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\begin{document}

\title{Effects of early-life exposure to dust mite allergen and endotoxin on
the development of asthma and wheezing: the Japan Environment and
Children's Study}



\author[1]{Hideki Hasunuma}%
\author[1]{Yoshiko Yoda}%
\author[1]{Narumi Tokuda}%
\author[1]{Naoko Taniguchi}%
\author[1]{Yasuhiro Takeshima}%
\author[1]{Masayuki Shima}%
\affil[1]{Hyogo College of Medicine}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: The effects of early-life exposure to house dust mite
allergen and endotoxin on the development of asthma are unclear in the
literature. We investigated the association of early-life exposure (0-36
months old) to house dust mite allergen and endotoxin with asthma
incidence. Methods: In this novel, large-scale, nationwide birth cohort
study, 5,017 participants were randomly selected from those who met the
eligibility criteria. House dust was vacuum-sampled from the children's
bed within homes and assayed for the presence of dust mite allergen (Der
1) and endotoxin. The participants were classified into four quartiles
(Q1-Q4) according to exposure levels. We defined the incidence of asthma
and wheezing using a questionnaire at 12, 24, and 36 months old. Odds
ratios (ORs) of the incidence of asthma and wheezing by age in Der 1 and
endotoxin exposure level were estimated using logistic regression.
Results: The cumulative incidence rates of asthma and wheezing during
0-36 months were 10.4\% and 38.1\%, respectively. Significant ORs were
observed in asthma onset during 12-24 months old, asthma onset during
24-36 months old, and wheezing onset during 0-12 months old in the Q4
Der 1 group. In the Q4 endotoxin group, significant positive
associations between endotoxin exposure and asthma (OR 2.00, 95\%
confidence interval {[}CI{]}: 1.03-3.85) and wheezing (OR 1.78, 95\% CI:
1.01-3.12) onset during 24-36 months old were found. Conclusions: Our
results indicated that high levels of early-life exposure to Der 1 and
endotoxin in mattresses may be involved in the development of asthma.%
\end{abstract}%



\sloppy


\section*{Introduction}

{\label{introduction}}

The prevalence of paediatric allergic diseases has recently increased in
developed countries. Adoption of Western lifestyle was noted as a
contributing factor, but it has not been sufficiently
verified.\textsuperscript{1,2} Western lifestyle is characterised by an
increase in the time spent indoors, highly insulated airtight houses,
and a more hygienic environment; in addition, it may also be
characterised by changes in the pattern of exposure to house dust mite
allergen and microorganisms that influence the prevalence of paediatric
allergic diseases.\textsuperscript{2}There is sufficient scientific
evidence for dust mite allergen as a factor aggravating asthma symptoms,
but there is no evidence that it is a risk factor for asthma
incidence.\textsuperscript{3,4} A European large-scale birth cohort
study showed that exposure to a high concentration of allergens
immediately after birth influences the development of allergic
sensitisation.\textsuperscript{5,6} Moreover, exposure to dust mite
allergens is associated with dust mite sensitisation.\textsuperscript{7}
However, intervention studies reported no associations between dust mite
exposure in infancy and asthma onset at 3 \textsuperscript{8} or 8 years
of age.\textsuperscript{9}Reports suggest no consistency in the
association between endotoxin, a cell wall component of gram-negative
rods and microorganism exposure marker, and asthma development.
Braun-Fahrl\selectlanguage{ngerman}änder et al. reported that in farming areas in Germany,
Austria, and Switzerland, the endotoxin concentration in bed dust was
higher in children of farmers than in that of other children, and
significantly fewer children of farmers had pollinosis, asthma, and
atopic sensitisation.\textsuperscript{10,11}Mendy et al. reported a
positive correlation between the endotoxin concentration in dust
(endotoxin concentration per gram of dust) and wheezing in infants and
toddlers (0-3 years old) but an inverse correlation with asthma after
they reached school age.\textsuperscript{12}Furthermore, recent studies
in adult Americans revealed that the influence on allergic disease
differs due to climate and lifestyle because of differences in exposure
to endotoxin and indoor allergens such as mites and
moulds.\textsuperscript{13,14} Therefore, it is necessary to clarify the
association of exposure to dust mite allergen and endotoxin with asthma
development in Asian regions under different climatic regions and
lifestyles in comparison with that in Western countries. Although
large-scale birth cohort studies have been conducted in Western
countries, there are few reports from Asia, including Japan. We
conducted a large-scale birth cohort study on the effects of early-life
exposure to dust mite allergen and endotoxin in mattresses on asthma and
wheezing development by 0-36 months old in Japan.

\section*{Methods}

{\label{methods}}

\subsection*{Participants}

{\label{participants}}

This is a sub-cohort study of the Japan Environment and Children's Study
(JECS). The study design and recruitment methods have been described in
detail previously.\textsuperscript{15,16} In brief, JECS is a
large-scale birth cohort study involving 100,000 pairs of parents and
children in 15 nationwide regions in Japan, wherein participants were
observed from the foetal period to the age of 13 years old. Participants
were recruited between January 2011 and March 2014; 103,060 pregnancies
were registered, of which 100,304 were live births. For the sub-cohort
study, 5,017 participants were randomly selected from those who met the
eligibility criteria and consented to participate in the sub-cohort
study.\textsuperscript{17} Data for this study were used from the
dataset released in October 2019 (dataset jecs-ta-20190930). A schematic
summarising the sample selection methodology is shown in Figure 1, and
detailed study methodology is provided in Supporting Information. The
JECS protocol was approved by the Institutional Review Board on
Epidemiological Studies of the Ministry of the Environment (IRB number:
100406001) and by the Ethics Committees of all participating
institutions. Written informed consent was obtained from all
participants.

\subsection*{Questionnaire}

{\label{questionnaire}}

Questionnaires were sent to the participants by post at the age of 12,
18, 24, and 36 months old, and the parents filled them out and sent them
back. Those questionnaires were a modified version of ISAAC for children
aged 6--7 years, with translation validation in
Japanese.\textsuperscript{18-20} Okabe et al. investigated the validity
of asthma symptoms based on ISAAC questionnaires involving
0-60-month-old infants by using asthma diagnosis made by allergists as
criteria,\textsuperscript{21} wherein the sensitivity and specificity of
asthma symptoms were 0.91 and 0.64 in 0-24-month-old children and 0.88
and 0.68 in 36-60-month-old children, respectively. In our study, asthma
and wheezing were defined according to the definition by Okabe et
al..\textsuperscript{21,22} The incidence rates of wheezing and asthma
by age (0-12, 12-24, and 24-36 months old) were defined as a ratio of
participants initially judged as having wheezing/asthma at each age
compared to the that of all participants. Information on lower
respiratory tract inflammation and respiratory syncytial virus (RSV)
infection was obtained from the questionnaires at the age of 12, 18, and
24 months. Information concerning the presence of indoor pets and
passive smoke after birth was obtained from the questionnaires at 18
months old.

\subsection*{Blood and urinal examination of
mothers}

{\label{blood-and-urinal-examination-of-mothers}}

Blood samples were obtained from mothers during the first trimester of
pregnancy. Total serum IgE titres, assayed using ImmunoCAP (Thermo
Fisher Scientific, Inc., Sweden), were analysed by an independent
clinical laboratory. Mothers with high levels of total serum IgE were
defined as those with a total IgE level of 214 IU/mL or
above.\textsuperscript{23}Maternal spot urine was collected at
mid-pregnancy, and the urinary cotinine level was measured. Sample
collection and measuring methods were described in detail
previously.\textsuperscript{24} The cut-off values for active smoking
and passive smoking during pregnancy were set at 36.8 and 0.31 \selectlanguage{greek}μ\selectlanguage{english}g/g
creatinine, respectively.\textsuperscript{24}

\subsection*{Blood examination of
children}

{\label{blood-examination-of-children}}

The mothers and children visited the test site when their children were
24 months old; blood samples were obtained from children. Total serum
IgE titres, assayed using ImmunoCAP (Thermo Fisher Scientific, Inc.,
Sweden), were analysed by an independent clinical laboratory. As the
cut-off value of high levels of total serum IgE in 24-month-old children
is unclear, it was set at the upper tenth percentile of the total IgE
level.

\subsection*{Exposure assessment (mite
allergen/endotoxin)}

{\label{exposure-assessment-mite-allergenendotoxin}}

Trained investigators visited homes when the children were 18 months old
and collected house dust from mattresses on which the children slept. A
50-cm \selectlanguage{ngerman}× 100-cm frame exclusive for sampling was placed on the mattress,
and a vacuum cleaner (Model DC61, Dyson, Japan) was run over the area in
the frame for 2 minutes (Supp Info Figure 1). The collected dust was
frozen until analysis.\emph{Dermatophagoides pteronyssinus} (Der p 1)
and\emph{Dermatophagoides farinae} (Der f 1) in the dust were measured
using an enzyme-linked immunosorbent assay kit (Indoor Biotechnologies
Ltd, UK). Endotoxin levels in the dust were measured by kinetic
chromogenic limulus amebocyte lysate assays (Kinetic-QCL), obtained from
Lonza Japan (Tokyo). There is high structural similarity in the
corresponding allergen between Der p 1 and Der f 1. Once sensitised,
there is strong cross-reactivity even when sensitised with either
type.\textsuperscript{25} Thus, we used the total exposure to both mite
species Der 1 (Der p 1 + Der f 1) in the analysis of the association
with health outcomes.

\subsection*{Statistical analysis}

{\label{statistical-analysis}}

Dust mite allergen and endotoxin concentrations were expressed as
`amount per m\textsuperscript{2} of dust sampling area' and `amount per
unit mass of dust',\textsuperscript{26} and Spearman's rank correlation
coefficient was calculated for these exposure indices. The correlation
coefficient for the `amount per gram of dust' and `amount per
m\textsuperscript{2} dust sampling area' of Der 1 was 0.86, and that in
endotoxin between the `amount per mg dust' and `amount per
m\textsuperscript{2} dust sampling area' was 0.79, demonstrating a high
correlation between these exposure indices. Thus, the `amount per
m\textsuperscript{2} dust sampling area' was analysed in the main
analysis thereafter. Analysis of the `amount per unit mass of dust' was
performed as a sensitivity analysis. If the data were lower than those
of the method detection limit, then a value of one-half was assigned. In
the analysis of the association with health outcomes, the participants
were classified into the first-to-fourth quartile (Q1-Q4) groups.
Regarding the association between the dust mite allergen or endotoxin
exposure level and health outcome, the odds ratios (ORs) of the exposure
level in the Q2-Q4 groups in comparison with the Q1 group were estimated
using a logistic regression model. Regarding health outcomes, the
incidence rates of asthma and wheezing by age (0-12, 12-24, 24-36, and
0-36 months old) were selected for the main analysis. Secondary analysis
was set to high levels of total serum IgE in children. High levels of
maternal total serum IgE, active smoking/passive smoking based on the
urinary cotinine level in mid-pregnancy, passive smoke after birth,
children's sex, lower respiratory tract inflammation, RSV infection,
presence of indoor pets, and climatic region were regarded as
covariates. Climatic region was classified into three areas based on
mean temperature and mean humidity (Supp Info Figure 2). A trend test
was then performed to confirm the quantitative relationship of health
outcomes with the exposure level. Regarding continuous variable x giving
1, 2, 3, and 4 points to the Q1-Q4 groups as an explanatory variable of
the statistical model, the significance level, p-value, of the OR of
increase per unit x was calculated. All statistical analyses were
performed using SAS ver9.4 (SAS Institute Inc., Cary, NC, USA).

\begin{CJK}{UTF8}{gbsn}\section*{　Results}\end{CJK}\selectlanguage{english}

{\label{results}}

\subsection*{Participant
characteristics}

{\label{participant-characteristics}}

The characteristics of the 2,934 participants included in the analysis
sets are shown in Table 1. The cumulative incidence rates of asthma and
wheezing were 10.4\% and 38.1\%, respectively. The incidence rates of
asthma by age were 2.7\%, 4.8\%, and 3.0\% at 0-12, 12-24, and 24-36
months old, respectively, and were highest at 12-24 months old. The
incidence rates of wheezing by age were 20.0\%, 13.6\%, and 4.6\%,
respectively, and were highest at 0-12 months old. The total serum IgE
level was measured at 24 months old in 2,900 children. The upper tenth
percentile of the total serum IgE level in 24-month-old children was 180
IU/mL, and the serum IgE level was high ([?]180 IU/mL) at 9.7\% and low
(\textless{}180 IU/mL) at 90.3\%. High levels of maternal serum IgE were
detected in 18.0\%, and the rates of maternal active smoking (36.8 \selectlanguage{greek}μ\selectlanguage{english}g/g
creatinine or higher) and passive smoking (0.31 36.8 \selectlanguage{greek}μ\selectlanguage{english}g/g-creatinine)
based on the urinary creatinine level were 5.7\% and 33.1\%,
respectively.

\subsection*{Mite allergen and
endotoxin}

{\label{mite-allergen-and-endotoxin}}

The levels of dust mite allergen and endotoxin in dust are shown in
Table 2. The geometric means of Der 1 per unit area and per gram dust
determined in the analysis were 64.95 \selectlanguage{greek}µ\selectlanguage{english}g/m\textsuperscript{2} and 3.89
\selectlanguage{greek}µ\selectlanguage{english}g/g, respectively. The geometric means of endotoxin were
2.71\selectlanguage{ngerman}×10\textsuperscript{5} EU/m\textsuperscript{2} and 16.0 EU/mg,
respectively.

\subsection*{Association of mite allergen with the incidence of asthma or
wheezing}

{\label{association-of-mite-allergen-with-the-incidence-of-asthma-or-wheezing}}

The adjusted ORs of dust mite allergen exposure level Der 1 in the Q2-Q4
groups in comparison with the Q1 group are shown in Fig. 2. The ORs of
the cumulative incidence rates of asthma and wheezing were significantly
higher in the Q4 group during 0-36 months old (OR: 1.85 (95\% CI:
1.30-2.63) and OR: 1.33 (95\% CI: 1.05-1.67), respectively). The ORs in
the Q2 and Q3 groups were not significant. A significant trend
relationship with Der 1 was noted in the cumulative incidence rates of
asthma and wheezing (Fig. 2). By onset age, a significant OR was noted
in asthma in the Q4 group at 12-24 months old (OR 1.91, 95\% CI:
1.17-3.12) and 24-36 months old (OR 1.99, 95\% CI: 1.07-3.70).
Similarly, a significant OR was noted in wheezing in the Q4 group during
0-12 months old (OR 1.60, 95\% CI: 1.18-2.16). A significant trend was
noted at all the aforementioned onset ages.

\subsection*{Association of endotoxin with the incidence of asthma or
wheezing}

{\label{association-of-endotoxin-with-the-incidence-of-asthma-or-wheezing}}

The adjusted ORs of the endotoxin exposure level are shown in Fig. 3.
The adjusted OR of the cumulative incidence rate of asthma during 0-36
months old was 1.59 (95\% CI: 1.03-2.44) in the Q4 group of the
endotoxin exposure level, indicating significance. The OR (95\% CI) of
the cumulative incidence rate of wheezing during 0-36 months old was
significant in the Q2 group of the endotoxin exposure level (OR: 1.28
{[}95\% CI: 1.04-1.57{]}). In the cumulative incidence rate of asthma, a
significant trend relationship with the exposure level was noted (Fig.
3). By onset age, the ORs for the onset of asthma (OR 2.00, 95\% CI:
1.03-3.85) and wheezing (OR 1.78, 95\% CI: 1.01-3.12) were the highest
and significant in the Q4 group during 24-36 months old.

\subsection*{Association of mite allergen or endotoxin with high levels
of total serum
IgE}

{\label{association-of-mite-allergen-or-endotoxin-with-high-levels-of-total-serum-ige}}

The adjusted ORs for high levels of total serum IgE at 24 months old in
the Q2-Q4 groups of the dust mite allergen exposure level and endotoxin
exposure level in comparison with the Q1 group are shown in Fig. 4. A
significant OR (95\% CI) was noted in both dust mite allergen and
endotoxin exposure levels in the Q4 group in comparison with the Q1
group (OR: 1.69 (95\% CI: 1.18-2.42) and OR: 1.66 (95\% CI: 1.07-2.57),
respectively, but the OR was not significant in the Q2 or Q3 group. A
significant trend was noted for both dust mite allergen and endotoxin
exposure levels. Results of sensitivity analysis of the `amount per unit
mass of dust' are shown in Supp Info Figure 3-5.

\section*{Discussion}

{\label{discussion}}

In this study, the risks of the cumulative incidence rates of asthma and
wheezing increased during 0-36 months old in children with a high level
of dust mite allergen exposure in bed dust. Regarding the endotoxin
exposure level, a significant positive OR was occasionally noted in the
onset of asthma and wheezing in the high exposure group. In the
simultaneous exposure to allergen, microorganisms, fungi, and air
pollutants, these were found to interact with each other and promote or
prevent allergic disease.\textsuperscript{27} As dust mite allergen and
endotoxin were simultaneously investigated in only limited
epidemiological studies, these are individually discussed below. In an
integrated analysis of five geographically different European birth
cohort studies, an association was noted between dust mite allergen
exposure (2-6 months old) and sensitisation to house dust mite (\selectlanguage{english}[?]6 years
old), but no association was observed with an increase in the risk of
asthma (\selectlanguage{english}[?]6 years old), suggesting that house dust mite exposure alone is
insufficient to increase the asthma risk and sensitisation with allergen
bridges allergen exposure and the development of
asthma.\textsuperscript{6,27} Similarly, in this study, the association
of an increase in the risk of high levels of total serum IgE with dust
mite allergen exposure was observed in the Q4 group and the OR was 1.58
(95\% CI: 1.08-2.31). In addition, a trend was observed, suggesting an
association between dust mite allergen exposure and sensitisation with
allergen. This finding is consistent with the results of the five
European birth cohort studies. On the contrary, no increase in the
asthma risk was noted in the European cohorts, but a trend was observed
between dust mite allergen exposure and the cumulative incidence rates
of asthma and wheezing in the present study. This may have been due to
differences in the dust mite allergen sampling point and the difference
in the unit of Der 1 (\selectlanguage{greek}μ\selectlanguage{english}g/m\textsuperscript{2}, \selectlanguage{greek}μ\selectlanguage{english}g/g). Regarding the
sampling point, dust on the floor of the children's bedroom was
collected in the European cohort studies, whereas dust mite allergen in
bed dust was evaluated in our study. The dust mite allergen
concentration is higher in bed dust than that in dust on the bedroom
floor and the dust mite allergen exposure level in children was also
higher.\textsuperscript{26} Although it is necessary to pay attention to
a simple comparison of the Der 1 concentration, the geometric mean was
within a range of 0.06-0.97 \selectlanguage{greek}μ\selectlanguage{english}g/g in the European cohort studies, but the
Der 1 concentration was higher at 3.88 \selectlanguage{greek}μ\selectlanguage{english}g/g in our study performed in
Japan. Regarding the second point, the difference in the unit of Der 1
level, \selectlanguage{greek}μ\selectlanguage{english}g/g was used in the European cohort studies, whereas it was
measured as \selectlanguage{greek}μ\selectlanguage{english}g/m\textsuperscript{2} in our study. The risk of mite
sensitisation markedly increased when the Der 1 level exceeded 2
\selectlanguage{greek}μ\selectlanguage{english}g/g.\textsuperscript{28} In the present study, based on analysis of the
amount per gram dust (unit: \selectlanguage{greek}μ\selectlanguage{english}g/g), no significant increase in total IgE
levels was noted in the Q3 (3.17-8.37 \selectlanguage{greek}μ\selectlanguage{english}g/g) or Q4 (8.37 \selectlanguage{greek}μ\selectlanguage{english}g/g or higher)
groups (Supp Info Figure 5), but in the evaluation of the amount per
dust sampling area, a marked increase in total IgE levels was noted in
the Q4 group (158.27 \selectlanguage{greek}μ\selectlanguage{english}g/m\textsuperscript{2} or larger) (Fig. 2),
suggesting that the evaluation of the amount per dust sampling area is
more important. In this study, a significant association with dust mite
allergen was observed for the development of wheezing during 0-12 months
old and the development of asthma during 0-24 months old in analysis by
the onset age. The most important steps towards the development of
mature systematic immune responses are taken in the first 1-2 years of
life.\textsuperscript{5,29} Therefore, avoidance of early-life exposure
to high concentrations of dust mite allergen may be important to prevent
allergic diseases, such as asthma. Previous studies have lacked
consistency in the association between endotoxin and asthma. For
example, a cohort study was performed in an urban area of Boston
involving children with parents having a medical history of atopy, in
which simultaneous exposure to dust mite allergen (in the children's
bed) and endotoxin (in family room dust) was investigated until 2-3
months after birth, and the associations with asthma, wheezing, and
atopy were surveyed until 7 years old. Endotoxin exposure was associated
with an increase in the risk of asthma at 7 years old and wheezing at
1-7 years old (no change in the risk due to children's age with
wheezing).\textsuperscript{26} In a meta-analysis of 19 studies
involving infants, the OR of wheezing in the high endotoxin exposure
group (endotoxin concentration per gram of dust) compared to that in the
low exposure group was 1.48 (95\% CI: 1.10-1.98), demonstrating a
positive correlation, whereas the OR of asthma after the participants
reached school age was 0.82 (95\% CI: 0.67-0.97), demonstrating an
inverse correlation.\textsuperscript{12} Shamosollahi et al.
systematically reviewed endotoxin exposure and health influence and
discussed that ``early life exposure to endotoxin at the environmental
level induces respiratory symptoms, including wheezing, because of
airway inflammation''. They reported that early-life environmental
exposure to endotoxin may induce inflammatory reaction, but continuous
exposure to endotoxin may activate immunity in healthy individuals and
prevent the later onset of asthma.\textsuperscript{30}In our study,
significantly positive ORs of asthma and wheezing onsets were
occasionally noted in the Q2-Q4 groups of the endotoxin exposure level
in comparison with the Q1 group, supporting the possibility that
environmental exposure to endotoxin in early life induces an
inflammatory reaction. This finding is consistent with the results of
the study in the urban area of Boston, the meta-analysis, and the
systematic review. As the evaluation was performed for up to 3 years in
our study, the risk of (or preventive effect on) endotoxin
exposure-induced asthma at school age will be investigated through a
follow-up survey in the future. The geometric mean endotoxin
concentration was 15.5 EU/mg in a survey conducted in the US (n=6,953),
and it was 2.5-76 EU/mg in other surveys performed in US inner
cities,\textsuperscript{31} whereas that in dust in the present study
was 16.0 EU/mg, which is within a range similar to that seen in US. The
main strength of this study was that this is the first large-scale birth
cohort study wherein an environmental measurement for both dust mite
allergen and endotoxin and health influence were evaluated using
standardised methods involving approximately 3,000 infants. The
cumulative incidence rate of wheezing and asthma in early life could be
evaluated by conducting annual questionnaire surveys. However, this
study has the following limitations. First, dust mite allergen and
endotoxin exposure were evaluated only at 18 months old. Exposure to
high concentrations of allergens immediately after birth strongly
influences the advancement of paediatric allergic
disease.\textsuperscript{5,6} Samples were not collected immediately
after birth in our study, but dust was collected at the age of 18 months
old. The association with allergic disease over school age after growth
will be investigated in a follow-up survey. Second, asthma symptoms were
investigated using self-administered questionnaires filled out by
mothers. However, a validation study of questionnaires that followed the
diagnostic criteria of asthma used by allergists reported that the
sensitivity and specificity of asthma symptoms were 0.91 and 0.64,
respectively, in 0-24-month-old children.\textsuperscript{21} Third,
total IgE levels at 24 months old alone were used as the cut-off for
outcome evaluation of high serum IgE levels, and the specific IgE level
could not be used. The associations with specific IgE and with changes
accompanying growth remain to be investigated.

\section*{Conclusion}

{\label{conclusion}}

Early-life exposure to dust mite allergen was associated with the onset
of wheezing during the age of 0-12 months old and asthma during the age
of 12-36 months old. Early-life exposure to endotoxin was also
associated with the onset of wheezing and asthma during the age of 24-36
months old. Our results indicated that high levels of early life
exposure to dust mite allergen and endotoxin may be involved in the
development of asthma.

\textbf{References}

1. Graham-Rowe D. Lifestyle: When allergies go west. Nature.
2011;479:S2-S4.

2. Lambrecht BN, Hammad H. The immunology of the allergy epidemic and
the hygiene hypothesis. Nat Immunol. 2017;18:1076-1083.

3. Dick S, Friend A, Dynes K, et al. A systematic review of associations
between environmental exposures and development of asthma in children
aged up to 9 years. BMJ Open. 2014;4:e006554.

4. Miller JD. The role of dust mites in allergy. Clin Rev Allergy
Immunol. 2019;57:312-329.

5. Illi S, von Mutius E, Lau S, Niggemann B, Gr\selectlanguage{ngerman}über C, Wahn U. Perennial
allergen sensitisation early in life and chronic asthma in children: a
birth cohort study. Lancet. 2006;368:763-770.

6. Casas L, Sunyer J, Tischer C, et al. Early-life house dust mite
allergens, childhood mite sensitization, and respiratory outcomes.
Allergy. 2015;70:820-827.

7. Kanchongkittiphon W, Mendell MJ, Gaffin JM, Wang G, Phipatanakul W.
Indoor environmental exposures and exacerbation of asthma: an update to
the 2000 review by the Institute of Medicine. Environ Health Perspect.
2015;123:6-20.

8. Woodcock A, Lowe LA, Murray CS, et al. Early life environmental
control: effect on symptoms, sensitization, and lung function at age 3
years. Am J Respir Crit Care Med. 2004;170:433-439.

9. Gehring U, de Jongste JC, Kerkhof M, et al. The 8-year follow-up of
the PIAMA intervention study assessing the effect of mite-impermeable
mattress covers. Allergy. 2012;67:248-256.

10. Braun-Fahrländer C, Riedler J, Herz U, et al. Environmental exposure
to endotoxin and its relation to asthma in school-age children. N Engl J
Med. 2002;347:869-877.

11. von Mutius E, Vercelli D. Farm living: effects on childhood asthma
and allergy. Nat Rev Immunol. 2010;10:861-868.

12. Mendy A, Gasana J, Vieira ER, et al. Endotoxin exposure and
childhood wheeze and asthma: a meta-analysis of observational studies. J
Asthma. 2011;48:685-693.

13. Mendy A, Wilkerson J, Salo PM, Cohn RD, Zeldin DC, Thorne PS.
Endotoxin predictors and associated respiratory outcomes differ with
climate regions in the U.S. Environ Int. 2018;112:218-226.

14. Mendy A, Wilkerson J, Salo PM, Zeldin DC, Thorne PS. Endotoxin
clustering with allergens in house dust and asthma outcomes in a U.S.
national study. Environ Health. 2020;19:35.

15. Kawamoto T, Nitta H, Murata K, et al. Rationale and study design of
the Japan environment and children's study (JECS). BMC Public Health.
2014;14:25.

16. Michikawa T, Nitta H, Nakayama SF, et al. Baseline profile of
participants in the Japan Environment and Children's Study (JECS). J
Epidemiol. 2018;28:99-104.

17. Sekiyama M, Yamazaki S, Michikawa T, et al. Study design and
participants' profile in the sub-cohort study in the Japan Environment
and Children's Study (JECS). J Epidemiol. 2020. doi:
10.2188/jea.JE20200448.

18. Asher MI, Keil U, Anderson HR, et al. International Study of Asthma
and Allergies in Childhood (ISAAC): rationale and methods. Eur Respir J.
1995;8:483-491.

19. Weiland SK, Björkstén B, Brunekreef B, Cookson WO, von Mutius E,
Strachan DP. Phase II of the International Study of Asthma and Allergies
in Childhood (ISAAC II): rationale and methods. Eur Respir J.
2004;24:406-412.

20. Ellwood P, Asher MI, Beasley R, Clayton TO, Stewart AW. The
international study of asthma and allergies in childhood (ISAAC): phase
three rationale and methods. Int J Tuberc Lung Dis. 2005;9:10-16.

21. Okabe Y, Adachi Y, Itazawa T, et al. Evaluation of the validity of
two written questionnaires for epidemiological study of asthma in
younger children. Nihon Shoni Arerugi Gakkaishi The Japanese Journal of
Pediatric Allergy and Clinical Immunology. 2010;24:705-712.

22. Soto-Ramírez N, Ziyab AH, Karmaus W, et al. Epidemiologic methods of
assessing asthma and wheezing episodes in longitudinal studies: measures
of change and stability. J Epidemiol. 2013;23:399-410.

23. Martins TB, Bandhauer ME, Bunker AM, Roberts WL, Hill HR. New
childhood and adult reference intervals for total IgE. J Allergy Clin
Immunol. 2014;133:589-591.

24. Nishihama Y, Nakayama SF, Tabuchi T, et al. Determination of urinary
cotinine cut-off concentrations for pregnant women in the Japan
Environment and Children's Study (JECS). International Journal of
Environmental Research and Public Health. 2020;17:5537.

25. Yasueda H, Mita H, Yui Y, Shida T. Comparative analysis of
physicochemical and immunochemical properties of the two major allergens
from Dermatophagoides pteronyssinus and the corresponding allergens from
Dermatophagoides farinae. Int Arch Allergy Appl Immunol.
1989;88:402-407.

26. Celedón JC, Milton DK, Ramsey CD, et al. Exposure to dust mite
allergen and endotoxin in early life and asthma and atopy in childhood.
J Allergy Clin Immunol. 2007;120:144-149.

27. Burbank AJ, Sood AK, Kesic MJ, Peden DB, Hernandez ML. Environmental
determinants of allergy and asthma in early life. J Allergy Clin
Immunol. 2017;140:1-12.

28. Leaderer BP, Belanger K, Triche E, et al. Dust mite, cockroach, cat,
and dog allergen concentrations in homes of asthmatic children in the
northeastern United States: impact of socioeconomic factors and
population density. Environ Health Perspect. 2002;110:419-425.

29. Holt PG, Jones CA. The development of the immune system during
pregnancy and early life. Allergy. 2000;55:688-697.

30. Shamsollahi HR, Ghoochani M, Jaafari J, Moosavi A, Sillanpää M,
Alimohammadi M. Environmental exposure to endotoxin and its health
outcomes: A systematic review. Ecotoxicol Environ Saf. 2019;174:236-244.

31. Thorne PS, Mendy A, Metwali N, et al. Endotoxin exposure: predictors
and prevalence of associated asthma outcomes in the United States. Am J
Respir Crit Care Med. 2015;192:1287-1297.

Table 1. Participant characteristics\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
& Characteristics & n & (\%)\tabularnewline
Sex & Sex & 3,089 &\tabularnewline
& Boy & 1,576 & (51.0)\tabularnewline
& Girl & 1,513 & (49.0)\tabularnewline
Asthma incidence & Asthma incidence & 2,934 &\tabularnewline
& Age 0-12 months & 78 & (2.7)\tabularnewline
& Age 12-24 months & 140 & (4.8)\tabularnewline
& Age 24-36 months & 88 & (3.0)\tabularnewline
& Age 0-36 months & 306 & (10.4)\tabularnewline
& None & 2,628 & (89.6)\tabularnewline
Wheezing incidence & Wheezing incidence & 2,934 &\tabularnewline
& Age 0-12 months & 586 & (20.0)\tabularnewline
& Age 12-24 months & 398 & (13.6)\tabularnewline
& Age 24-36 months & 134 & (4.6)\tabularnewline
& Age 0-36 months & 1,118 & (38.1)\tabularnewline
& None & 1,816 & (61.9)\tabularnewline
Total serum IgE level in 2-year-old children & Total serum IgE level in
2-year-old children & 2,900 &\tabularnewline
& High: [?]180 IU/mL & 282 & (9.7)\tabularnewline
& Low: \textless{}180 IU/mL & 2,618 & (90.3)\tabularnewline
Total serum IgE level during pregnancy & Total serum IgE level during
pregnancy & 3,089 &\tabularnewline
& High: [?]214 IU/mL & 557 & (18.0)\tabularnewline
& Low: \textless{}214 IU/mL & 2,532 & (82.0)\tabularnewline
Urinary cotinine level in mid-pregnancy & Urinary cotinine level in
mid-pregnancy & 3,089 &\tabularnewline
& \textgreater{}36.8 \selectlanguage{greek}μ\selectlanguage{english}g/g creatinine & 175 & (5.7)\tabularnewline
& 0.31--36.8 \selectlanguage{greek}μ\selectlanguage{english}g/g creatinine & 1,022 & (33.1)\tabularnewline
& \textless{}0.31 \selectlanguage{greek}μ\selectlanguage{english}g/g creatinine & 1,892 & (61.2)\tabularnewline
Passive smoke after birth & Passive smoke after birth & 3,089
&\tabularnewline
& Yes & 863 & (27.9)\tabularnewline
& No & 2,226 & (72.1)\tabularnewline
Presence of indoor pets & Presence of indoor pets & 3,089
&\tabularnewline
& Yes & 543 & (17.6)\tabularnewline
& No & 2,546 & (82.4)\tabularnewline
Lower respiratory tract inflammation & Lower respiratory tract
inflammation & 3,089 &\tabularnewline
& Yes & 471 & (15.2)\tabularnewline
& No & 2,618 & (84.8)\tabularnewline
Respiratory syncytial virus infection & Respiratory syncytial virus
infection & 3,089 &\tabularnewline
& Yes & 606 & (19.6)\tabularnewline
& No & 2,483 & (80.4)\tabularnewline
Area & Area & 3,089 &\tabularnewline
& Cold area & 1,025 & (33.2)\tabularnewline
& Warm low-humidity area & 1,202 & (38.9)\tabularnewline
& Warm high-humidity area & 862 & (27.9)\tabularnewline
\bottomrule
\end{longtable}

Table 2. Concentration of dust mite allergen and endotoxin\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& & n & GM & 25th & 50th & 75th\tabularnewline
Der1 (Der p 1 + Der f 1) & Der1 (Der p 1 + Der f 1) & & & &
&\tabularnewline
& Der 1, \selectlanguage{greek}μ\selectlanguage{english}g/m\textsuperscript{2} & 3,089 & 64.95 & 22.52 & 54.07 &
158.76\tabularnewline
& Der 1, \selectlanguage{greek}μ\selectlanguage{english}g/g & 3,089 & 3.89 & 1.57 & 3.17 & 8.37\tabularnewline
Endotoxin & Endotoxin & & & & &\tabularnewline
& Endotoxin, EU/m\textsuperscript{2} \selectlanguage{ngerman}× 10\textsuperscript{5} & 2,941 &
2.71 & 1.51 & 2.84 & 5.02\tabularnewline
& Endotoxin, EU/mg & 2,941 & 16.0 & 9.80 & 16.0 & 25.0\tabularnewline
\bottomrule
\end{longtable}

Der p 1, \emph{Dermatophagoides pteronyssinus}; Der f
1,\emph{Dermatophagoides farinae}; EU, endotoxin unit, GM, geometric
mean.\textbf{Figure LegendsFigure 1.} Flowchart of the selection of
study participants\textbf{Figure 2.} Associations among dust mite
allergen, Der 1 {[}\selectlanguage{greek}μ\selectlanguage{english}g/m\textsuperscript{2}{]}, and incidence of asthma
or wheezing using a logistic regression model adjusted for high levels
of maternal total serum IgE, active smoking/passive smoking based on the
urinary cotinine level in mid-pregnancy, passive smoke after birth,
children's sex, lower respiratory inflammation, respiratory syncytial
virus (RSV) infection, presence of indoor pets, and climatic
region\textbf{Figure 3.} Associations between endotoxin
{[}EU/m\textsuperscript{2}{]} and the incidence of asthma or wheezing
using a logistic regression model adjusted for high levels of maternal
total serum IgE, active smoking/passive smoking based on the urinary
cotinine level in mid-pregnancy, passive smoke after birth, children's
sex, lower respiratory inflammation, respiratory syncytial virus (RSV)
infection, presence of indoor pets, and climatic region\textbf{Figure
4.} Associations between dust mite allergen, Der 1
{[}\selectlanguage{greek}μ\selectlanguage{english}g/m\textsuperscript{2}{]}, or endotoxin
{[}EU/m\textsuperscript{2}{]} and high serum IgE levels in children aged
24 months old using a logistic regression model adjusted for high levels
of maternal total serum IgE, active smoking/passive smoking based on the
urinary cotinine level in mid-pregnancy, passive smoke after birth,
children's sex, lower respiratory inflammation, respiratory syncytial
virus (RSV) infection, presence of indoor pets, and climatic
region\textbf{Appendices} Members of the JECS Group as of 2021:
Michihiro Kamijima (principal investigator, Nagoya City University,
Nagoya, Japan), Shin Yamazaki (National Institute for Environmental
Studies, Tsukuba, Japan), Yukihiro Ohya (National Center for Child
Health and Development, Tokyo, Japan), Reiko Kishi (Hokkaido University,
Sapporo, Japan), Nobuo Yaegashi (Tohoku University, Sendai, Japan),
Koichi Hashimoto (Fukushima Medical University, Fukushima, Japan),
Chisato Mori (Chiba University, Chiba, Japan), Shuichi Ito (Yokohama
City University, Yokohama, Japan), Zentaro Yamagata (University of
Yamanashi, Chuo, Japan), Hidekuni Inadera (University of Toyama, Toyama,
Japan), Takeo Nakayama (Kyoto University, Kyoto, Japan), Hiroyasu Iso
(Osaka University, Suita, Japan), Masayuki Shima (Hyogo College of
Medicine, Nishinomiya, Japan), Youichi Kurozawa (Tottori University,
Yonago, Japan), Narufumi Suganuma (Kochi University, Nankoku, Japan),
Koichi Kusuhara (University of Occupational and Environmental Health,
Kitakyushu, Japan), and Takahiko Katoh (Kumamoto University, Kumamoto,
Japan).

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