International Journal of Pediatric Otorhinolaryngology 77 (2013) 1065–1071

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International Journal of Pediatric Otorhinolaryngology

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Review

The pathophysiology of the hygiene hypothesis

Emmanuel Prokopakis a,*, Alexios Vardouniotis a, Hideyuki Kawauchi b, Glenis Scadding c,
Christos Georgalas d, Peter Hellings e, George Velegrakis a, Livije Kalogjera f
a
Department of Otorhinolaryngology, University of Crete School of Medicine, Crete, Greece
b
Department of Otorhinolaryngology, University of Shimane School of Medicine, Shimane, Japan
c
Department of Allergy and Rhinology, Royal National TNE Hospital, London, Gr. Britain, UK
d
Endoscopic Skull Base Centre, Academic Medical Centre, Amsterdam, The Netherlands
e
Department of Otorhinolaryngology, Katholic University of Leuven, Leuven, Belgium
f
Department of Otorhinolaryngology, University Hospital Centre ‘‘Sestre milosrdnice’’, Zagreb, Croatia

A R T I C L E I N F O A B S T R A C T

Article history: There has been a considerable increase in the diagnosis of allergic diseases over the last decades.
Received 11 February 2013 Prevalence of allergies in high-income countries and urban areas appears higher than in rural
Received in revised form 24 April 2013 environments. While environmental factors like pollution or nutrition can be important, it is more likely
Accepted 27 April 2013
that in the end they have a small association with allergies. Childhood infections and exposure to certain
Available online 20 May 2013
microbial antigens on the other hand seem to present a strong negative correlation with allergies, and
therefore the increase of the allergic burden in the Western world has been frequently related to a
Keywords:
decline of childhood infections giving birth to the ‘‘Hygiene Hypothesis’’. We address the issue with
Hygiene hypothesis
Allergy
emphasis on the associated pathophysiology tightrope walking between the skepticism of the critics,
Asthma which cast doubt on it, and the pilgrims’ belief of having discovered allergy’s Holy Grail.
Allergic rhinitis ß 2013 Elsevier Ireland Ltd. All rights reserved.
Endotoxins
Epigenetics

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1065
2. Hygiene hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
3. Pathophysiological route of the allergic infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
4. The toll-like receptor system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067
5. Epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067
6. Family and hygiene hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1068
7. Endotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1068
8. Hygiene hypothesis and auto-immune diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1068
9. Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1068
10. Microbial and virus infections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069
11. ‘‘Western’’ way of life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069
12. Vitamin D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069
13. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070

1. Introduction

Allergy as known is considered to be a disturbance of the

immune regulation, but the exact level or levels of this malfunction
* Corresponding author at: University Hospital of Crete, Building A 3rd Floor,
remain until today partially identified. During the last decades as
University Avenue, 71110 Heraklion, Crete, Greece. Tel.: +30 6932 237622. the number of patients with allergies is constantly increasing,
E-mail address: eprokopakis@gmail.com (E. Prokopakis). several theories have been developed, in order to provide a

0165-5876/$ – see front matter ß 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijporl.2013.04.036
1066 E. Prokopakis et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1065–1071
sufficient explanation of this phenomenon. Going back to the early
60s all started with immunoglobin E and its relationship to allergy,
while in the 70s, IgE gave its place to mast cells and eosinophiles,
succeeded by environmental allergy in the 80s, and the microbio-
logical background of allergy during the 90s. All these theories
had–to one point or another- as common denominator the
differentiation of T-cells towards Th1 or Th2. Hygiene hypothesis
has been presented as the rightful heir of the former theories,
claiming the role of a protagonist in the allergy series, by
attempting to resolve ongoing issues, and answering long lasting
questions about allergy.
The concept at first focused on allergic disorders, but since in
the last decades in developed countries several categories of
chronic inflammatory disorders have become much more preva-
lent, it extended to autoimmunity and inflammatory diseases as
well. It is estimated that in the United States alone fifty million
patients are affected from some kind of allergy, such as allergic
rhinitis, eczema or asthma, and that eight more million suffer from
some kind of self immune disease, such as systemic lupus
erythematosus, rheumatoid arthritis and insulin depended diabe-
tes. Hygiene hypothesis presents with the assumption that
reduced exposure to microorganisms leads to a disordered
regulation of the immune system in general and hence in a raise
of certain inflammatory disorders. Based on the fact that the most
critical period in the development of the immune system is during
infancy, any kind of intervention at the time, as for instance a
tonsillectomy, could alter the response leading to increased
susceptibility to allergic disease, due to the tonsils’ participation
in the defense against airborne and alimentary micro organisms
and therefore impaired cellular and humoral stimulation.
2. Hygiene hypothesis
The initial definition of Hygiene Hypothesis as proposed by the
epidemiologist D. Strachan in 1989 [1], quoted: ‘‘The apparent rise
in the prevalence of allergic diseases could be explained if allergic
diseases were prevented by infection in early childhood, transmit-
ted by unhygienic contact with older siblings, or acquired
prenatally. Over the past century declining family size, improved
household amenities and higher standards of personal cleanliness
have reduced opportunities for cross-infection in young families.
This may have resulted in more widespread clinical expression of
atopic disease’’
His research collected epidemiological data for allergic rhinitis
and eczema from a national sample of 17.414 children from
schools in Great Britain during a period from their birth–during
one week in March 1958- until they had reached the age of 23. The
three parameters that were studied were the patient’s statement of
allergy at the age of 23, their parents’ testimony of allergic rhinitis
at the age of 11 and their history of eczema at the age of 1, as stated
again from their parents.
The outcome of this research depicted a decrease in the
prevalence of allergic rhinitis and eczema as the number of older
children in the family increased and did not relate to the
socioeconomic status of the family. In order to explain these
rather unprecedented findings, Strachan made the assumption
that the presence of older children or generally siblings in a large
family had a protective effect against allergic sensitization.
Initially, the hypothesis was based on epidemiological evidence
and the obvious at the time explanation was the larger amount of
infections in these families that seemed to somehow lower the
manifestation of atopy. Hygiene hypothesis has been since then
supported from a large number of studies that also proved the
beneficiary effect of having older children in the family, as well as
in children attending day care units, especially during the first
months of life, resulting in less patients with allergic rhinitis,
asthma or other allergic diseases [2,3].
The new theory was greeted with enthusiasm and a rapid
uptake over the years to follow and new evidence from
epidemiological, biological and genetic studies significantly
enlarged the scope of the hypothesis. However, more recent
research has come up with contradictory evidence that argues
against certain facts attributed to hygiene’s basic points. In
contrast to what would be expected–in a survey conducted in
children in Australia between 2000 and 2005- asthma prevalence
has declined whereas eczema prevalence has increased. Further-
more, immigrant and non-immigrant children showed a similar
asthma trend, which is opposite to the statement that immuno-
logical responsiveness during childhood is established within the
first year of life or even earlier, as proposed by hygiene hypothesis
[4].
3. Pathophysiological route of the allergic infection
It is necessary to mention some facts that are highly related
with hygiene hypothesis, and the regulatory role of T-cells through
the production of cytokines. The regulation of the allergic reaction
is performed by the interaction between cells and chemical
transmitters. The initial exposure to an allergic antigen produces
antibodies IgE from B-cell lymphocytes, which in consequence
provokes an allergic reaction to every new exposure to the specific
antigen that is regulated by T-cell lymphocytes. The antigen after
entering the human body is captured by macrophages and
dendritic cells and presented to T-helpers cells (Th0). Th0 cells
have the ability of differentiating either to Th1 type that participate
in immunological reactions by inducing macrophages and creating
inflammatory tissue, or to Th2 type that induce atopy and increase
the production of IgE, mast cells and eosinophiles participating in
allergic reactions [5–8].
The absence of balance between these two types of cells seems
to be the cause of various diseases, and constitutes the basis of the
hygiene hypothesis theory. The immunological definition of this
theory came afterwards and is based on the fact that children
coming from multimember families have increased possibilities of
infections, which lead their immune system mainly towards the
direction of Th1 cell type differentiation, in order to confront
infections from viruses and microorganisms. On the other hand,
children growing up in small families, facing less inflammatory
conditions in comparison, develop a larger number of Th2 cells
instead of Th1, which leads to an increased development of allergic
responses [9].
After the initial sensitization, the differentiation of primitive T-
helper cells to Th1 or Th2 depends on the presence of interleukins
IL-12 and IL-4 correspondently. Th2 lymphocytes produce a
number of interleukins including IL-4 and IL-13 that provoke
proliferation of B-cell lymphocytes. B-cell lymphocytes produce
antibodies that attach to organ-tissue ‘‘targets’’ and when exposed
to the antigen for the second time, bond with it and begin the
immediate phase of allergic reaction with the degranulation of
mast cells. Other cytokines like IL-3 and IL-5 have a similar effect
on the proliferation and stimulating of eosinophiles that determine
the delayed phase of the allergic reaction [10,11]. Additionally, Th1
lymphocytes produce IFN-g and IL-2 that participate in immune
reactions like sarcoidosis, Chron’s disease etc. Furthermore IL-4
inhibits the expression of the Th1 lymphocytes, while IFN-g
inhibits the expression of Th2 lymphocytes respectively. However,
Th-2 hypothesis for asthma based on experimental studies in mice,
where Th-1/Th2 polarization is clear, may be too simple to directly
imply on human airway disease, where interaction of Th-1 and Th-
2 profile cytokines may both play a role in the severity of the
disease [12]. It came clear more than a decade ago that application
 E. Prokopakis et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1065–1071
of biological agents, like anti-Il-5, and Il-12, shift towards Th-1
response, although decreasing circulating eosinophiles, did not
prevent early and late allergic response or bronchial reactivity
[13,14].
4. The toll-like receptor system
The innate immune system is a pivotal defense mechanism in
humans. Even though its function is based only on a limited
number of receptors – expressed on the surface of and within cells
– it is capable of recognizing an abundant number of pathogen-
associated molecular patterns (PAMPs), and initiating immune
responses. Major components of these innate immunity pathogen
recognition receptors are the toll-like receptors (TLRs). Backed up
by hygiene hypothesis, which suggests that allergies appear
because of a change in microbial exposure and associated immune
signals early in life, it has been speculated that alterations in TLRs
signaling could influence allergy development. Therefore, besides
their role in inflammation and infection, TLR genes, and their
genetic variations as well as their association with allergy and
atopic diseases were investigated over the past years.
In humans, 11 TLRs have been identified [15,16]. With regard to
their cellular localization, TLRs can be divided into two subgroups:
TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, and TLR11 are expressed on
the cell surface, while TLR3, TLR7, TLR8, and TLR9 are localized in
intracellular vesicles. TLR2 is involved in the recognition of a wide
range of PAMPs derived from bacteria, fungi, parasites, and viruses.
It generally forms a heterodimer with TLR1 and TLR6; TLR1- TLR2
respond to the bacterial triacylated lipopeptide, whereas TLR2-
TLR6 recognize the mycobacterial diacylated lipopeptide. TLR4 is
essential for responses to lipopolysaccharides (LPS), a major
constituent of the outer membrane of Gram-negative bacteria.
TLR5 recognizes flagellin, a protein component of bacterial flagella.
TLR3 responds to viral double-stranded RNA, while TLR7 and TLR8
preferentially recognize single-stranded RNA. TLR9 was originally
identified to respond to specific DNA motifs that are frequently
present in bacteria [17]. The knowledge regarding TLR10 and 11
ligands is still incomplete, even though recent studies have
demonstrated that TLR10 shares some common agonists with TLR1
(and TLR2) [18].
Ligand binding on TLRs leads to an activation of several
intracellular signaling that induces expression of costimulatory
molecules (CD80 and CD86) and proinflammatory cytokines (TNFa,
type-1 interferons, IL-1, IL-6, IL-10, and IL-12) that mainly favors
Th1 differentiation [19–22]. According to the hygiene hypothesis,
TLRs need to be stimulated in the early period of life to drive the
Th-cell populations from the in utero-shaped Th2-bias into a Th1-
direction [23]. Some experiments, mainly in animal models of
experimental asthma, showed pronounced Th2 responses and
elevated allergic parameters as a reaction on LPS application
[24,25] whereas others reported reduced asthma phenotypes after
LPS application [26,27]. However, these discrepancies can be
sufficiently explained by dose-dependent effects [28]. In the ALEX
study blood cells from farmers’ children expressed significantly
higher amounts of toll-like receptor 2 than those from non-
farmers’ children, indicating that these TLR might also be involved
in the ‘‘farming effect’’ [29].
TLRs undoubtfully play an important role in the prevention in
allergic disorders. Furthermore, there are certain TLR gene
polymorphisms that are associated with an increased prevalence
of allergic diseases [30]. For example, allergic diseases in farmer’s
children could be contributed to a significantly elevated preva-
lence of a polymorphism found in the TLR2-coding gene [31].
Results from a Swedish study indicated that a polymorphism in the
TLR4-gene is associated with asthma characterized by a decreased
1067
IL-12 production by antigen presenting cells (APCs) after LPS
stimulation [32].
In the GABRIEL Advanced Studies) [33] conducted in rural
regions of Austria, Germany, and Switzerland, 79,888 school-aged
children categorized as ‘‘farmers’’, ‘‘visiting farms’’ and ‘‘non-
farmers’’, answered a recruiting questionnaire. Afterwards a
stratified random subsample of 8419 children answered another
detailed questionnaire on farming environment. A broad definition
of asthma comprising symptoms, diagnosis, or treatment ever was
used and blood samples and specific IgE levels were available for
7682 of these children. The conclusion was that those children
living on a farm were at significantly reduced risk of asthma, hay
fever, atopic dermatitis, and atopic sensitization compared to
children visiting farms and nonfarm children (asthma: 11%, 16%,
18% respectively, hay fever: 5%, 11%, 15% respectively).
It seems that a specific type of traditional farming (i.e., with
cows and cultivation) was protective against asthma, hay fever,
and atopy, aligning with the results of a similar study [34]
comparing the prevalence of allergic sensitization in a population
of Amish children (157 families) to children both living in farms in
Switzerland (3000 families) and nonfarm children (11,000 fami-
lies). Based on parental reporting, the prevalence of ever having
asthma was 5.2%, 6.8%, and 11.3% among Amish, Swiss farm, and
Swiss nonfarm children, respectively. The corresponding figures
for allergic sensitization were 7.2%, 25.2%, and 44.2%, confirming
previous observations.
Even though, in the GABRIEL Studies the farm effect on asthma
could be explained by specific farm characteristics, there is still a
link missing for hay fever and atopy, whereas in the Amish
population study, since the Amish are of Swiss descent and
therefore presenting a genetically similar background to Swiss
children, the exceedingly low level of sensitization of 7.2% among
Amish children, implicates that there may be additional protective
factors in this population.
5. Epigenetics
The possible link between all the above observations is the
effect of environmental factors upon gene expression in man-.
Epigenetics is the study of heritable changes in gene expression or
cellular phenotype caused by mechanisms other than changes in
the underlying DNA sequence–hence the name epi- (Greek: epí-
over, above, outer) -genetics [35]. It refers to functionally relevant
modifications to the genome such as DNA methylation and histone
modification, that do not involve a change in the nucleotide
sequence [35]. These changes may remain through cell divisions
for the remainder of the cell’s life and may also last for multiple
generations. However, there is no change in the underlying DNA
sequence of the organism; instead, non-genetic factors cause the
organism’s genes to behave (or ‘‘express themselves’’) differently.
Recent data about the effect of diet on gene methylation offers an
example of how epigenetic mechanisms might affect gene-
environment interactions [36]. In the case of allergic disease,
epigenetics could explain the discordances observed between
monozygous twins as well as sporadic cases, incomplete pene-
trance, variable expression, gender and progenitor effects. The
hygiene hypothesis is of great relevance since it integrates genetic
and epidemiological data in the context of environmental
exposures [36].
Environmental factors able to regulate epigenetic changes
include diet: folic acid and vitamin B12 influence the availability of
methyl groups and co-factors for the formation of S-adenosyl-
methionine, essential for de novo DNA methylation. Fish oil-rich
diets could induce epigenetic effects. Other important factors are
the microbial composition of the gut flora [37].
1068 E. Prokopakis et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1065–1071
The information on the epigenetic mechanism possibly
underlying the relationship between such factors and allergic
diseases is still incomplete but is an area of rapid expansion and
interest [37].
6. Family and hygiene hypothesis
With the use of skin prick tests (SPT) and the levels of specific
IgE immunoglobins, an inverse relation is detected between
allergic rhinitis and the size of families [38,39]. The protective
effect against atopic reactions in multimember families appears to
be stronger as the age of older children increases and stronger in
boys than girls [40,41]. The presence of older children in the family
decreases the possibility of allergic rhinitis and eczema, while the
presence of younger children is responsible for the decrease in
asthma [42]. The close contact among children that is more likely
to occur in a multimember family, as in families were more than
one children share the same bedroom, appears to offer protection
against the risk of developing atopic diseases since the possibility
of exposure to microorganisms or infections is high [43].
Similarly, children raised with older siblings or attending
daycare are more likely to develop wheezing at the age of 2 years,
but increasingly less likely at older ages [44]. However, all these
studies are mainly based on epidemiological data and cannot easily
tackle the criticism of crudely assessing their subject without being
able to prove their point beyond dispute, and therefore not
sufficing to explain the effects related to the hygiene hypothesis.
7. Endotoxins
In international literature great interest is demonstrated for the
differences between civil and rural populations in the developing
of allergic diseases. Recent studies have depicted the fact of living
in farms and not generally living in the countryside as the main
protective factor against allergy. For example, there is a decrease in
the incidence of allergic rhinitis in farmers’ children in particular,
in comparison to those of other rural populations [45,46].
A lot of effort has been made, in order to explain the
immunological background of this difference, most of which is
focused on bacterial endotoxins [46,47]. Endotoxins are lipopo-
lysaccharides (LPS) that consist part of the external structure of the
cellular wall of Gram-negative bacteria. The immune system has a
highly sensitive system of adaptors that detect the presence of LPS.
Endotoxins have the ability of connecting with adaptor CD14,
which leads to an increased production of IL-12 through antigen
presenting cells, inducing Th1 differentiation that is not connected
with the development of allergy [48,49]. The precise mechanism is
not yet totally clear for as if the exposure to endotoxins regards
airborne or food related antigens.
In South Germany and Switzerland [50], it was proven that the
concentrations of endotoxins in house dust from kitchens and
mattresses of children of rural families are significantly higher than
those of non-rural families. This lead to an even larger study in
Germany, Austria and Switzerland that presented an inverse
relation between the level of endotoxins in domestic dust, and the
developing later on of allergic rhinitis and asthma. Other studies
that measured endotoxins levels in non-rural environments [51]
with the use of skin prick tests in infants aged from 9 to 24 months,
presented that the higher levels of endotoxins in domestic dust
were associated to a lower rate of allergic sensitization. In Estonia,
where there is a low prevalence of allergy, higher levels of
endotoxins were found in domestic dust in comparison to those in
Sweden [52], where allergy is generally higher. This fact is based on
the development of atopic disease during the two first years of life
only in children from Sweden.
Important factors that seem to determine the outcome of
endotoxin exposure in allergic diseases include the existence or
absence of exposure in early life, dose and frequency of the
endotoxins exposure, concurrent exposures, and the genetic
profile that influences immune responsiveness to endotoxins
[53]. The issue is that even though all the above-mentioned studies
can suggest association, they cannot prove cause and effect, leaving
space for rejection to blossom. For example, one could point out
that children with atopic asthma have their beddings washed more
often or even avoid spending time in barns, thus minimizing
exposure and decreasing mattress dust endotoxin. Nevertheless,
there is skepticism for the part that endotoxins seem to have, not
for their undisputed participation, as for the fact that they might
just be an easily measurable second factor of the same microbial
element [54,55]. Studies which combine measuring endotoxin
exposure and evaluating genetic background suggest that allergy
and asthma result from a complex interaction between genetic and
environmental factors. Although increasing endotoxin exposure is
associated with reduced risk of allergic sensitization in children
with the CC genotype at -159 of the CD14 gene, in the same
genotype increased risk of nonatopic wheeze was demonstrated. It
is suggested, that complex impact of environmental endotoxin
may be enhanced in individuals with this genotype of CD14 gene,
while other alleles were not affected [56].
8. Hygiene hypothesis and auto-immune diseases
Many studies over the past years have focused their research on
the existence of possible links between limited microbial exposure
and an increased rate of immunological diseases, which have
similarly followed the rise of allergies during the last decades. The
deficit of the immunoregulatory mechanisms to terminate
inappropriate inflammatory responses can lead to simultaneous
upraise in diverse types of pathology as depicted in XLAAD
syndrome (X-linked autoimmunity-allergic dysregulation syn-
drome [57]. Apparently, there is a positive relation between
various positive markers of microbial exposure and Th1 regulated
inflammatory diseases, such as juvenile diabetes [58–60], rheu-
matoid arthritis [61], Chron’s disease, and leukemia [62–64].
On the other hand, once again other studies propose that a
reciprocal relationship between Th1 versus Th2 immune develop-
ment and allergy, autoimmunity and inflammatory diseases is far
too simplistic when based only on the epidemiological data of the
hygiene hypothesis. As a result, they do not prove any kind of
relationship with these markers of microbial exposure [65–67].
9. Antibiotics
The possibility of a relationship between the use of antibiotics
and the appearance of asthma or other atopic reactions is a modern
subject of interest, even though it is hard to define whether the
reactions are due to the infection or the antibiotic itself [68].
Furthermore, in Great Britain any kind of drug use before the age of
two is related to double the possibility of developing allergic
rhinitis and eczema, especially when the medication contained
cephalosporins and macrolides [69]. It has been suggested that the
effect of antibiotics may be related to the effect on bacterial
colonization of the gastrointestinal system at the early stages of
development [70]. This is according with the results of another
study with laboratory mice, in which the antibiotic induced
changes in the intestinal system provoked the response of the
immune system to common allergens in the lungs [71]. Another
interesting analysis in ninety-nine centers of twenty-eight
countries depicts a positive relation between antibiotic sales per
capita and the prevalence of the symptoms of allergic rhinitis,
asthma and eczema [72].
 E. Prokopakis et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1065–1071
However, this is only one way of looking at things. Since in
many countries asthma, particularly at a young age, is still treated
with antibiotics, an association between the use of these drugs and
asthma must become positive. Studies that take in to consideration
the actual beginning of asthma and the timing of drug use are rare,
and these studies do not so far show a convincing effect of
antibiotic use on the development of asthma and allergic illnesses
[73,74]. Furthermore, it is demonstrated that the use of antibiotics,
not only in human treatment, but also in the livestock feed, may
significantly increase the risk of asthma in children living on farms
where antibiotics are used in the feed, compared to those where
they are not used [75].
10. Microbial and virus infections
The best evidence of the inverse relationship between exposure
to a particular pathogen and atopy originated from data regarding
Hepatitis A, an infection associated with large families and low
socioeconomic level, in a study conducted in Italy. In students of
military academies, it was found that the increased prevalence of
specific IgE immunoglobins to airborne allergens was decreased in
half when there was evidence of an earlier HAV infection. These
findings are independent from age, family size, birth line, and area
of residence and the educational level of the parents [76]. Two
more studies present that seropositivity for HAV in the general
population is related to a decrease in allergic reactions by 40% and
37% respectively [77,78]. In air force cadets in Italy that suffered
from various allergic diseases, significantly lower levels of
antibodies to Toxoplasma gondii, Helicobacter pylori and HAV were
measured, in comparison to non allergic subjects [79]. The receptor
for HAV on human lymphocytes is T-cell immunoglobulin- and
mucin-domain- containing molecules (TIM-1), which is involved in
the regulation of T-cell subsets including regulatory T cells and Th2
cells. Exposure to HAV might selectively remove Th2 cells, or alter
the balance of T-cell subsets, leading to the conclusion that
premature exposure to microorganisms through oral and gastro-
intestinal routes, offer protection against infections of the
respiratory system, and that the ‘‘healthy, westernized, sterile
nutrition’’ can facilitate the appearance of allergic reactions
through the effect of microorganisms that stimulate the gastroin-
testinal lymphoid tissue.
However, in some other studies there are populations that do
not present any relationship between Helicobacter pylori or HAV,
and the developing of allergic diseases [80–82]. Furthermore,
common childhood infections like measles, mumps and chick-
enpox seem to be unable to protect from allergic disorders [83,84],
leaving HAV as possibly the only resisting stronghold of hygiene
hypothesis.
11. ‘‘Western’’ way of life
The drastic effect that modern life style has on the impressive
rise of allergic diseases and the role that environmental agents
play, are characteristically demonstrated in a epidemiological
study conducted in New Zealand regarding a population that was
forced to change residence, and the effect that this change had to
the frequency of allergic reactions. In particular, in 1966, after a
catastrophic hurricane that stroke the small island Tokelau, in the
Pacific Ocean, New Zealand’s government that had the island under
its jurisdiction, relocated 1950 inhabitants to New Zealand.
Fourteen years later the children that moved from the almost
primitive conditions to modern conditions of living, presented in
comparison to the children that remained on the island, a
significantly higher prevalence of allergic rhinitis (28–14%) and
asthma (25–10%) [85].
1069
The abundance of aseptic products and practices that promote a
‘virgin’ environment for people in general and for young children in
particular, may have decreased our exposure to incoming ‘danger’
signals from microbes and germs that has been part of our allergic
alert for centuries. One suggestion is that since the immune
system’s primary driving force is the need to detect and protect
against danger, and that it does not do the job alone, but receives
positive and negative communications from an extended network
of other bodily tissues, the lack of these signals due to the ‘‘clean
Western’’ way of life, adds to the problem [86]. The exact way that
urbanization and westernization influences allergic diseases is an
ongoing investigation [87]–in a recent study even having a
tonsillectomy before 7 years of age seemed to increase the risk
of early onset allergic rhinitis (OR = 1.7, 95% CI 1.2–2.5) [88]. The
mechanism for the effect of tonsillectomy is open to discussion,
since possibilities include its significance as a marker of antibiotic
use or, alternatively, a marker of severe, repeated, upper
respiratory infection resulting in inflammation and, therefore,
increased risk of sensitization via increased mucosal permeability.
However, since long-term data are lacking, one can only speculate
that short-term changes might proceed into immunological
imbalance. Regardless, there is no doubt that genetic disposition
is only one of the many driving forces in allergy as represented in
the changes of the allergic profile from patients in East Germany
only 20 years after the reunification [89]. It is of note, that the
increased prevalence of allergic sensitization and concurrent
allergic diseases may be one of the factors associated with the
rise of chronic rhinosinusitis [90].
12. Vitamin D
More recently, variations in vitamin D status and intake have
also been implicated in allergy development and considered as one
of a number of explanations for epidemiological and immunologi-
cal associations [91], based on its role in the development and
maintenance of lung structure and function and in immunity
[92,93], even reaching the point were one has started to ask
whether correcting vitamin D levels affects the incidence and the
course of allergic disease [94].
Aside from skeletal health vitamin D regulates the activity of
various immune cells, including monocytes, dendritic cells (DCs), T
and B lymphocytes, as well as immune functions of epithelial cells
[95]. Furthermore, some immune cells express vitamin D-
activating enzymes facilitating local conversion of inactive vitamin
D into active calcitriol with subsequent paracrine and autocrine
effects [96,97]. In particular, vitamin D inhibits the expression of
pattern recognition receptors, which activate innate immune
responses such as the Toll-like receptors on monocytes and
suppresses TLR mediated inflammation [98], decreases immune
receptor expression also on monocyte-derived DCs, reduces the
function of these cells (e.g. chemotaxis, antigen presentation,
maturation) [99,100] and furthermore, induces autophagy in
human macrophages, which possibly contributes to the defense
against opportunistic infections [101].
Besides its effect on the innate immune system, vitamin D is a
major player in adaptive immunity, since it inhibits T-cell
proliferation through decreased Th1 cytokine secretion [102],
increases IL-10 and decreases IL-2 production, thereby inducing a
state of hypo-responsiveness in T regulatory cells, similar to the
one produced by corticosteroids or allergen immunotherapy in
anti-allergic therapies [103].
Therefore, the impact of vitamin D on immune functions might
be particularly critical as prevalence of hypovitaminosis D is high
with up to 30% in the adult Western population and up to 70% in
the elderly or institutionalized, easily awarded the title of a
‘‘pandemia’’ [104,105].
1070 E. Prokopakis et al. / International Journal of Pediatric Otorhinolaryngology 77 (2013) 1065–1071
Even though the allergy society anxiously anticipates the
moment of ‘‘Eureka’’, the evidence for beneficial effects of vitamin
D on allergic diseases is primarily based on observational and often
retrospective studies, and while experimental and pre-clinical data
point towards a protective role of vitamin D, the clinical results
available to date draw a less clear picture [106]. Asthmatic patients
might be the only ones who seem to benefit from vitamin D
supplementation [107], and childhood wheezing is the disease that
is probably most likely to be prevented by sufficient vitamin D
during pregnancy [108], not excluding however the need for larger
multicentric prospective studies to clarify whether vitamin D has
any role whatsoever in preventing allergic diseases.
13. Conclusion
Various pieces of the jigsaw regarding allergy have been added
over the years but a truly unifying concept is still missing. The birth
of the hygiene hypothesis filled the medical community with great
expectations, but, after a classic adolescence with better and worse
days, now even though matured, it still has not managed to fill in
the gap of the missing link considering all the questions of allergic
diseases. Since epidemiological evidence mainly comes from cross-
sectional studies, which are not able to elucidate cause–effect
relationships, the hygiene hypothesis will inevitably be conflicted
by opposite results. The fact that repeated stimulation of the
immune system by pathogens is central to the development of a
balanced response rather than a Th1-Th2 dichotomy remains
perhaps the best point that hygiene hypothesis has to offer to
future diagnostic and therapeutic strategies considering allergy.
Besides a potentially protective role of early exposure to bacterial
antigens, it is important to emphasize that exposure to both gram
negative (LPS) and gram positive (super antigens) bacterial
antigens in already sensitized individuals may promote inflam-
matory responses leading to increased severity of allergic disease
and asthma. It is undeniable that the route and the period of
exposure, the dose and the genetic background of each patient
determine the outcome of immune responses. The phenomenon of
epigenetics could provide the link between diet, bacterial
exposure, Toll like receptors, Th1/2balance, vitamin D and the
effects of antibiotics and thus ‘‘the quest for the Holy Grail
continues. . .’’
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