL-1▲IFNγ▲ in maternal
blood
IFNγ ▲
IL-10-no difference
eczema, ARC, asthma and
allergic asthma at 16 yrs.
30th gw delivery
First 4 m of
lactation
asthma▼
allergic asthma▼ at 16yrs
S-IgE, SPT, eczema up to 5
years
Allergy questionnaire
Lymphocyte cytokine responses
to allergen stimulation
6 months of age
or at weaning -5
years.
Cooking oil
from gw 36
T cell PKC zeta▲
SPT to egg at 1 year▼
SCORAD▼
Wheeze at 18 m ▼
Cough in atopic children at 3
years ▼
Cord blood plasma IL-13▼
MC response of IL-10 to cat
allergen▼
20th gw delivery
Plasma cytokine concentrations
in cord blood.
Cord blood MC cytokine
responses to allergens
SPT,
SCORAD 1year
Findings in ω-3 suppl. group
Exposure period Outcome measured
Table 9. Omega-3 supplementation in pregnancy and/or lactation. Clinical and immunological outcomes .
2007
2005
2008
2003-2006
Year
published
2003-2007
(273)
(212)
(198)
(199, 201)
(8, 9, 99)
ref
Phospholipid fatty acids in relation to allergic disease
In order to assess compliance, fatty acid status in serum/plasma phosholipids
were analysed in mothers and children. The dose and length of supplementation
varied in spite of our instructions to the mothers (Fig. 8) but the analysis of fatty
acid proportions in blood allowed us to relate maternal and infant ω-3 fatty acid
status to the clinical outcomes regardless of intervention group. We found an
inverse dose-response relationship between both maternal DHA status at
delivery and infant DHA status at 12 months and the prevalence of infant IgE
associated disease (Fig.10). This dose-response relationship supports causality
between ω-3 LCPUFA status and allergic disease according to the criteria by
Bradford-Hill (276). Still, it does not tell us the appropriate dose to prevent IgE
associated disease or if the dose should be given in pregnancy and/or lactation.
A previous study by Mirshahi et al investigated plasma levels of ω-3 fatty acids
in relation to allergic outcomes after ω-3 supplementation in early childhood. No
dose dependent relationship could be found but levels of infant plasma ω-3 fatty
acids in the higher quintiles at 18 months of age were associated with a reduced
prevalence of some asthma symptoms (199). During the last four weeks of
pregnancy and breastfeeding the mothers in the fish oil intervention group used
cooking oils and spreads based on canola oil to reduce the ω-6 LCPUFA intake
and the children were supplemented with 500 mg tuna fish oil daily, starting at
weaning or at 6 months, at the latest, and continuing through childhood. The
dose of ω-3 was low compared to our study and DHA and EPA were not
analysed separately. Furthermore, Mirshahi et al analysed fatty acid levels in the
infants at 18 months and not in maternal blood. These factors may explain the
different results.
- 92 -
AA/
EPA
EPA
DHA
0
20
40
60
0
20
40
60
0
20
40
60
80 100 %
0
20
40
60
24
0.4-1.0
- 93 -
0.4-1.0
80 100 %
20
22
25
1.0-4.6
1.1-4.6
22
27
4.6-8.9
80 100 %
21
0.2-0.4
4.6-9.0
80 100 %
22
22
0.6-0.8
0.5-0.6
20
0.8-2.6
80 100 %
21
#
60
40
9.0- 21.4
40
20
26
20
0
9.0- 21.4
0
29
0.5-0.6
25
24
0.6-0.8
0.2-0.5
24
80 100 %
0.8-2.6
#
60
21
2.0-3.1
26
2.5-5.0
22
3.1-3.9
20
n
26
5.0-6.3
#
22
5.0-8.6
Mol %
Infant PL at 12 months
of age
4.0-4.9
25
6.3-7.9
n
25
#
8.0- 11.3
Mol %
Maternal PL one week
after delivery
2.4-9.9
9.9-12
12.1-15
15.1-33
0.3-0.6
0.6-0.7
0.7-0.9
0.9-3.7
2.0-3.3
3.3-3.8
3.8-4.4
4.4-8.8
Mol %
0
0
0
20
20
20
40 60
40 60
40 60
80 100 %
21
20
19
21
#= Chi-2 for linear trend
within black bars, p<0.01.
Thus, the outcomes of allergic
disease and/or allergic
sensitisation in all children in
each quartile are represented.
Figure 10. Mother-infant
pairs divided into four groups
according to quartiles of
n
DHA, EPA proportions and
AA/EPA ratios in maternal
19
plasma phospholipids (PL)
one week after delivery, in
20
infant plasma PL at 12
months and in infant plasma
21
PL at 24 months.
Black bars: Infants with IgE
21
associated disease (allergic
80 100 % symptoms combined with
positive SPT and/or
circulating specific IgE).
Striped bars: Infants with
22
allergic symptoms but no
positive SPTs or detectable
19
specificIgE.
Dotted bars: Infants with
19
detectable IgE antibodies or
positive SPT without allergic
21
symptoms.
80 100 % White bars: Infants without
any allergic symptoms or
sensitisation.
Infant PL at 24 months
of age
The ω-3 LCPUFA seem to have an impact on development of allergic
sensitisation early in life but what is the immediate clinical consequence of this
result? There were no significant differences between the intervention groups in
terms of severity of allergic disease i.e. SCORAD (discussed in paper III) or
number of allergic symptoms. However, the infants with more than one
symptom of allergic disease had significantly lower serum/plasma proportions
of DHA and EPA and higher AA/EPA ratios at several timepoints than the
children with no symptoms (Table 4, paper III). In agreement with this, the risk
reduction associated with regular fish consumption at age 1 in an observational
study, appeared particularly pronounced for more severe allergic disease
measured as multiple allergic disease, i.e. two or three diseases (aOR 0.56, 0.35–
0.89) (190). We also found an association between sensitisation and number of
allergic symptoms (p= 0.001, paper III). This indicates that IgE associated
disease (atopic disease) presents itself with a more severe phenotype than
allergic disease without detectable sensitisation. A previous study has found that
children with severe eczema are sensitised to common allergens to a larger
extent than children with milder forms of eczema, which agrees with our
findings (277).
Possible gene-by-environment interaction
Children with allergies in the immediate family were recruited to this study in
order to get the expected incidence of allergic disease according to the power
calculation. Hence, both allergic and non-allergic mothers were included, even
though the study was not designed to find out differences between these two
groups of mothers. It was striking, however, how the differences between the
intervention groups in terms of infant sensitisation and IgE associated disease
were significant in the rather small group of non-allergic mothers while it was
not at all as evident in the group of allergic mothers (Table 11).
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Table 11. The main clinical outcomes from the study divided into groups
regarding maternal allergic history.
Cumulative
incidence
0-24 m, infants.
No maternal history of allergy
ω-3
n (%)
Placebo
n (%)
Maternal history of allergy
p‡
ω-3
n (%)
Placebo
n (%)
p‡
Any pos SPT*
2/14(14)
11/22(50)
0.03
8/38(21)
11/39(28)
0.6
Any detectable IgE*
4/13(31)
5/13(38)
1.0
7/31(23)
10/32(31)
0.6
Allergic symptoms¶
3/14(21)
12/24(50)
0.10
16/40(39)
16/41(40)
0.9
Food allergy
0/14(0)
8/24(33)
0.017
3/40(25)
6/41(15)
0.5
IgE mediated eczema
0/14(0)
7/24(29)
0.033
5/40(12)
8/39(21)
0.4
IgE associated disease
1/14(7)
9/23(39)
0.06
5/40(12)
10/39(26)
0.14
*: towards egg, milk, wheat, birch, timothy, cat.
¶: eczema, food reactions, asthma or rhinoconjunctivitis.
‡ : Chi-2 test (Fisher’s exact test if expected values were less than 5).
This is in agreement with the observational studies reporting that fish in the diet
during pregnancy offers protection from allergic sensitisation in children of non
allergic mothers (278) or without allergic heredity (190). Further, children of
non-allergic, but not of allergic mothers had a protective effect of breastfeeding
(supposedly through its content of LCPUFA) on eczema at two years of age
(167).
Our immunological findings follow the same pattern: the decrease in LPSinduced PGE2 secretion in the ω-3 LCPUFA-treated mothers was more
pronounced among non-atopic mothers (Fig. 1B, paper I). Further, the
correlation between changes in maternal LPS-induced PGE2 production during
intervention and changes in serum/plasma phospholipid EPA, was stronger in
non-atopic compared to atopic mothers (Fig. 2B, paper I). In children without,
but not with, maternal history of allergy, the ω-3 supplementation was related to
lower CCL17/CCL11 ratios (p<0.05, paper IV). When the cord blood samples in
the study by Krauss-Etschmann et al was stratified for maternal allergic disease,
the decrease of IL-13 mRNA levels in the fish oil group was more pronounced
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in cord blood samples from non-allergic mothers, which agrees with our results
(273). In the study by Dunstan, where the mothers receiving ω-3
supplementation (3.7 g/day) were all atopic, there was still a slight effect on
sensitisation to egg the first year of life and corroborating immunological
changes were noted (8, 9). The question is whether the allergic mothers need a
larger dose of EPA and/or DHA in order to get a protective effect in the child,
due to a FADS1/FADS2 genotype variant that has an impact on PUFA
metabolism (279). However, in disagreement with this we did not see any
differences in serum/plasma LCPUFA levels between allergic and non-allergic
mothers in our study (dns).
Furthermore in the non-allergic, but not in the allergic children, ω-3
supplementation was associated with higher Th1-associated CXCL11 levels
(p<0.05), as well as increased IgG titres to diphtheria (p=0.01) and tetanus
(p=0.05) toxins compared to placebo (paper IV). This could be representing a
modification of the immune responses toward more Th1 dominance in the nonallergic ω-3 supplemented children.
Even though these results should be interpreted carefully it may strengthen the
idea that the allergic status of the individual has an impact on the balancing
effect of ω-3 supplementation towards a less Th2 dominated immune response.
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CONCLUSIONS
This thesis was based on a double-blind placebo controlled trial, supplementing
women with ω-3 LCPUFA in pregnancy and lactation. Allergic disease in the
offspring in addition to maternal and infant immunological markers were
analysed.
Major observations:
I. Maternal lipopolysaccharide-induced PGE2 production was decreased in a
majority of the ω-3 LCPUFA supplemented mothers in contrast to the placebo
group who displayed an increase in the majority of cases (p=0.002). No effect
on lipopolysaccharide induced cytokine secretion was observed.
II. Maternal ω-3 fatty acid supplementation decreased the risk of food allergy
(i.e.IgE mediated food allergy) and IgE-associated eczema during the first year
of life in infants with a family history of allergic disease (p=0.01 and <0.05
respectively). No effect was seen on the prevalence of allergic symptoms per se
and the benefit for the families may be doubtful. However the decreased
incidence of sensitisation in the eczematous children may indicate a lower risk
of asthma later in childhood.
III. Maternal ω-3 fatty acid supplementation decreased the risk of any IgE
associated disease (asthma, rhinoconjunctivitis, eczema and /or food allergy)
during the two first years of life in infants with a family history of allergic
disease (p=0.01). The effect on eczema and food allergy seen the first year
remained during the second year. Maternal and infant serum/plasma
phospholipid ω-3 LCPUFA proportions were related to infant IgE associated
disease in an inversed dose response manner. Low ω-3 LCPUFA status in
mother and infant was related to increased severity of allergic disease expressed
- 97 -
as number of allergic manifestations. This indicates a clinical benefit for the
infants from the increased ω-3 LCPUFA proportions, which may be achieved
with maternal ω-3 LCPUFA supplementation.
IV. The levels of the circulating Th2 chemokine CCL17 were higher in infants
with eczema or any IgE associated disease but there were no significant
differences in the Th1 or Th2 chemokines or vaccine induced responses between
the ω-3 and the placebo group. Nevertheless, in the group of non-allergic
mothers there were decreased infant levels of CCL17 after ω-3 supplementation
and in the non-allergic children the immune responses against tetanus and
diphtheria were strengthened in the ω-3 group.
Thus, the prospect of balancing the infant immune system towards a less Th2
dominated response, by maternal ω-3 fatty acid supplementation, seems to be
influenced by allergic status. This is also supported by the findings in paper I-III
where maternal allergic status seemed to influence the effect of the ω-3
LCPUFA supplementation on maternal eicosanoid production as well as clinical
outcomes in the infants.
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FUTURE PERSPECTIVES
Analysis of fatty acids in breast milk is the next step in this study and it is
performed at present.
A follow-up of the children in this study at 8-9 years of age, focusing on
respiratory disease and sensitisation towards inhalants, is currently being
planned.
S-IgA and cytokines in faeces and saliva are interesting to analyse in the future
and relate to clinical outcomes.
It would also be interesting to analyse maternal FADS1/FADS2 polymorphisms
in relation to maternal allergy and clinical outcomes in the children.
Reports, mostly from animal studies, indicate that the anti-viral immune
responses are depressed by ω-3 fatty acids (280). To investigate this further it
would be appealing to assess vaccine-induced responses to morbilli, rubella and
paramyxo viruses in this study group.
- 99 -
- 100 -
ACKNOWLEDGEMENT
I am grateful to everyone who has contributed to this thesis:
All the children, mothers and fathers who participated in this study. Thank you
for your time and endurance.
My supervisor Karel Duchén for inviting me to participate in this project and for
invaluable encouragement along the way in the clinical as well as in the
scientific work.
My supervisor professor Karin Fälth-Magnusson for your endless support and
patience. Thank you for meticulously reading my manuscripts and for all the
scientific and clinical advice.
The midwives at the antenatal department “Kvinnohälsan” and at the maternal
health centre “Storken” who recruited the families.
The research nurses Lena Lindell, Kicki Helander, Nina Timelin and Linnea
Andersson for keeping track of our study-families, supporting and caring for
them in an outstanding way.
Kristina Warstedt, my co-worker, for sharing the mysteries of laboratory work
and leading the way through the PhD studies. Thank you for very important
contributions to our publications and for being “on call” for the births in our
study, day and night.
Malin Fagerås, co-author and supervisor, for lots of thoughtful ideas, and for
your support at our conference trips to Barcelona and Gothenburg.
Johanna Andersson, co-author, for the clinical assessment in Jönköping and for
always mailing me the data I need.
Maria Jenmalm, co-author, for enthusiasm and great knowledge.
Mats Fredriksson, co-author and statistician who never hesitates to answer
questions, explain and explain again the secrets of statistics.
Professor Johnny Ludvigsson for support and wise comments on my work
through the years.
Ammi Fornander for guidance in the laboratory and for performing some of the
laboratory works in this study
- 101 -
Lennart Nilsson and Thomas Abrahamsson for thoughtful comments on this
work throughout the years.
Nina Nelson, my former boss, for making it possible for me to finish this work
in a feasible way and for providing an outstanding PhD course on research in
children.
Christina Johansson, my boss at the paediatric clinic, for patience and support
during the final part of this work.
All friends at KEF for good discussions and scheduled coffee breaks.
All my friends, of all professions, working at the paediatric clinic, for believing
in me, supporting me and making me enjoy clinical work. Special thanks to
Farhad Vahedi and Emma Olsson and all co-workers at “BAVA”, for being
patient with my absence.
My parents Eva and Jan for being role models and for actively sharing the
delight of our children.
My mother-in-law Elisabeth for all the care you give our family.
Our children: Karin, Johan and Gustav for patiently sharing my computer with
me these past years. You and baby David fill my life with joy!
My husband Jörgen for love, happiness and encouragement always.
This work was supported by grants from Medical Research Council of SouthEast Sweden (FORSS), Östergötland County Council, The Ekhaga Foundation,
Swedish Asthma and Allergy association,The Research Council for
Environmental, Agricultural Sciences and Spatial Planning (FORMAS), The
Swedish Society of Medicine, The Swedish Medical Research Council (VR)
Glaxo Smith Kline and Lions Club Gnesta. The fish oil capsules were supplied
by Pharma Nord Denmark, at a reduced cost.
- 102 -
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
van Odijk J, Hulthen L, Ahlstedt S, Borres MP. Introduction of food during the
infant's first year: a study with emphasis on introduction of gluten and of egg, fish and
peanut in allergy-risk families. Acta Paediatr 2004;93:464-70.
Asher MI, Montefort S, Bjorksten B, Lai CK, Strachan DP, Weiland SK, et al.
Worldwide time trends in the prevalence of symptoms of asthma, allergic
rhinoconjunctivitis, and eczema in childhood: ISAAC Phases One and Three repeat
multicountry cross-sectional surveys. Lancet 2006;368:733-43.
Weiland SK, von Mutius E, Husing A, Asher MI. Intake of trans fatty acids and
prevalence of childhood asthma and allergies in Europe. ISAAC Steering Committee.
Lancet 1999;353:2040-1.
Black PN. The prevalence of allergic disease and linoleic acid in the diet. J Allergy
Clin Immunol 1999;103:351-2.
Krause TG, Koch A, Poulsen LK, Kristensen B, Olsen OR, Melbye M. Atopic
sensitization among children in an arctic environment. Clin Exp Allergy 2002;32:36772.
Krause T, Koch A, Friborg J, Poulsen LK, Kristensen B, Melbye M. Frequency of
atopy in the Arctic in 1987 and 1998. Lancet 2002;360:691-2.
Woods RK, Thien FC, Abramson MJ. Dietary marine fatty acids (fish oil) for asthma
in adults and children. Cochrane Database Syst Rev 2002:CD001283.
Dunstan JA, Mori TA, Barden A, Beilin LJ, Taylor AL, Holt PG, et al. Maternal fish
oil supplementation in pregnancy reduces interleukin-13 levels in cord blood of
infants at high risk of atopy. Clin Exp Allergy 2003;33:442-8.
Dunstan JA, Mori TA, Barden A, Beilin LJ, Taylor AL, Holt PG, et al. Fish oil
supplementation in pregnancy modifies neonatal allergen-specific immune responses
and clinical outcomes in infants at high risk of atopy: a randomized, controlled trial. J
Allergy Clin Immunol 2003;112:1178-84.
Johansson SG, Bieber T, Dahl R, Friedmann PS, Lanier BQ, Lockey RF, et al.
Revised nomenclature for allergy for global use: Report of the Nomenclature Review
Committee of the World Allergy Organization, October 2003. J Allergy Clin Immunol
2004;113:832-6.
Hahn EL, Bacharier LB. The atopic march: the pattern of allergic disease development
in childhood. Immunol Allergy Clin North Am 2005;25:231-46, v.
Scadding GK. Further marches: allergic and non-allergic. Clin Exp Allergy
2007;37:485-7.
Illi S, von Mutius E, Lau S, Nickel R, Gruber C, Niggemann B, et al. The natural
course of atopic dermatitis from birth to age 7 years and the association with asthma. J
Allergy Clin Immunol 2004;113:925-31.
Mölne JW, A. Inflammation. first ed. Nacka: Liber; 2007.
Schmid-Grendelmeier P, Simon D, Simon HU, Akdis CA, Wuthrich B. Epidemiology,
clinical features, and immunology of the "intrinsic" (non-IgE-mediated) type of atopic
dermatitis (constitutional dermatitis). Allergy 2001;56:841-9.
Mandic Z, Marusic M, Boranic M. Low levels of immunoglobulin A in children with
intrinsic asthma: a possible protection against atopy. Med Hypotheses 2004;62:600-4.
Eller E, Kjaer HF, Host A, Andersen KE, Bindslev-Jensen C. Food allergy and food
sensitization in early childhood: results from the DARC cohort. Allergy
2009;64:1023-9.
- 103 -
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
Arshad SH, Tariq SM, Matthews S, Hakim E. Sensitization to common allergens and
its association with allergic disorders at age 4 years: a whole population birth cohort
study. Pediatrics 2001;108:E33.
Kjaer HF, Eller E, Andersen KE, Host A, Bindslev-Jensen C. The association between
early sensitization patterns and subsequent allergic disease. The DARC birth cohort
study. Pediatr Allergy Immunol 2009;20:726-34.
Lowe AJ, Hosking CS, Bennett CM, Carlin JB, Abramson MJ, Hill DJ, et al. Skin
prick test can identify eczematous infants at risk of asthma and allergic rhinitis. Clin
Exp Allergy 2007;37:1624-31.
Hattevig G, Kjellman B, Bjorksten B. Appearance of IgE antibodies to ingested and
inhaled allergens during the first 12 years of life in atopic and non-atopic children.
Pediatr Allergy Immunol 1993;4:182-6.
Kulig M, Bergmann R, Tacke U, Wahn U, Guggenmoos-Holzmann I. Long-lasting
sensitization to food during the first two years precedes allergic airway disease. The
MAS Study Group, Germany. Pediatr Allergy Immunol 1998;9:61-7.
Benhamou AH, Schappi Tempia MG, Belli DC, Eigenmann PA. An overview of
cow's milk allergy in children. Swiss Med Wkly 2009;139:300-7.
Sicherer SH, Sampson HA. 9. Food allergy. J Allergy Clin Immunol 2006;117:S4705.
Sampson HA. Food allergy. Part 2: diagnosis and management. J Allergy Clin
Immunol 1999;103:981-9.
Sicherer SH. Food allergy: when and how to perform oral food challenges. Pediatr
Allergy Immunol 1999;10:226-34.
Wood RA. The natural history of food allergy. Pediatrics 2003;111:1631-7.
Savage JH, Matsui EC, Skripak JM, Wood RA. The natural history of egg allergy. J
Allergy Clin Immunol 2007;120:1413-7.
Host A, Halken S. A prospective study of cow milk allergy in Danish infants during
the first 3 years of life. Clinical course in relation to clinical and immunological type
of hypersensitivity reaction. Allergy 1990;45:587-96.
Östblom E, Lilja G, Pershagen G, van Hage M, Wickman M. Phenotypes of food
hypersensitivity and development of allergic diseases during the first 8 years of life.
Clin Exp Allergy 2008;38:1325-32.
Chehade M, Mayer L. Oral tolerance and its relation to food hypersensitivities. J
Allergy Clin Immunol 2005;115:3-12; quiz 13.
van Wijk F, Knippels L. Initiating mechanisms of food allergy: Oral tolerance versus
allergic sensitization. Biomed Pharmacother 2007;61:8-20.
Abrahamsson T. Dissertation no 1126: Can Lactobacillus reuteri prevent allergic
disease in early childhood. Linköping, Sweden: Faculty of Health Sciences, Linköping
University; 2009.
Halkjaer LB, Loland L, Buchvald FF, Agner T, Skov L, Strand M, et al. Development
of atopic dermatitis during the first 3 years of life: the Copenhagen prospective study
on asthma in childhood cohort study in high-risk children. Arch Dermatol
2006;142:561-6.
Oranje AP. Development of childhood eczema and its classification. Pediatr Allergy
Immunol 1995;6 Suppl 7:31-5.
Hanifin JM RG. Diagnostic features of atopic dermatitis. Acta Derm Venereol
(Stockh). 1980;92(suppl):44-47.
Seymour JL, Keswick BH, Hanifin JM, Jordan WP, Milligan MC. Clinical effects of
diaper types on the skin of normal infants and infants with atopic dermatitis. J Am
Acad Dermatol 1987;17:988-97.
- 104 -
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
Johnke H, Vach W, Norberg LA, Bindslev-Jensen C, Host A, Andersen KE. A
comparison between criteria for diagnosing atopic eczema in infants. Br J Dermatol
2005;153:352-8.
Palmer CN, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, et al.
Common loss-of-function variants of the epidermal barrier protein filaggrin are a
major predisposing factor for atopic dermatitis. Nat Genet 2006;38:441-6.
Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell death in the
skin. Nat Rev Mol Cell Biol 2005;6:328-40.
Mischke D, Korge BP, Marenholz I, Volz A, Ziegler A. Genes encoding structural
proteins of epidermal cornification and S100 calcium-binding proteins form a gene
complex ("epidermal differentiation complex") on human chromosome 1q21. J Invest
Dermatol 1996;106:989-92.
Weidinger S, Illig T, Baurecht H, Irvine AD, Rodriguez E, Diaz-Lacava A, et al. Lossof-function variations within the filaggrin gene predispose for atopic dermatitis with
allergic sensitizations. J Allergy Clin Immunol 2006;118:214-9.
Lowe AJ, Abramson MJ, Hosking CS, Carlin JB, Bennett CM, Dharmage SC, et al.
The temporal sequence of allergic sensitization and onset of infantile eczema. Clin
Exp Allergy 2007;37:536-42.
Almqvist C, Li Q, Britton WJ, Kemp AS, Xuan W, Tovey ER, et al. Early predictors
for developing allergic disease and asthma: examining separate steps in the 'allergic
march'. Clin Exp Allergy 2007;37:1296-302.
van den Oord RA, Sheikh A. Filaggrin gene defects and risk of developing allergic
sensitisation and allergic disorders: systematic review and meta-analysis. Bmj
2009;339:b2433.
Brand PL, Baraldi E, Bisgaard H, Boner AL, Castro-Rodriguez JA, Custovic A, et al.
Definition, assessment and treatment of wheezing disorders in preschool children: an
evidence-based approach. Eur Respir J 2008;32:1096-110.
Lau S, Nickel R, Niggemann B, Gruber C, Sommerfeld C, Illi S, et al. The
development of childhood asthma: lessons from the German Multicentre Allergy
Study (MAS). Paediatr Respir Rev 2002;3:265-72.
Garde J, Hervas D, Marco N, Milan JM, Dolores Martos M. Calculating the
prevalence of atopy in children. Allergol Immunopathol (Madr) 2009;37:129-34.
Calder PC, Kew S. The immune system: a target for functional foods? Br J Nutr
2002;88 Suppl 2:S165-77.
Liu AH. Innate microbial sensors and their relevance to allergy. J Allergy Clin
Immunol 2008;122:846-58; quiz 858-60.
Gould HJ, Sutton BJ, Beavil AJ, Beavil RL, McCloskey N, Coker HA, et al. The
biology of IGE and the basis of allergic disease. Annu Rev Immunol 2003;21:579628.
Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for
costimulation and inhibition of immune responses. Annu Rev Immunol 2002;20:2953.
Commins SP, Borish L, Steinke JW. Immunologic messenger molecules: cytokines,
interferons, and chemokines. J Allergy Clin Immunol;125:S53-72.
Corthay A. How do regulatory T cells work? Scand J Immunol 2009;70:326-36.
Zhu J, Paul WE. CD4 T cells: fates, functions, and faults. Blood 2008;112:1557-69.
Paul WE, Zhu J. How are T(H)2-type immune responses initiated and amplified? Nat
Rev Immunol;10:225-35.
Enelow R, Baramki DF, Borish LC. Inhibition of effector T lymphocytes mediated
through antagonism of IL-4. J Allergy Clin Immunol 2004;113:560-2.
- 105 -
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
Schleimer RP, Sterbinsky SA, Kaiser J, Bickel CA, Klunk DA, Tomioka K, et al. IL-4
induces adherence of human eosinophils and basophils but not neutrophils to
endothelium. Association with expression of VCAM-1. J Immunol 1992;148:1086-92.
Larche M. Regulatory T cells in allergy and asthma. Chest 2007;132:1007-14.
Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the
expanding diversity of effector T cell lineages. Annu Rev Immunol 2007;25:821-52.
Saeki H, Tamaki K. Thymus and activation regulated chemokine (TARC)/CCL17 and
skin diseases. J Dermatol Sci 2006;43:75-84.
Pease JE, Williams TJ. Chemokines and their receptors in allergic disease. J Allergy
Clin Immunol 2006;118:305-18; quiz 319-20.
Sandberg M, Frykman A, Ernerudh J, Berg G, Matthiesen L, Ekerfelt C, et al. Cord
blood cytokines and chemokines and development of allergic disease. Pediatr Allergy
Immunol 2009;20:519-27.
Fujisawa T, Fujisawa R, Kato Y, Nakayama T, Morita A, Katsumata H, et al. Presence
of high contents of thymus and activation-regulated chemokine in platelets and
elevated plasma levels of thymus and activation-regulated chemokine and
macrophage-derived chemokine in patients with atopic dermatitis. J Allergy Clin
Immunol 2002;110:139-46.
Kakinuma T, Nakamura K, Wakugawa M, Mitsui H, Tada Y, Saeki H, et al. Thymus
and activation-regulated chemokine in atopic dermatitis: Serum thymus and
activation-regulated chemokine level is closely related with disease activity. J Allergy
Clin Immunol 2001;107:535-41.
Terada N, Nomura T, Kim WJ, Otsuka Y, Takahashi R, Kishi H, et al. Expression of
C-C chemokine TARC in human nasal mucosa and its regulation by cytokines. Clin
Exp Allergy 2001;31:1923-31.
Hartl D, Griese M, Nicolai T, Zissel G, Prell C, Konstantopoulos N, et al. Pulmonary
chemokines and their receptors differentiate children with asthma and chronic cough. J
Allergy Clin Immunol 2005;115:728-36.
Singh UP, Singh S, Iqbal N, Weaver CT, McGhee JR, Lillard JW, Jr. IFN-gammainducible chemokines enhance adaptive immunity and colitis. J Interferon Cytokine
Res 2003;23:591-600.
Fulkerson PC, Zimmermann N, Hassman LM, Finkelman FD, Rothenberg ME.
Pulmonary chemokine expression is coordinately regulated by STAT1, STAT6, and
IFN-gamma. J Immunol 2004;173:7565-74.
Platts-Mills TA. The role of immunoglobulin E in allergy and asthma. Am J Respir
Crit Care Med 2001;164:S1-5.
Hales BJ, Martin AC, Pearce LJ, Laing IA, Hayden CM, Goldblatt J, et al. IgE and
IgG anti-house dust mite specificities in allergic disease. J Allergy Clin Immunol
2006;118:361-7.
Stapel SO, Asero R, Ballmer-Weber BK, Knol EF, Strobel S, Vieths S, et al. Testing
for IgG4 against foods is not recommended as a diagnostic tool: EAACI Task Force
Report. Allergy 2008;63:793-6.
Aalberse RC, Stapel SO, Schuurman J, Rispens T. Immunoglobulin G4: an odd
antibody. Clin Exp Allergy 2009;39:469-77.
Tomicic S, Norrman G, Falth-Magnusson K, Jenmalm MC, Devenney I, Bottcher MF.
High levels of IgG4 antibodies to foods during infancy are associated with tolerance to
corresponding foods later in life. Pediatr Allergy Immunol 2009;20:35-41.
Linneberg A. Are we getting enough allergens? Int Arch Allergy Immunol
2008;147:93-100.
Woof JM, Kerr MA. The function of immunoglobulin A in immunity. J Pathol
2006;208:270-82.
- 106 -
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
Bochner BS, Gleich GJ. What targeting eosinophils has taught us about their role in
diseases. J Allergy Clin Immunol;126:16-25; quiz 26-7.
Boyce JA. Mast cells: beyond IgE. J Allergy Clin Immunol 2003;111:24-32; quiz 33.
Herz U, Petschow B. Perinatal events affecting the onset of allergic diseases. Curr
Drug Targets Inflamm Allergy 2005;4:523-9.
Watanabe M, Iwatani Y, Kaneda T, Hidaka Y, Mitsuda N, Morimoto Y, et al.
Changes in T, B, and NK lymphocyte subsets during and after normal pregnancy. Am
J Reprod Immunol 1997;37:368-77.
Warner JO. The early life origins of asthma and related allergic disorders. Arch Dis
Child 2004;89:97-102.
Barrett EG. Maternal influence in the transmission of asthma susceptibility. Pulm
Pharmacol Ther 2008;21:474-84.
Raghupathy R. Th1-type immunity is incompatible with successful pregnancy.
Immunol Today 1997;18:478-82.
Mjosberg J, Svensson J, Johansson E, Hellstrom L, Casas R, Jenmalm MC, et al.
Systemic reduction of functionally suppressive CD4dimCD25highFoxp3+ Tregs in
human second trimester pregnancy is induced by progesterone and 17beta-estradiol. J
Immunol 2009;183:759-69.
Amoudruz P, Minang JT, Sundstrom Y, Nilsson C, Lilja G, Troye-Blomberg M, et al.
Pregnancy, but not the allergic status, influences spontaneous and induced interleukin1beta (IL-1beta), IL-6, IL-10 and IL-12 responses. Immunology 2006;119:18-26.
Sandberg M, Frykman A, Jonsson Y, Persson M, Ernerudh J, Berg G, et al. Total and
allergen-specific IgE levels during and after pregnancy in relation to maternal allergy.
J Reprod Immunol 2009;81:82-8.
Chaouat G. The Th1/Th2 paradigm: still important in pregnancy? Semin
Immunopathol 2007;29:95-113.
Papadogiannakis N. Traffic of leukocytes through the maternofetal placental interface
and its possible consequences. Curr Top Microbiol Immunol 1997;222:141-57.
Hanson LA, Korotkova M, Lundin S, Haversen L, Silfverdal SA, Mattsby-Baltzer I, et
al. The transfer of immunity from mother to child. Ann N Y Acad Sci 2003;987:199206.
Holloway JA, Warner JO, Vance GH, Diaper ND, Warner JA, Jones CA. Detection of
house-dust-mite allergen in amniotic fluid and umbilical-cord blood. Lancet
2000;356:1900-2.
Casas R, Bjorksten B. Detection of Fel d 1-immunoglobulin G immune complexes in
cord blood and sera from allergic and non-allergic mothers. Pediatr Allergy Immunol
2001;12:59-64.
Jones AC, Miles EA, Warner JO, Colwell BM, Bryant TN, Warner JA. Fetal
peripheral blood mononuclear cell proliferative responses to mitogenic and allergenic
stimuli during gestation. Pediatr Allergy Immunol 1996;7:109-16.
Thornton CA, Upham JW, Wikstrom ME, Holt BJ, White GP, Sharp MJ, et al.
Functional maturation of CD4+CD25+CTLA4+CD45RA+ T regulatory cells in
human neonatal T cell responses to environmental antigens/allergens. J Immunol
2004;173:3084-92.
Prescott SL, Macaubas C, Holt BJ, Smallacombe TB, Loh R, Sly PD, et al.
Transplacental priming of the human immune system to environmental allergens:
universal skewing of initial T cell responses toward the Th2 cytokine profile. J
Immunol 1998;160:4730-7.
Rowe J, Kusel M, Holt BJ, Suriyaarachchi D, Serralha M, Hollams E, et al. Prenatal
versus postnatal sensitization to environmental allergens in a high-risk birth cohort. J
Allergy Clin Immunol 2007;119:1164-73.
- 107 -
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
Holt PG, Clough JB, Holt BJ, Baron-Hay MJ, Rose AH, Robinson BW, et al. Genetic
'risk' for atopy is associated with delayed postnatal maturation of T-cell competence.
Clin Exp Allergy 1992;22:1093-9.
Herberth G, Heinrich J, Roder S, Figl A, Weiss M, Diez U, et al. Reduced IFNgamma- and enhanced IL-4-producing CD4+ cord blood T cells are associated with a
higher risk for atopic dermatitis during the first 2 yr of life. Pediatr Allergy
Immunol;21:5-13.
Langrish CL, Buddle JC, Thrasher AJ, Goldblatt D. Neonatal dendritic cells are
intrinsically biased against Th-1 immune responses. Clin Exp Immunol 2002;128:11823.
Prescott SL, Irvine J, Dunstan JA, Hii C, Ferrante A. Protein kinase Czeta: a novel
protective neonatal T-cell marker that can be upregulated by allergy prevention
strategies. J Allergy Clin Immunol 2007;120:200-6.
Jones CA, Vance GH, Power LL, Pender SL, Macdonald TT, Warner JO.
Costimulatory molecules in the developing human gastrointestinal tract: a pathway for
fetal allergen priming. J Allergy Clin Immunol 2001;108:235-41.
Bonnelykke K, Pipper CB, Bisgaard H. Sensitization does not develop in utero. J
Allergy Clin Immunol 2008;121:646-51.
Bertino E, Bisson C, Martano C, Coscia A, Fabris C, Monti G, et al. Relationship
between maternal- and fetal-specific IgE. Pediatr Allergy Immunol 2006;17:484-8.
Nambu M, Shintaku N, Ohta S. Relationship between cord blood level of IgE specific
for Dermatophagoides pteronyssinus and allergic manifestations in infancy. Biol
Neonate 2003;83:102-6.
von Mutius E, Radon K. Living on a farm: impact on asthma induction and clinical
course. Immunol Allergy Clin North Am 2008;28:631-47, ix-x.
Ege MJ, Bieli C, Frei R, van Strien RT, Riedler J, Ublagger E, et al. Prenatal farm
exposure is related to the expression of receptors of the innate immunity and to atopic
sensitization in school-age children. J Allergy Clin Immunol 2006;117:817-23.
Jones CA, Holloway JA, Warner JO. Does atopic disease start in foetal life? Allergy
2000;55:2-10.
Flicker S, Marth K, Kofler H, Valenta R. Placental transfer of allergen-specific IgG
but not IgE from a specific immunotherapy-treated mother. J Allergy Clin Immunol
2009;124:1358-60 e1.
Ruiz RG, Richards D, Kemeny DM, Price JF. Neonatal IgE: a poor screen for atopic
disease. Clin Exp Allergy 1991;21:467-72.
Croner S. Prediction and detection of allergy development: influence of genetic and
environmental factors. J Pediatr 1992;121:S58-63.
Lin YC, Wen HJ, Lee YL, Guo YL. Are maternal psychosocial factors associated with
cord immunoglobulin E in addition to family atopic history and mother
immunoglobulin E? Clin Exp Allergy 2004;34:548-54.
Kaan A, Dimich-Ward H, Manfreda J, Becker A, Watson W, Ferguson A, et al. Cord
blood IgE: its determinants and prediction of development of asthma and other allergic
disorders at 12 months. Ann Allergy Asthma Immunol 2000;84:37-42.
Bjerke T, Hedegaard M, Henriksen TB, Nielsen BW, Schiotz PO. Several genetic and
environmental factors influence cord blood IgE concentration. Pediatr Allergy
Immunol 1994;5:88-94.
Liu CA, Wang CL, Chuang H, Ou CY, Hsu TY, Yang KD. Prediction of elevated cord
blood IgE levels by maternal IgE levels, and the neonate's gender and gestational age.
Chang Gung Med J 2003;26:561-9.
Magnusson CG. Cord serum IgE in relation to family history and as predictor of
atopic disease in early infancy. Allergy 1988;43:241-51.
- 108 -
115.
116.
117.
118.
119.
120.
121.
122.
123.
124.
125.
126.
127.
128.
129.
130.
131.
132.
133.
134.
Miyake Y, Sasaki S, Tanaka K, Hirota Y. Consumption of vegetables, fruit, and
antioxidants during pregnancy and wheeze and eczema in infants. Allergy.
Romieu I, Torrent M, Garcia-Esteban R, Ferrer C, Ribas-Fito N, Anto JM, et al.
Maternal fish intake during pregnancy and atopy and asthma in infancy. Clin Exp
Allergy 2007;37:518-25.
Willers SM, Devereux G, Craig LC, McNeill G, Wijga AH, Abou El-Magd W, et al.
Maternal food consumption during pregnancy and asthma, respiratory and atopic
symptoms in 5-year-old children. Thorax 2007;62:773-9.
Braback L, Kjellman NI, Sandin A, Bjorksten B. Atopy among schoolchildren in
northern and southern Sweden in relation to pet ownership and early life events.
Pediatr Allergy Immunol 2001;12:4-10.
Koplin J, Allen K, Gurrin L, Osborne N, Tang ML, Dharmage S. Is caesarean delivery
associated with sensitization to food allergens and IgE-mediated food allergy: a
systematic review. Pediatr Allergy Immunol 2008;19:682-7.
Bager P, Wohlfahrt J, Westergaard T. Caesarean delivery and risk of atopy and
allergic disease: meta-analyses. Clin Exp Allergy 2008;38:634-42.
Stene LC, Nafstad P. Relation between occurrence of type 1 diabetes and asthma.
Lancet 2001;357:607-8.
Rook GA, Rosa-Brunet L, Hunt J. Cat allergen and sensitisation. Lancet
2001;357:2137-8.
Cottrez F, Hurst SD, Coffman RL, Groux H. T regulatory cells 1 inhibit a Th2-specific
response in vivo. J Immunol 2000;165:4848-53.
Bergmann RL, Edenharter G, Bergmann KE, Guggenmoos-Holzmann I, Forster J,
Bauer CP, et al. Predictability of early atopy by cord blood-IgE and parental history.
Clin Exp Allergy 1997;27:752-60.
Björkstén B, Kjellman NI. Perinatal factors influencing the development of allergy.
Clin Rev Allergy 1987;5:339-47.
Halken S. Prevention of allergic disease in childhood: clinical and epidemiological
aspects of primary and secondary allergy prevention. Pediatr Allergy Immunol
2004;15 Suppl 16:4-5, 9-32.
Miller RL, Ho SM. Environmental epigenetics and asthma: current concepts and call
for studies. Am J Respir Crit Care Med 2008;177:567-73.
Romieu I, Moreno-Macias H, London SJ. Gene by environment interaction and
ambient air pollution. Proc Am Thorac Soc;7:116-22.
Ober C, Thompson EE. Rethinking genetic models of asthma: the role of
environmental modifiers. Curr Opin Immunol 2005;17:670-8.
Nafstad P, Magnus P, Gaarder PI, Jaakkola JJ. Exposure to pets and atopy-related
diseases in the first 4 years of life. Allergy 2001;56:307-12.
Nicolai T, Illi S, von Mutius E. Effect of dampness at home in childhood on bronchial
hyperreactivity in adolescence. Thorax 1998;53:1035-40.
Hagendorens MM, Ebo DG, Bridts CH, Van de Water L, De Clerck LS, Stevens WJ.
Prenatal exposure to house dust mite allergen (Der p 1), cord blood T cell phenotype
and cytokine production and atopic dermatitis during the first year of life. Pediatr
Allergy Immunol 2004;15:308-15.
Hamelmann E, Herz U, Holt P, Host A, Lauener RP, Matricardi PM, et al. New
visions for basic research and primary prevention of pediatric allergy: an iPAC
summary and future trends. Pediatr Allergy Immunol 2008;19 Suppl 19:4-16.
Kramer MS, Kakuma R. Maternal dietary antigen avoidance during pregnancy or
lactation, or both, for preventing or treating atopic disease in the child. Cochrane
Database Syst Rev 2006;3:CD000133.
- 109 -
135.
136.
137.
138.
139.
140.
141.
142.
143.
144.
145.
146.
147.
148.
149.
150.
151.
Falth-Magnusson K, Kjellman NI. Allergy prevention by maternal elimination diet
during late pregnancy--a 5-year follow-up of a randomized study. J Allergy Clin
Immunol 1992;89:709-13.
Lilja G, Dannaeus A, Foucard T, Graff-Lonnevig V, Johansson SG, Oman H. Effects
of maternal diet during late pregnancy and lactation on the development of atopic
diseases in infants up to 18 months of age--in-vivo results. Clin Exp Allergy
1989;19:473-9.
Prescott SL, Smith P, Tang M, Palmer DJ, Sinn J, Huntley SJ, et al. The importance of
early complementary feeding in the development of oral tolerance: concerns and
controversies. Pediatr Allergy Immunol 2008;19:375-80.
Host A, Halken S, Muraro A, Dreborg S, Niggemann B, Aalberse R, et al. Dietary
prevention of allergic diseases in infants and small children. Pediatr Allergy Immunol
2008;19:1-4.
Brand PL, Vlieg-Boerstra BJ, Dubois AE. Dietary prevention of allergic disease in
children: are current recommendations really based on good evidence? Pediatr Allergy
Immunol 2007;18:475-9.
Strachan DP, Cook DG. Health effects of passive smoking.5. Parental smoking and
allergic sensitisation in children. Thorax 1998;53:117-23.
Gilliland FD, Li YF, Gong H, Jr., Diaz-Sanchez D. Glutathione s-transferases M1 and
P1 prevent aggravation of allergic responses by secondhand smoke. Am J Respir Crit
Care Med 2006;174:1335-41.
Melen E, Nyberg F, Lindgren CM, Berglind N, Zucchelli M, Nordling E, et al.
Interactions between glutathione S-transferase P1, tumor necrosis factor, and trafficrelated air pollution for development of childhood allergic disease. Environ Health
Perspect 2008;116:1077-84.
Gilliland FD, Li YF, Dubeau L, Berhane K, Avol E, McConnell R, et al. Effects of
glutathione S-transferase M1, maternal smoking during pregnancy, and environmental
tobacco smoke on asthma and wheezing in children. Am J Respir Crit Care Med
2002;166:457-63.
Segerstrom SC, Miller GE. Psychological stress and the human immune system: a
meta-analytic study of 30 years of inquiry. Psychol Bull 2004;130:601-30.
Garn H, Renz H. Epidemiological and immunological evidence for the hygiene
hypothesis. Immunobiology 2007;212:441-52.
von Mutius E, Pearce N, Beasley R, Cheng S, von Ehrenstein O, Bjorksten B, et al.
International patterns of tuberculosis and the prevalence of symptoms of asthma,
rhinitis, and eczema. Thorax 2000;55:449-53.
Bieli C, Eder W, Frei R, Braun-Fahrlander C, Klimecki W, Waser M, et al. A
polymorphism in CD14 modifies the effect of farm milk consumption on allergic
diseases and CD14 gene expression. J Allergy Clin Immunol 2007;120:1308-15.
Gurka MJ, Blackman JA, Heymann PW. Risk of childhood asthma in relation to the
timing of early child care exposures. J Pediatr 2009;155:781-787 e1.
Cramer C, Link E, Bauer CP, Hoffmann U, von Berg A, Lehmann I, et al. Association
between attendance of day care centres and increased prevalence of eczema in the
German birth cohort study LISAplus. Allergy.
Hoffjan S, Nicolae D, Ostrovnaya I, Roberg K, Evans M, Mirel DB, et al. Geneenvironment interaction effects on the development of immune responses in the 1st
year of life. Am J Hum Genet 2005;76:696-704.
Björkstén B, Sepp E, Julge K, Voor T, Mikelsaar M. Allergy development and the
intestinal microflora during the first year of life. J Allergy Clin Immunol
2001;108:516-20.
- 110 -
152.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
164.
165.
166.
167.
Abrahamsson TR, Jakobsson T, Bottcher MF, Fredrikson M, Jenmalm MC, Bjorksten
B, et al. Probiotics in prevention of IgE-associated eczema: a double-blind,
randomized, placebo-controlled trial. J Allergy Clin Immunol 2007;119:1174-80.
Kalliomaki M, Salminen S, Poussa T, Arvilommi H, Isolauri E. Probiotics and
prevention of atopic disease: 4-year follow-up of a randomised placebo-controlled
trial. Lancet 2003;361:1869-71.
Korppi M, Piippo-Savolainen E, Korhonen K, Remes S. Respiratory morbidity 20
years after RSV infection in infancy. Pediatr Pulmonol 2004;38:155-60.
Valkonen H, Waris M, Ruohola A, Ruuskanen O, Heikkinen T. Recurrent wheezing
after respiratory syncytial virus or non-respiratory syncytial virus bronchiolitis in
infancy: a 3-year follow-up. Allergy 2009;64:1359-65.
Thomsen SF, van der Sluis S, Stensballe LG, Posthuma D, Skytthe A, Kyvik KO, et
al. Exploring the association between severe respiratory syncytial virus infection and
asthma: a registry-based twin study. Am J Respir Crit Care Med 2009;179:1091-7.
Singh AM, Moore PE, Gern JE, Lemanske RF, Jr., Hartert TV. Bronchiolitis to
asthma: a review and call for studies of gene-virus interactions in asthma causation.
Am J Respir Crit Care Med 2007;175:108-19.
Thomas M, Custovic A, Woodcock A, Morris J, Simpson A, Murray CS. Atopic
wheezing and early life antibiotic exposure: a nested case-control study. Pediatr
Allergy Immunol 2006;17:184-8.
Kummeling I, Stelma FF, Dagnelie PC, Snijders BE, Penders J, Huber M, et al. Early
life exposure to antibiotics and the subsequent development of eczema, wheeze, and
allergic sensitization in the first 2 years of life: the KOALA Birth Cohort Study.
Pediatrics 2007;119:e225-31.
Wickens K, Ingham T, Epton M, Pattemore P, Town I, Fishwick D, et al. The
association of early life exposure to antibiotics and the development of asthma,
eczema and atopy in a birth cohort: confounding or causality? Clin Exp Allergy
2008;38:1318-24.
Yang YW, Tsai CL, Lu CY. Exclusive breastfeeding and incident atopic dermatitis in
childhood: a systematic review and meta-analysis of prospective cohort studies. Br J
Dermatol 2009;161:373-83.
Gdalevich M, Mimouni D, David M, Mimouni M. Breast-feeding and the onset of
atopic dermatitis in childhood: a systematic review and meta-analysis of prospective
studies. J Am Acad Dermatol 2001;45:520-7.
Kull I, Bohme M, Wahlgren CF, Nordvall L, Pershagen G, Wickman M. Breastfeeding reduces the risk for childhood eczema. J Allergy Clin Immunol 2005;116:65761.
Kull I, Melen E, Alm J, Hallberg J, Svartengren M, van Hage M, et al. Breast-feeding
in relation to asthma, lung function, and sensitization in young schoolchildren. J
Allergy Clin Immunol;125:1013-9.
Sears MR, Greene JM, Willan AR, Taylor DR, Flannery EM, Cowan JO, et al. Longterm relation between breastfeeding and development of atopy and asthma in children
and young adults: a longitudinal study. Lancet 2002;360:901-7.
Bergmann RL, Diepgen TL, Kuss O, Bergmann KE, Kujat J, Dudenhausen JW, et al.
Breastfeeding duration is a risk factor for atopic eczema. Clin Exp Allergy
2002;32:205-9.
Snijders BE, Thijs C, Dagnelie PC, Stelma FF, Mommers M, Kummeling I, et al.
Breast-feeding duration and infant atopic manifestations, by maternal allergic status,
in the first 2 years of life (KOALA study). J Pediatr 2007;151:347-51, 351 e1-2.
- 111 -
168.
169.
170.
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
181.
182.
183.
184.
185.
186.
187.
188.
Martindale S, McNeill G, Devereux G, Campbell D, Russell G, Seaton A. Antioxidant
intake in pregnancy in relation to wheeze and eczema in the first two years of life. Am
J Respir Crit Care Med 2005;171:121-8.
Fogarty A, Lewis S, Weiss S, Britton J. Dietary vitamin E, IgE concentrations, and
atopy. Lancet 2000;356:1573-4.
Miller RL. Prenatal maternal diet affects asthma risk in offspring. J Clin Invest
2008;118:3265-8.
Swedish Pediatric Society sfca. Faktorer av betydelse för uppkomsten av astma och
allergisjukdom hos barn. 2010 [cited; Available from:
http://www.barnallergisektionen.se/stenciler_nya06/a1_prevention2010.pdf
Hansen AE, Knott EM, et al. Eczema and essential fatty acids. Am J Dis Child
1947;73:1-18.
Hamosh M, Salem N, Jr. Long-chain polyunsaturated fatty acids. Biol Neonate
1998;74:106-20.
Glaser C, Heinrich J, Koletzko B. Role of FADS1 and FADS2 polymorphisms in
polyunsaturated fatty acid metabolism. Metabolism 2009.
Becker W. Dietary guidelines and patterns of food and nutrient intake in Sweden. Br J
Nutr 1999;81 Suppl 2:S113-7.
Sioen IA, Pynaert I, Matthys C, De Backer G, Van Camp J, De Henauw S. Dietary
intakes and food sources of fatty acids for Belgian women, focused on n-6 and n-3
polyunsaturated fatty acids. Lipids 2006;41:415-22.
Meyer BJ, Mann NJ, Lewis JL, Milligan GC, Sinclair AJ, Howe PR. Dietary intakes
and food sources of omega-6 and omega-3 polyunsaturated fatty acids. Lipids
2003;38:391-8.
Sanders TA. Polyunsaturated fatty acids in the food chain in Europe. Am J Clin Nutr
2000;71:176S-8S.
Burdge GC, Calder PC. Dietary alpha-linolenic acid and health-related outcomes: a
metabolic perspective. Nutr Res Rev 2006;19:26-52.
Arterburn LM, Hall EB, Oken H. Distribution, interconversion, and dose response of
n-3 fatty acids in humans. Am J Clin Nutr 2006;83:1467S-1476S.
Brenna JT. Efficiency of conversion of alpha-linolenic acid to long chain n-3 fatty
acids in man. Curr Opin Clin Nutr Metab Care 2002;5:127-32.
Burdge GC, Wootton SA. Conversion of alpha-linolenic acid to eicosapentaenoic,
docosapentaenoic and docosahexaenoic acids in young women. Br J Nutr
2002;88:411-20.
Williard DE, Nwankwo JO, Kaduce TL, Harmon SD, Irons M, Moser HW, et al.
Identification of a fatty acid delta6-desaturase deficiency in human skin fibroblasts. J
Lipid Res 2001;42:501-8.
Dutta-Roy AK. Transport mechanisms for long-chain polyunsaturated fatty acids in
the human placenta. Am J Clin Nutr 2000;71:315S-22S.
Innis SM. Perinatal biochemistry and physiology of long-chain polyunsaturated fatty
acids. J Pediatr 2003;143:S1-8.
Rum P, Hornstra G. The n-3 and n-6 polyunsaturated fatty acid composition of plasma
phospholipids in pregnant women and their infants. relationship with maternal linoleic
acid intake. Clin Chem Lab Med 2002;40:32-9.
Hornstra G, Al MD, van Houwelingen AC, Foreman-van Drongelen MM. Essential
fatty acids in pregnancy and early human development. Eur J Obstet Gynecol Reprod
Biol 1995;61:57-62.
Al MD, van Houwelingen AC, Kester AD, Hasaart TH, de Jong AE, Hornstra G.
Maternal essential fatty acid patterns during normal pregnancy and their relationship
to the neonatal essential fatty acid status. Br J Nutr 1995;74:55-68.
- 112 -
189.
190.
191.
192.
193.
194.
195.
196.
197.
198.
199.
200.
201.
202.
203.
204.
Nafstad P, Nystad W, Magnus P, Jaakkola JJ. Asthma and allergic rhinitis at 4 years
of age in relation to fish consumption in infancy. J Asthma 2003;40:343-8.
Kull I, Bergstrom A, Lilja G, Pershagen G, Wickman M. Fish consumption during the
first year of life and development of allergic diseases during childhood. Allergy
2006;61:1009-15.
Calder PC, Kremmyda LS, Vlachava M, Noakes PS, Miles EA. Is there a role for fatty
acids in early life programming of the immune system? Proc Nutr Soc 2010;69:37380.
Duchén K, Casas R, Fagerås-Böttcher M, Yu G, Björkstén B. Human milk
polyunsaturated long-chain fatty acids and secretory immunoglobulin A antibodies
and early childhood allergy. Pediatr Allergy Immunol 2000;11:29-39.
Wallace FA, Miles EA, Calder PC. Comparison of the effects of linseed oil and
different doses of fish oil on mononuclear cell function in healthy human subjects. Br
J Nutr 2003;89:679-89.
Krauss-Etschmann S, Shadid R, Campoy C, Hoster E, Demmelmair H, Jimenez M, et
al. Effects of fish-oil and folate supplementation of pregnant women on maternal and
fetal plasma concentrations of docosahexaenoic acid and eicosapentaenoic acid: a
European randomized multicenter trial. Am J Clin Nutr 2007;85:1392-400.
van Houwelingen AC, Sorensen JD, Hornstra G, Simonis MM, Boris J, Olsen SF, et
al. Essential fatty acid status in neonates after fish-oil supplementation during late
pregnancy. Br J Nutr 1995;74:723-31.
Dunstan JA, Mori TA, Barden A, Beilin LJ, Holt PG, Calder PC, et al. Effects of n-3
polyunsaturated fatty acid supplementation in pregnancy on maternal and fetal
erythrocyte fatty acid composition. Eur J Clin Nutr 2004;58:429-37.
Endres S, Ghorbani R, Kelley VE, Georgilis K, Lonnemann G, van der Meer JW, et
al. The effect of dietary supplementation with n-3 polyunsaturated fatty acids on the
synthesis of interleukin-1 and tumor necrosis factor by mononuclear cells. N Engl J
Med 1989;320:265-71.
Olsen SF, Osterdal ML, Salvig JD, Mortensen LM, Rytter D, Secher NJ, et al. Fish oil
intake compared with olive oil intake in late pregnancy and asthma in the offspring: 16
y of registry-based follow-up from a randomized controlled trial. Am J Clin Nutr
2008;88:167-75.
Mihrshahi S, Peat JK, Webb K, Oddy W, Marks GB, Mellis CM. Effect of omega-3
fatty acid concentrations in plasma on symptoms of asthma at 18 months of age.
Pediatr Allergy Immunol 2004;15:517-22.
Mihrshahi S, Peat JK, Marks GB, Mellis CM, Tovey ER, Webb K, et al. Eighteenmonth outcomes of house dust mite avoidance and dietary fatty acid modification in
the Childhood Asthma Prevention Study (CAPS). J Allergy Clin Immunol
2003;111:162-8.
Peat JK, Mihrshahi S, Kemp AS, Marks GB, Tovey ER, Webb K, et al. Three-year
outcomes of dietary fatty acid modification and house dust mite reduction in the
Childhood Asthma Prevention Study. J Allergy Clin Immunol 2004;114:807-13.
Marks GB, Mihrshahi S, Kemp AS, Tovey ER, Webb K, Almqvist C, et al. Prevention
of asthma during the first 5 years of life: a randomized controlled trial. J Allergy Clin
Immunol 2006;118:53-61.
Toelle BG, Ng KK, Crisafulli D, Belousova EG, Almqvist C, Webb K, et al. Eightyear outcomes of the Childhood Asthma Prevention Study. J Allergy Clin
Immunol;126:388-9, 389 e1-3.
Meydani SN, Endres S, Woods MM, Goldin BR, Soo C, Morrill-Labrode A, et al.
Oral (n-3) fatty acid supplementation suppresses cytokine production and lymphocyte
proliferation: comparison between young and older women. J Nutr 1991;121:547-55.
- 113 -
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
217.
218.
219.
220.
221.
Caughey GE, Mantzioris E, Gibson RA, Cleland LG, James MJ. The effect on human
tumor necrosis factor alpha and interleukin 1 beta production of diets enriched in n-3
fatty acids from vegetable oil or fish oil. Am J Clin Nutr 1996;63:116-22.
Molvig J, Pociot F, Worsaae H, Wogensen LD, Baek L, Christensen P, et al. Dietary
supplementation with omega-3-polyunsaturated fatty acids decreases mononuclear cell
proliferation and interleukin-1 beta content but not monokine secretion in healthy and
insulin-dependent diabetic individuals. Scand J Immunol 1991;34:399-410.
Fujikawa M, Yamashita N, Yamazaki K, Sugiyama E, Suzuki H, Hamazaki T.
Eicosapentaenoic acid inhibits antigen-presenting cell function of murine splenocytes.
Immunology 1992;75:330-5.
Weatherill AR, Lee JY, Zhao L, Lemay DG, Youn HS, Hwang DH. Saturated and
polyunsaturated fatty acids reciprocally modulate dendritic cell functions mediated
through TLR4. J Immunol 2005;174:5390-7.
Kim DN, Schmee J, Thomas WA. Dietary fish oil added to a hyperlipidemic diet for
swine results in reduction in the excessive number of monocytes attached to arterial
endothelium. Atherosclerosis 1990;81:209-16.
Harbige LS. Fatty acids, the immune response, and autoimmunity: a question of n-6
essentiality and the balance between n-6 and n-3. Lipids 2003;38:323-41.
Gold DR, Willwerth BM, Tantisira KG, Finn PW, Schaub B, Perkins DL, et al.
Associations of cord blood fatty acids with lymphocyte proliferation, IL-13, and IFNgamma. J Allergy Clin Immunol 2006;117:931-8.
Lauritzen L, Kjaer TM, Fruekilde MB, Michaelsen KF, Frokiaer H. Fish oil
supplementation of lactating mothers affects cytokine production in 2 1/2-year-old
children. Lipids 2005;40:669-76.
Dunstan JA, Roper J, Mitoulas L, Hartmann PE, Simmer K, Prescott SL. The effect of
supplementation with fish oil during pregnancy on breast milk immunoglobulin A,
soluble CD14, cytokine levels and fatty acid composition. Clin Exp Allergy
2004;34:1237-42.
Hawkes JS, Bryan DL, Makrides M, Neumann MA, Gibson RA. A randomized trial of
supplementation with docosahexaenoic acid-rich tuna oil and its effects on the human
milk cytokines interleukin 1 beta, interleukin 6, and tumor necrosis factor alpha. Am J
Clin Nutr 2002;75:754-60.
Jones CA, Holloway JA, Popplewell EJ, Diaper ND, Holloway JW, Vance GH, et al.
Reduced soluble CD14 levels in amniotic fluid and breast milk are associated with the
subsequent development of atopy, eczema, or both. J Allergy Clin Immunol
2002;109:858-66.
Field CJ, Thomson CA, Van Aerde JE, Parrott A, Euler A, Lien E, et al. Lower
proportion of CD45R0+ cells and deficient interleukin-10 production by formula-fed
infants, compared with human-fed, is corrected with supplementation of long-chain
polyunsaturated fatty acids. J Pediatr Gastroenterol Nutr 2000;31:291-9.
Blumer N, Renz H. Consumption of omega3-fatty acids during perinatal life: role in
immuno-modulation and allergy prevention. J Perinat Med 2007;35 Suppl 1:S12-8.
Sampath H, Ntambi JM. Polyunsaturated fatty acid regulation of genes of lipid
metabolism. Annu Rev Nutr 2005;25:317-40.
Gottrand F. Long-chain polyunsaturated fatty acids influence the immune system of
infants. J Nutr 2008;138:1807S-1812S.
Yaqoob P. Fatty acids as gatekeepers of immune cell regulation. Trends Immunol
2003;24:639-45.
Fan YY, McMurray DN, Ly LH, Chapkin RS. Dietary (n-3) polyunsaturated fatty
acids remodel mouse T-cell lipid rafts. J Nutr 2003;133:1913-20.
- 114 -
222.
223.
224.
225.
226.
227.
228.
229.
230.
231.
232.
233.
234.
235.
236.
237.
238.
239.
Prescott SL, Dunstan JA. Prenatal fatty acid status and immune development: the
pathways and the evidence. Lipids 2007;42:801-10.
Miles EA, Allen E, Calder PC. In vitro effects of eicosanoids derived from different
20-carbon Fatty acids on production of monocyte-derived cytokines in human whole
blood cultures. Cytokine 2002;20:215-23.
Serhan CN. Systems approach with inflammatory exudates uncovers novel antiinflammatory and pro-resolving mediators. Prostaglandins Leukot Essent Fatty Acids
2008;79:157-63.
Harris SG, Padilla J, Koumas L, Ray D, Phipps RP. Prostaglandins as modulators of
immunity. Trends Immunol 2002;23:144-50.
Roper RL, Graf B, Phipps RP. Prostaglandin E2 and cAMP promote B lymphocyte
class switching to IgG1. Immunol Lett 2002;84:191-8.
Ohmori H, Hikida M, Takai T. Prostaglandin E2 as a selective stimulator of antigenspecific IgE response in murine lymphocytes. Eur J Immunol 1990;20:2499-503.
Moore ML, Peebles RS, Jr. Update on the role of prostaglandins in allergic lung
inflammation: separating friends from foes, harder than you might think. J Allergy
Clin Immunol 2006;117:1036-9.
Harizi H, Corcuff JB, Gualde N. Arachidonic-acid-derived eicosanoids: roles in
biology and immunopathology. Trends Mol Med 2008;14:461-9.
Lee TH, Hoover RL, Williams JD, Sperling RI, Ravalese J, 3rd, Spur BW, et al. Effect
of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro
neutrophil and monocyte leukotriene generation and neutrophil function. N Engl J
Med 1985;312:1217-24.
Serhan CN. Novel eicosanoid and docosanoid mediators: resolvins, docosatrienes, and
neuroprotectins. Curr Opin Clin Nutr Metab Care 2005;8:115-21.
Calder PC. Polyunsaturated fatty acids and inflammatory processes: New twists in an
old tale. Biochimie 2009;91:791-5.
Stafford AN, Rider SH, Hopkin JM, Cookson WO, Monaco AP. A 2.8 Mb YAC
contig in 11q12-q13 localizes candidate genes for atopy: Fc epsilon RI beta and CD20.
Hum Mol Genet 1994;3:779-85.
Schaeffer L, Gohlke H, Muller M, Heid IM, Palmer LJ, Kompauer I, et al. Common
genetic variants of the FADS1 FADS2 gene cluster and their reconstructed haplotypes
are associated with the fatty acid composition in phospholipids. Hum Mol Genet
2006;15:1745-56.
Xie L, Innis SM. Genetic variants of the FADS1 FADS2 gene cluster are associated
with altered (n-6) and (n-3) essential fatty acids in plasma and erythrocyte
phospholipids in women during pregnancy and in breast milk during lactation. J Nutr
2008;138:2222-8.
Koletzko B, Demmelmair H, Schaeffer L, Illig T, Heinrich J. Genetically determined
variation in polyunsaturated fatty acid metabolism may result in different dietary
requirements. Nestle Nutr Workshop Ser Pediatr Program 2008;62:35-44; discussion
44-9.
Caspi A, Williams B, Kim-Cohen J, Craig IW, Milne BJ, Poulton R, et al. Moderation
of breastfeeding effects on the IQ by genetic variation in fatty acid metabolism. Proc
Natl Acad Sci U S A 2007;104:18860-5.
Yu G, Björkstén B. Serum levels of phospholipid fatty acids in mothers and their
babies in relation to allergic disease. Eur J Pediatr 1998;157:298-303.
Becker W. Befolkningens kostvanor och näringsintag i Sverige 1989. Metod- och
resultatanalys. 1994 [cited; Available from: http://www.slv.se/sv/grupp1/Mat-ochnaring/Matvanor---undersokningar/Vuxna/Riksmaten-1997-98/Referenser--Riksmaten-1997-98/
- 115 -
240.
241.
242.
243.
244.
245.
246.
247.
248.
249.
250.
251.
252.
253.
254.
255.
256.
257.
258.
Duchén K. Are human milk polyunsaturated fatty acids (PUFA) related to atopy in the
mother and her child? Allergy 2001;56:587-92.
Trebble TM, Wootton SA, Miles EA, Mullee M, Arden NK, Ballinger AB, et al.
Prostaglandin E2 production and T cell function after fish-oil supplementation:
response to antioxidant cosupplementation. Am J Clin Nutr 2003;78:376-82.
Olsen SF, Sorensen JD, Secher NJ, Hedegaard M, Henriksen TB, Hansen HS, et al.
Randomised controlled trial of effect of fish-oil supplementation on pregnancy
duration. Lancet 1992;339:1003-7.
Onwude JL, Lilford RJ, Hjartardottir H, Staines A, Tuffnell D. A randomised double
blind placebo controlled trial of fish oil in high risk pregnancy. Br J Obstet Gynaecol
1995;102:95-100.
Eritsland J. Safety considerations of polyunsaturated fatty acids. Am J Clin Nutr
2000;71:197S-201S.
Szajewska H, Horvath A, Koletzko B. Effect of n-3 long-chain polyunsaturated fatty
acid supplementation of women with low-risk pregnancies on pregnancy outcomes
and growth measures at birth: a meta-analysis of randomized controlled trials. Am J
Clin Nutr 2006;83:1337-44.
Makrides M, Duley L, Olsen SF. Marine oil, and other prostaglandin precursor,
supplementation for pregnancy uncomplicated by pre-eclampsia or intrauterine growth
restriction. Cochrane Database Syst Rev 2006;3:CD003402.
Olsen SF, Secher NJ. Low consumption of seafood in early pregnancy as a risk factor
for preterm delivery: prospective cohort study. Bmj 2002;324:447.
Blonk MC, Bilo HJ, Nauta JJ, Popp-Snijders C, Mulder C, Donker AJ. Dose-response
effects of fish-oil supplementation in healthy volunteers. Am J Clin Nutr 1990;52:1207.
Sampson HA. Pathogenesis of eczema. Clin Exp Allergy 1990;20:459-67.
Vlaardingerbroek H, Hornstra G. Essential fatty acids in erythrocyte phospholipids
during pregnancy and at delivery in mothers and their neonates: comparison with
plasma phospholipids. Prostaglandins Leukot Essent Fatty Acids 2004;71:363-74.
Matorras R, Ruiz JI, Perteagudo L, Barbazan MJ, Diaz A, Valladolid A, et al.
Longitudinal study of fatty acids in plasma and erythrocyte phospholipids during
pregnancy. J Perinat Med 2001;29:293-7.
Oldaeus G, Anjou K, Bjorksten B, Moran JR, Kjellman NI. Extensively and partially
hydrolysed infant formulas for allergy prophylaxis. Arch Dis Child 1997;77:4-10.
Daly LE. Confidence limits made easy: interval estimation using a substitution
method. Am J Epidemiol 1998;147:783-90.
Kankaanpaa P, Nurmela K, Erkkila A, Kalliomaki M, Holmberg-Marttila D, Salminen
S, et al. Polyunsaturated fatty acids in maternal diet, breast milk, and serum lipid fatty
acids of infants in relation to atopy. Allergy 2001;56:633-8.
Otto SJ, van Houwelingen AC, Badart-Smook A, Hornstra G. Comparison of the
peripartum and postpartum phospholipid polyunsaturated fatty acid profiles of
lactating and nonlactating women. Am J Clin Nutr 2001;73:1074-9.
Jorgensen MH, Nielsen PK, Michaelsen KF, Lund P, Lauritzen L. The composition of
polyunsaturated fatty acids in erythrocytes of lactating mothers and their infants.
Matern Child Nutr 2006;2:29-39.
Helland IB, Saarem K, Saugstad OD, Drevon CA. Fatty acid composition in maternal
milk and plasma during supplementation with cod liver oil. Eur J Clin Nutr
1998;52:839-45.
Calder PC, Miles EA. Fatty acids and atopic disease. Pediatr Allergy Immunol
2000;11 Suppl 13:29-36.
- 116 -
259.
260.
261.
262.
263.
264.
265.
266.
267.
268.
269.
270.
271.
272.
273.
274.
275.
Kompauer I, Demmelmair H, Koletzko B, Bolte G, Linseisen J, Heinrich J.
Association of fatty acids in serum phospholipids with hay fever, specific and total
immunoglobulin E. Br J Nutr 2005;93:529-35.
Newson RB, Shaheen SO, Henderson AJ, Emmett PM, Sherriff A, Calder PC.
Umbilical cord and maternal blood red cell fatty acids and early childhood wheezing
and eczema. J Allergy Clin Immunol 2004;114:531-7.
Yu G, Duchen K, Bjorksten B. Fatty acid composition in colostrum and mature milk
from non-atopic and atopic mothers during the first 6 months of lactation. Acta
Paediatr 1998;87:729-36.
Yehuda S, Rabinovitz S, Mostofsky DI. Mixture of essential fatty acids lowers test
anxiety. Nutr Neurosci 2005;8:265-7.
Hamazaki T, Sawazaki S, Nagasawa T, Nagao Y, Kanagawa Y, Yazawa K.
Administration of docosahexaenoic acid influences behavior and plasma
catecholamine levels at times of psychological stress. Lipids 1999;34 Suppl:S33-7.
Fontani G, Corradeschi F, Felici A, Alfatti F, Migliorini S, Lodi L. Cognitive and
physiological effects of Omega-3 polyunsaturated fatty acid supplementation in
healthy subjects. Eur J Clin Invest 2005;35:691-9.
Hibbeln JR. Seafood consumption, the DHA content of mothers' milk and prevalence
rates of postpartum depression: a cross-national, ecological analysis. J Affect Disord
2002;69:15-29.
Freeman MP. Omega-3 fatty acids and perinatal depression: A review of the literature
and recommendations for future research. Prostaglandins Leukot Essent Fatty Acids
2006;75:291-7.
Bergman G, Borgstrom E, Lundell B, Sonesson SE. [Improved prenatal diagnosis of
congenital heart defects. A follow-up study of prenatal ultrasound screening].
Lakartidningen 2008;105:899-903.
Hilkens CM, Snijders A, Snijdewint FG, Wierenga EA, Kapsenberg ML. Modulation
of T-cell cytokine secretion by accessory cell-derived products. Eur Respir J Suppl
1996;22:90s-94s.
Larque E, Demmelmair H, Berger B, Hasbargen U, Koletzko B. In vivo investigation
of the placental transfer of (13)C-labeled fatty acids in humans. J Lipid Res
2003;44:49-55.
Peng YM, Zhang TY, Wang Q, Zetterstrom R, Strandvik B. Fatty acid composition in
breast milk and serum phospholipids of healthy term Chinese infants during first 6
weeks of life. Acta Paediatr 2007;96:1640-5.
Roper RL, Brown DM, Phipps RP. Prostaglandin E2 promotes B lymphocyte Ig
isotype switching to IgE. J Immunol 1995;154:162-70.
Novembre E, Cianferoni A, Lombardi E, Bernardini R, Pucci N, Vierucci A. Natural
history of "intrinsic" atopic dermatitis. Allergy 2001;56:452-3.
Krauss-Etschmann S, Hartl D, Rzehak P, Heinrich J, Shadid R, Del Carmen RamirezTortosa M, et al. Decreased cord blood IL-4, IL-13, and CCR4 and increased TGFbeta levels after fish oil supplementation of pregnant women. J Allergy Clin Immunol
2008;121:464-470 e6.
Holt PG, Rudin A, Macaubas C, Holt BJ, Rowe J, Loh R, et al. Development of
immunologic memory against tetanus toxoid and pertactin antigens from the
diphtheria-tetanus-pertussis vaccine in atopic versus nonatopic children. J Allergy Clin
Immunol 2000;105:1117-22.
Linnamaa P, Savolainen J, Koulu L, Tuomasjukka S, Kallio H, Yang B, et al.
Blackcurrant seed oil for prevention of atopic dermatitis in newborns: a randomized,
double-blind, placebo-controlled trial. Clin Exp Allergy;40:1247-55.
- 117 -
276.
277.
278.
279.
280.
Harris WS. The omega-3 index: from biomarker to risk marker to risk factor. Curr
Atheroscler Rep 2009;11:411-7.
Guillet G, Guillet MH. Natural history of sensitizations in atopic dermatitis. A 3-year
follow-up in 250 children: food allergy and high risk of respiratory symptoms. Arch
Dermatol 1992;128:187-92.
Calvani M, Alessandri C, Sopo SM, Panetta V, Pingitore G, Tripodi S, et al.
Consumption of fish, butter and margarine during pregnancy and development of
allergic sensitizations in the offspring: role of maternal atopy. Pediatr Allergy
Immunol 2006;17:94-102.
Lattka E, Illig T, Heinrich J, Koletzko B. FADS gene cluster polymorphisms:
important modulators of fatty acid levels and their impact on atopic diseases. J
Nutrigenet Nutrigenomics 2009;2:119-28.
Lindskog M. Omega 3-supplementation should probably be avoided during the
influenza pandemia. Läkartidningen, Organ for The Swedish Medical Association
2009;106:2274.
- 118 -