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\begin{document}

\title{Association between serum zonulin level and severity of house dust mite
allergic asthma}



\author[1]{Shereen Baioumy}%
\author[1]{Shereen Ibrahim}%
\author[2]{Aya Elgendy}%
\author[2]{Shaimaa Fouad}%
\affil[1]{Zagazig University Faculty of Human Medicine}%
\affil[2]{Ain Shams University Faculty of Medicine}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Increased intestinal permeability, either due to the
exposure to antigens in asthmatic patients or due to a barrier defect,
play a critical role in susceptibility to environmental allergens. House
dust mites allergy occurs more commonly than any other allergens among
Egyptian asthmatic patients. Aim: To assess the relation between serum
zonulin level as a marker of increased intestinal permeability and the
severity of house dust mites allergic asthma. Methods: A case control
study which included 96 subjects attending the allergy and immunology
unit, microbiology and immunology department, Faculty of medicine,
Zagazig University. They were divided into 48 house dust mites allergic
asthma and 48 healthy control subjects. Results: On comparing the 2
studied groups, there was a statistically significant difference between
the 2 groups concerning serum IgE and serum zonulin levels ( p=0.000,
0.000 respectively)The mean serum zonulin was equal to 258.3\selectlanguage{ngerman}±153.01
ng/ml in the asthmatic group and 80±13 ng/ml in the control group. Serum
zonulin level significantly increased with the increase of asthma
severity (p\selectlanguage{english}<0.001). The cut off value of serum zonulin was [?] 198 ng/ml,
and the area under the curve was 0.76. It displayed sensitivity equal to
80\% and specificity equal to 71.4\%. Its negative predictive value was
equal to 83.3\%. Conclusion: Intestinal barrier dysfunction contributes
in the pathogenesis of allergic asthma. Serum zonulin level reflects an
increase in intestinal permeability and acts as prognostic factor of
severity in Asthma. Correction of the gut barrier defect may be an
additional novel approach for Asthma.%
\end{abstract}%



\sloppy


\textbf{Association between serum zonulin level and severity of house
dust mite allergic asthma}

\textbf{Authors:} \textbf{Shereen A. Baioumy}
\textsuperscript{1}\textbf{, Shereen Mahmoud Ibrahim\textsuperscript{3},
Aya Elgendy \textsuperscript{2} and Shaimaa Fouad\textsuperscript{2}}

\textbf{Running Title: Zonulin- Intestinal permeability marker - is a
predictor of Asthma severity}

\textbf{Correspondence:} Shaimaa Hani Fouad, Department of Internal
Medicine/ Allergy and Clinical Immunology, Ain Shams University, 11566
Abbasia, Cairo, Egypt.

Phone/Fax: +(202) 24346888 Mobile: +(2) 01116110004

E-mail: shaimaahani@med.asu.edu.eg

\textbf{Authors details:}

1-Department of Microbiology and Immunology, Faculty of Medicine,
Zagazig University, Egypt. 2-Department of Internal Medicine / Allergy
and Clinical Immunology, Faculty of Medicine, Ain Shams University,
Cairo,Egypt. 3-Department of Parasitology , Faculty of Medicine, Zagazig
University, Egypt.

\textbf{Word count:} 4300

\textbf{Abstract}

\textbf{Background:}

Increased intestinal permeability, either due to the exposure to
antigens in asthmatic patients or due to a barrier defect, play a
critical role in susceptibility to environmental allergens. House dust
mites allergy occurs more commonly than any other allergens among
Egyptian asthmatic patients.

\textbf{Aim:}

To assess the relation between serum zonulin level as a marker of
increased intestinal permeability and the severity of house dust mites
allergic asthma.

\textbf{Methods:}

A case control study which included 48 house dust mites allergic asthma
and 48 healthy control subjects attending the allergy and immunology
unit, microbiology and immunology department, Faculty of medicine,
Zagazig University.

\textbf{Results:}

On comparing the 2 studied groups, there was a statistically significant
difference between the 2 groups concerning serum IgE and serum zonulin
levels ( p=0.000, 0.000 respectively)The mean serum zonulin was equal to
258.3\selectlanguage{ngerman}±153.01 ng/ml in the asthmatic group and 80±13 ng/ml in the control
group. Serum zonulin level significantly increased with the increase of
asthma severity (p\selectlanguage{english}<0.001). The cut off value of serum zonulin was [?] 198
ng/ml, and the area under the curve was 0.76. It displayed sensitivity
equal to 80\% and specificity equal to 71.4\%. Its negative predictive
value was equal to 83.3\%.

\textbf{Conclusion:}

Intestinal barrier dysfunction contributes in the pathogenesis of
allergic asthma. Serum zonulin level reflects an increase in intestinal
permeability and acts as prognostic factor of severity in Asthma.
Correction of the gut barrier defect may be an additional novel approach
for Asthma.

\textbf{Keywords:}

\emph{Asthma; Zonulin; Asthma grade; Severity; House dust mites;
Intestinal Barrier.}

\textbf{Introduction}

Asthma is a common chronic airway disease characterized by airway
inflammation, hyperresponsiveness, and variable airway obstruction,
which is often attributed to gene-environment interactions {[}1{]}.

Epidemiologic studies have shown that sensitization to indoor allergens
is an important risk factor for the occurrence of acute attacks of
asthma{[}2{]}.

The most prevalent indoor allergens include house dust mites (HDMs),
animal dander, moulds and cockroaches. Of these, HDMs, especially D.
pteronyssinus and D. farinae, are considered the major perennial indoor
allergen sources inducing allergic sensitization worldwide {[}3{]}.

Environmental factors, microbiome, epithelial cells and immune cells
show a dynamic cross talk at the skin and mucosal barriers in the
development of atopic dermatitis, allergic rhinitis, eosinophilic
esophagitis and asthma.{[}4{]}

Recent trends in targeting allergic disorders are directed towards
personalized medicine approach which necessitates novel developments in
the area of disease phenotyping , endotyping, and the development and
application of reliable biomarkers.{[}4{]}

Increased intestinal permeability due to the exposure to antigens in
asthmatic patients may play a role in susceptibility to environmental
allergens. Therefore, correction of the gut barrier defect may be an
additional novel approach for asthma treatment.{[}34{]}

Gut mucosal barrier depends on efficient tight junctions which are
regulated by over 50 proteins. Zonulin is a 47 kDa protein that
increases the permeability of the small intestine. It has a role in the
intestinal mucosal innate immunity and it is the only physiological
protein proven to reversibly modulate intestinal permeability. High
serum levels of zonulin have been found in several autoimmune diseases,
e.g., celiac disease as a marker of impaired intestinal
barrier.{[}5{]}Chronic inflammation develops in response to
environmental stimuli, it leads to up-regulated expression of zonulin
and serum zonulin was found to be significantly associated with presence
and severity of atopic dermatitis.{[}6{]}

\textbf{Patients and methods:}

This is a case control study which included 96 subjects attending the
allergy and immunology unit, microbiology and immunology department,
Faculty of medicine, Zagazig University.

\textbf{Ethical approval:}

The study was approved by the institutional review board (IRB) no
\#6856/14-12-2020, Faculty of medicine, Zagazig University. An informed
written consent was obtained from all parents at time of recruitment.
This study was conducted in accordance with the Declaration of Helsinki.

\textbf{Sample size:}

Assuming that serum zonulin level in case group versus control group is
(11.3 \selectlanguage{ngerman}± 3.7 ng/ml) and (9.3 ± 3.3 ng/ml) respectively with confidence
level 95\% and power 80\%. So the sample size is 96 (48 in every group)

They will be divided into 2 groups.

\emph{Group 1 (Control group):} includes 48 healthy controls who
fulfilled the same exclusion criteria as the patients, and never had
asthma or eczema.

\emph{Group 2 (Asthmatic patients):} includes 48 adult (\selectlanguage{english}[?]18 years old)
subjects with allergic asthma according to Global Initiative for asthma
consensus report \textbf{(GINA), 2020} . {[}7{]}

\textbf{Inclusion Criteria:}

Allergic asthma patients were selected as having:

Obstructive pattern; Low FEV1/FVC on pulmonary function test:

Allergy as positive skin prick test for house dust mites.

\textbf{Exclusion Criteria:}

1-Evidence of digestive disease

2-Any other condition associated with increased intestinal permeability,
such as cystic fibrosis,

3- Food allergy or patients having positive skin test against common
food allergens.

The control subjects fulfilled the same exclusion criteria as the
patients, and never had asthma or eczema.

4- Patient refusal to participate.

The control subjects fulfilled the same exclusion criteria as the
patients, and never had asthma or eczema.

\textbf{Each patient was subjected to the following:}

1-Full detailed history and examination to exclude any comorbid
condition that may affect the result of the study.

2-Full detailed allergic history and clinical examination of the
respiratory system

3-Chest x ray was done to exclude other lung pathology.

4- Pulmonary function tests with special emphasis on FEV1, FEV1/FVC
using SPIROMETRICS

5- Total serum IgE was done for each patient using enzyme-linked
immunosorbent assay (ELISA).

6- Skin prick testing to common allergens.

7-Asthma severity score was calculated according to GINA, 2020. {[}7{]}

8- Serum zonulin level detection by ELISA

\textbf{Asthma severity scoring:}

Asthma was divided into grades I--IV (I being the mildest and IV the
most severe). Grading was based on events that occurred over the past
six months; including day and night symptoms, effect of asthma on daily
activity, use of steroids, and peak expiratory flow rate as defined
by\textbf{GINA guidelines 2020.} Pulmonary function tests were performed
for all patients using a fully computerized Spirometer (Jaeger
MasterScreen\selectlanguage{english} IOS, version 5.2 manufactured by VIASYS Healthcare GmbH,
Hoechberg, Germany) Pulmonary functions were assessed using forced
expiratory volume in 1 second (FEV1), forced vital capacity (FVC) and
the FEV1/FVC ratio, measured and expressed as a percentage of predicted
values with a ratio higher than 0.8 being normal.

\textbf{Skin testing:}

\begin{itemize}
\tightlist
\item
  Skin testing was performed according \textbf{to \emph{Bernstein et
  al., 2008}} \emph{.} {[}8{]}
\item
  Allergen extracts of skin testing were standardized allergen extracts
  provided by \textbf{Hamilton (Omega, Allergy OVERSEAS consultant Inc.,
  Canada).} The test was performed using the following allergens: for
  aeroallergens: (house dust mite; Dermatophagoides (D.)pteronyssinus
  and Dermatophagoides (D.) farnae mites; grass, mixed pollens; mixed
  molds, tobacco, cotton, wool, cockroach, hay dust) and food allergens:
  (egg, fish, peanuts, cows' milk).
\item
  Histamine dihydrochloride (10 mg/ml) was used as a positive control,
  while saline was used as a negative control.
\item
  Subjects were asked to stop antihistamines a week before skin testing.
\item
  The largest diameter of the wheal was measured, and it was considered
  positive if it was \selectlanguage{english}[?] 3 mm (\textbf{ASCIA guidelines, 2020).} {[}9{]}
\item
  Those with positive tests for food allergy were excluded from the
  study.
\end{itemize}

\textbf{Sample collection:}

Five ml blood were collected by venipuncture under complete aseptic
conditions. Samples were left to clot then centrifuged at 1000 xg for 15
minutes. Sera were collected and stored at -20 degC. Sera were used to
measure serum levels of total IgE, and zonulin.

\textbf{Serum level of total IgE:}

Quantitative measurement of serum level of total IgE was done using a
commercially available sandwich enzyme-linked immunosorbent assay
(ELISA) Kit supplied by \textbf{Chemux Bioscience, Inc. (CA, USA, Cat
No.10602)} according to the manufacturer's instructions. The results
were expressed in IU/mL. The total IgE level in a normal, allergy-free
adult is less than 150 IU/ml of serum. The minimum detectable
concentration of IgE by this assay is estimated to be 5.0 IU / ml. The
Absorbance of standards and samples were measured at 450 nm using a
microtiter plate ELISA reader \textbf{(Biotek, USA).}

\textbf{Measurement of serumzonulin level:}

Quantitative measurement of serum level of zonulin was done using a
commercially available sandwich enzyme-linked immunosorbent assay
(ELISA) Kit supplied by \textbf{MyBiosource, Inc. (USA, Cat No:
MBS774026)} according to the manufacturer's instructions. The results
were expressed in ng/ml. Absorbance of standards and samples were
measured at 450 nm using a microtiter plate ELISA reader\textbf{(Biotek,
USA).} The kit detection range was 20ng/ml - 800ng/ml.

\textbf{Statistical analysis:}

The collected data were processed and coded before being analyzed using
SPSS program version 23. Quantitative data were presented as minimum,
maximum, mean and SD.

Qualitative data were presented as count and percentage.Student t test
was used to compare quantitative data between two groups and One Way
ANOVA test was used to compare more than two groups.Pearson's
correlation was used to measure linear relationship between two
continuous variables.ROC curve was used to measure diagnostic validity
of quantitative variables and linear regression test was used to measure
independent effect of some factors on quantitative outcome variable.P
value\textless{}0.05 was considered statistically significant.

\textbf{Results:}

The current study was a case control study which included 96 subjects
attending the allergy and immunology unit, microbiology and immunology
department, Faculty of medicine, Zagazig University during the year
2021.They were divided into 2 groups, group 1 included healthy controls
with a median age of 28 {[} n=48{]} and group 2 included patients with
allergic asthma according to Global Initiative for asthma consensus
report (GINA), 2020 with a median age of 30 {[} n=48{]}.

Regarding the asthmatic patients, the mean value of age was
30.67+-15.609, most of the patients were female (62.5\%) and most of them
lived in rural areas (54.16\%). 39 patients (81.3\%) had positive family
history of atopy. All patients displayed positive skin prick test
results to house dust mites, most of the patients (70.8\%) were
sensitive to D. pteronyssinus and 60.4\% were sensitive to D. farina. 14
patients were mono sensitized to a single type of house dust mites and
34 patients were polysensitized to both types. As for the grade of
asthma severity, 4 patients (8.3\%) had grade 1severity, 24 patients
(50\%) had grade 2 asthma severity, 12 (25\%) patients had grade 3
severity, and 8 patients (16.7\%) had grade 3 asthma severity. (Table 1)

Concerning the control group, the mean value of age was 32.7+- 11.2, most
patients were males (85.3\%) and 70.8\% of them lived in rural areas. As
per the family history of atopy, only 11 (22.9\%) patients had positive
family history of atopy and all of them displayed negative skin prick
test results.

On comparing serum total immunoglobulin E (IgE) level between asthmatic
patients and control subjects, there was a highly statistically
significant difference between both groups (p=0.000). Asthmatic patients
displayed a higher statistically significant level of serum total IgE.
The mean serum IgE was equal to 233.3+-103.4IU/ml in the asthmatic group
and 84.4+-18.6 IU/ml in the control group. (Table 2)

Similarly, there was a high statistically significant difference between
both groups as regards serum zonulin level. Asthmatic patients displayed
a higher statistically significant level of serum zonulin than control
subjects. The mean serum zonulin was equal to 258.3+-153.01 ng/ml in the
asthmatic group and 80+-13 ng/ml in the control group. (Table 2)

Factors affecting serum total IgE level were male sex, asthma grade, and
positive skin prick test. Male patients displayed a statistically
significant higher serum total IgE level (p=0.03). The mean value of
serum total IgE was 272.89+-141.33 among male patients and was
193.53+-56.38 IU/ml among female patients. Besides, post hoc test showed
a significant difference between grade 1 vs grade 2 as regards serum
total IgE. As the asthma severity increased there was a significant
corresponding increase in the serum total IgE (p=0.02). Moreover, there
was a highly statistically significant difference as regards the mean
value of serum total IgE between patients displaying positive skin prick
test results and those displaying negative skin prick test results
(p=0.002) The mean value of serum total IgE among patients displaying
positive skin prick test results was 244.41+-114.85 IU/ml. (Table 3)

With regard to the correlation between serum zonulin level and seum
total IgE, it didn't reach statistical significance (p=0.34) . (Table 4)
Figure 2

As for the factors affecting serum zonulin level, they were residence
and asthma grade. There was a statistically significant difference in
the serum zonulin level between patients living in Urban areas and those
living in rural areas. (p=0.002) The mean value of serum zonulin was
significantly higher in patients living in urban areas and it equaled to
315.55+-157.34 ng/ml. Post hoc test showed a highly statistically
significant difference between grade 1 vs grade 2, grade 1 vs grade 3,
grade 1 vs grade 4, grade 2 vs grade 4 and grade 3 vs grade 4 as regards
serum zonulin level (p\selectlanguage{english}<0.001). (Table 4) Figure 2

Grade of asthma had independent significant effect on zonulin level
(p\textless{}0.001) while serum total IgE had no significant effect on
zonulin level via linear regression analysis ( p= 0.684 NS). ( Table 5)

Roc curve was done to assess the validity of zonulin level for
differentiation between (grade 1 or 2) vs (grade 3 or 4) (p=0.002). It
displayed sensitivity equal to 80\% and specificity equal to 71.4\%. Its
positive predictive value was equal to 66.7\% and negative predictive
value was equal to 83.3\%. The cut off value of serum zonulin was [?] 198
ng/ml. The area under the curve was 0.76. (Table 6) Figure 3

\textbf{Discussion:}

Asthma is a chronic inflammatory lung disease which is characterized by
airway inflammation, intermittent airflow obstruction, and bronchial
hyper-responsiveness.{[}10{]} Allergic asthma can be sparked by allergen
sensitization in which house dust mites (HDM) are one of the most
prevalent indoor allergens.{[}11{]} The prevalence of allergic
sensitization is rising in developing countries, a phenomenon related to
rapid urbanization and an adult's migration from a rural to an urban
area increases their risk of sensitization to mite allergens. It also
could contribute to a rapid increase of chronic respiratory diseases
(CRD). Exposure to dust mite allergens is a risk factor for clinical
asthma in sensitized subjects.{[}12{]}

The relationship between allergic asthma and intestinal permeability is
a subject of research interest. An increase in the intestinal
permeability may result in facilitating the entry of allergenic proteins
from the intestinal lumen into the systemic circulation. Consequently,
activation of the adaptive immune system, allergen sensitization and/or
extra-intestinal inflammation occurs.{[}13{]}

The current study is a case control study that aimed at assessing the
relation between serum zonulin level as a marker of increased intestinal
permeability and the severity of house dust mites allergic asthma. Our
study included 96 patients; 48 asthmatic patients and 48 control
subjects. The mean age of the asthmatic patients (n=48) was
30.67\selectlanguage{ngerman}±15.609, 30 ( 62.5\%) were females and 18 ( 37.5\%) were males and
most of them, 26 patients, lived in rural areas (54.16\%). 39 patients
(81.3\%) had a positive family history of atopy. All patients displayed
positive skin prick test results to house dust mites, most of the
patients (70.8\%) were sensitive to D. pteronyssinus and 60.4\% were
sensitive to D. farina . 14 patients were mono sensitized to a single
type of house dust mites and 34 patients were polysensitized to both
types. As for the grade of asthma severity, 4 patients (8.3\%) had grade
1 severity, 24 patients (50\%) had grade 2 asthma severity , 12 (25\%)
patients had grade 3 severity, and 8 patients (16.7\%) had grade 3
asthma severity.

Several studies supported these current results indicating that
bronchial asthma was associated with females with an obvious sex
bias.\textbf{Ricciardolo FL et al., 2020} conducted a cross-sectional
study on 499 asthmatic patients to assess the potential difference
between asthmatic males and females in a real-life setting. Their study
also displayed a female predominance in which 301 patients 60.32\% were
females.{[}14{]} In addition, \textbf{Sabry, 2011} reported that
20--40\% of asthmatic females of reproductive age suffer from worsening
of their symptoms during their menstrual period, suggesting that sex
hormones may have a major role in the biologic sex difference.
{[}15{]}\textbf{.}

These results could be attributed to hormonal factors, environmental
exposure, and the presence of comorbidities. Since estrogen receptors
(ERs) are highly expressed in the lungs and their smooth muscles with a
special role in bronchoconstriction/dilatation, this explains the gender
bias. Moreover, another possible hormonal factor is the lack of male sex
hormone which plays an integral role in downregulation of innate and
adaptive immune response.{[}16{]} Genetic factors may also be involved.
The Cyclooxygenase (COX) pathways play an important role in the course
of bronchial asthma and Cyclooxygenase-2(COX-2) gene homozygosity is
related to females.{[}17{]}

In fact, rural residence has higher prevalence of parental smoking,
higher number of siblings, advanced overcrowding rates, higher humidity
levels and greater exposure to chemicals and farm animals.{[}18{]}
Moreover, hot climatic conditions and high population density are
crucial for the development and reproduction of mites{[}19{]} . Hence,
the geographical situation of Egypt and its favorable climatic
conditions together with its overpopulation contribute to the abundance
of HDM especially in rural areas.{[}21{]}

The current results displayed that asthma patients were more commonly
living in rural areas (54\%vs 45.84\% urban asthmatics) and all of them
had positive skin prick test response to house dust mites, DP;Df
(70\%,60\% respectively). \textbf{Lawson et al., 2017} likewise reported
that children with asthma who lived in rural areas were more likely to
wheeze or have more severe symptoms of wheeze. {[}20{]} Furthermore,
this also agreed with \textbf{Müsken et al., 2002} study which concluded
that sensitization to storage mites in Germany was more frequently
sensitive to D. pteronyssinus and the prevalence of positive skin test
results to storage mites was greater in rural than in city dwellers.
Also, In vitro sensitization to B. tjibodas mites was also significantly
greater in rural than in city dwellers.{[}42{]} Besides, \textbf{Hassan
and Hagrass 2017} reported that house dust mites (HDM) allergy occurs
more commonly than any other allergens among Egyptian asthmatic
patients. {[}21{]}

Several studies disclosed that the high frequency of family history of
allergy is common among asthmatics. Familial assemblage of asthma and
allergic disease has frequently been observed, indicating that a
positive familial history of atopy may be considered as an identifiable
risk factor of asthma. This may be due to the evidence hinting that
genetics play an essential role in the pathogenesis of asthma. In
alliance, \textbf{Antonios et al. 2012} reported 60\% of cases had a
positive family history and displayed 95\% positive reactions to both D.
pteronyssinus and D. farinae allergens using SPT among asthmatic
patients in Gharbia Governorate, Egypt. {[}22{]} Similar results were
reached by \textbf{Haggag MG et al, 2017} .Their study revealed that
62.5\% of patients had positive family history.{[}23{]}

A detailed clinical history and physical examination followed by the
detection of total serum IgE level and IgE immunoreactivity against
specific allergens still represents the cornerstone in approaching
allergic disorders.{[}4{]}

On comparing serum total immunoglobulin E (IgE) level between asthmatic
patients and control subjects in the present study, asthmatic patients
displayed a higher statistically significant level of serum total IgE.
It was statistically significantly higher among male asthmatic patients.
There was a positive correlation between total serum IgE level and
asthma severity grade and skin prick test results.

Several studies disclosed high serum IgE levels among asthmatic
patients. This is in alliance with \textbf{Kim et al. 2013} who stated
that the total IgE levels were significantly higher in
males.{[}24{]}\textbf{Fereidouni M et al.,2009} study uncovered that the
mean total IgE serum was significantly high among asthmatics. Males had
higher mean total IgE values than females (305 vs 252 IU/mL, P = 0.6),
but the difference was not significant.{[}25{]}

On the contrary, \textbf{Somani ,2008} demonstrated that total IgE level
is higher in female patients and they attributed their results to the
possibility that total levels of IgE might be inheritable, especially in
females, because of the existence of two alleles at the X-linked
locus.{[}26{]}

The disparity in the development of total IgE between males and females
is the consequence of the fact that the levels of total IgE depend on
many other factors; such as parasitic infestations, smoking, pollution,
local diet and different genetic background in which males are at
greater risk of exposure.{[}25{]}

IgE is an antibody linked to allergic reactions and airway inflammation
which is associated with asthma severity. IgE is also linked to airway
hyperresponsiveness, and lower pulmonary function. Additionally, the use
of monoclonal anti-IgE has resulted in decreasing asthma severity.

Concerning the correlation between level of total IgE and asthma
severity, \selectlanguage{polish}\textbf{Kovač K et al,2007}\selectlanguage{english} also discerned that the greater
the asthma severity the greater the total serum total IgE level
(\textgreater{}288.0 kIU/L) and specific IgE to D pteronyssinus
(\textgreater{}44.1 kIUA/L). They conducted a study to evaluate the
correlation between serum total IgE level and asthma severity in
asthmatic children sensitive to D pteronyssinus.{[}27{]}

\textbf{Kenawy et al} ., \textbf{2017} found a highly significant
increase in serum level of IgE in patient with severe asthma than those
with mild asthma with P values \textless{} 0.001.{[}28{]} Likewise, a
study done by \textbf{Rotsides and his coworkers (2010)} reported a
strong positive association between high IgE level and asthma severity
in children and they proposed that serum IgE level is a strong predictor
for allergy in asthmatic children. {[}29{]}

Regarding the relation between serum IgE level and the positivity of
skin prick test, \textbf{Baldacci S et al., 1996} concluded that the
higher IgE levels the greater the positivity of SPT regardless the
gender .{[}30{]} \textbf{Rose et al., 1996} likewise assumed that there
is some inverse relationship between the quantitative level of IgE
antibody and the level of allergen necessary to cause symptoms of
asthma.{[}31{]}

The impairment of the epithelial barrier is a corner stone in the
development of allergic diseases. Increased intestinal permeability,
either due to the exposure to antigens in asthmatic patients or due to a
barrier defect, play a critical role in susceptibility to environmental
allergens.{[}32{]} The mechanisms responsible for increased intestinal
permeability in asthma remain unclear. It seems that intestinal
permeability may be a ''bronchus to gut'' consequence of bronchial
inflammatory process because a correlation with the severity of the
disease, airflow obstruction, could be demonstrated {[}33{]} This
supports that a primary general mucosal defect could be present
simultaneously in several organs and clinically expressed in a single
organ after an exogenous stimulation, such as antigenic or environmental
factors {[}41{]}. Duodenal histological changes mimicking those observed
in bronchial mucosa have been shown. In addition, gastrointestinal
abnormalities have been also reported in patients with asthma.{[}5{]}

Therefore, correction of the gut barrier defect may be an additional
novel approach for asthma treatment {[}34{]} via reduction of epithelial
susceptibility to damage and consequent inflammatory and remodeling
responses. In addition to growth factors, several other peptides have
been identified that have the ability to restore barrier function. One
such peptide is AT-1001, a peptide inhibitor of zonulin {[}35{]}.
Zonulin is a prehaptoglobulin protein and a biomarker for gut barrier
leakiness that downregulates tight junction function, and it has been
proposed to play a role in several autoimmune diseases {[}8{]} and
asthma in children {[}32{]}. It would therefore be of interest to
examine zonulin expression in airways and its regulation in patients
with asthma.{[}36{]}

The present study disclosed that asthmatic patients displayed a higher
statistically significant level of serum zonulin than control subjects.
Patients living in rural areas had a significantly higher levels on
serum zonulin. Furthermore, there was a significant correlation between
zonulin level and asthma grade . The higher the zonulin level the
greater the grade of asthma severity. Nevertheless, we found that there
was no difference in serum levels of zonulin subjects regarding gender
and age. The cut of value of serum zonulin level to deffrentiate between
grade 1-2 asthma severity and grade 3-4 asthma severity. In addition,
serum zonulin showed a sensitivity equal to 80\%, a specificity equal to
71.4\%, a positive predictive value equal to 66.7\% and a negative
predictive value equal to 83.3\%. There was no statistically significant
correlation to serum total IgE concentration. Grade of asthma had
independent significant effect on zonulin level while serum total IgE
had no significant effect on zonulin level via linear regression
analysis.

\textbf{Benard and colleagues} in France were the first study in the
literature on intestinal permeability in asthma. They showed increased
intestinal permeability in adult patients with bronchial asthma compared
with patients with chronic obstructive pulmonary disease and healthy
control subjects. In their study, they used radioactive material
(CrEDTA) which was administered orally, and estimated its urinary
recovery. They noticed that both patients with allergic asthma and those
with nonallergic asthma were significantly different from the control
groups and reported that intestinal permeability was not correlated with
the severity of asthma as judged by FEV\textasciitilde{} measurement.
Unlike the current results, they disclosed that intestinal permeability
did not significantly vary according to asthma severity score or steroid
treatment {[}37{]} This may be due to difference in method used to
assess intestinal permeability.

Furthermore, a study performed by \selectlanguage{ngerman}\textbf{Cervantes-García D and
collaborators} aiming at assessing the outcome of oral Lactococcus
lactis NZ9000 use on airway inflammation and lung remodeling in
asthmatic rats and its relation to the preservation of the intestinal
barrier, concluded that oral L. lactis could be used for asthma
prevention through its maintenance of an adequately functioning
intestinal barrier.{[}38{]}

Preliminary data suggest that a subset of asthmatic patients have
increased serum zonulin levels and increased intestinal permeability
{[}39{]}. These data suggest that both the lung and intestinal mucosa
may be routes through which specific antigens can gain access to the
submucosa with subsequent exposure to the immune system leading to lung
inflammation {[}40{]}.

Moreover, a study performed by \textbf{Yamaide F et al. 2020} to examine
the differences between serum zonulin level among allergic children and
non- allergic children concluded that serum zonulin was significantly
greater in children with allergy (Food allergy, Bronchial Asthma). In
addition, it was significantly higher in patients with food allergy than
in bronchial asthma patients. However, their results are not completely
reliable as food allergy can be the main reason for increased intestinal
permeability and this explains the higher increase in zonulin level
among food allergy patients than among asthmatic ones. {[}32{]}

Up to the present moment no studies have been conducted to assess the
relationship between serum zonulin level and asthma severity or to
assess its correlation to different residential distribution. A study
performed by Sheen et al to assess serum zonulin level in atopic
dermatitis (AD) and its correlation to disease severity. They found that
AD group had a greater median serum zonulin level than the control group
and serum zonulin level had significantly positive correlations with age
and the SCORAD index, but not with total IgE, total eosinophil count
(TEC), or the number of allergens to which a child is sensitized. And
concluded that each 1 ng/mL increase in serum zonulin was associated
with a 15\% greater risk of moderate-severe AD {[}6{]}. Their results
agree with the current results as regards the correlation between serum
zonulin level and total serum IgE level. Both revealed no statistically
significant correlation.

\textbf{Conclusion:}

Intestinal barrier dysfunction contributes in the pathogenesis of
allergic asthma. Serum zonulin level reflects an increase in intestinal
permeability and acts as prognostic factor of severity in Asthma.
Correction of the gut barrier defect may be an additional novel approach
for Asthma.

\textbf{Funding:} none.

\textbf{Conflicts of interest:} none.

\textbf{Authors' contributions}

Shereen A. Baioumy, Shereen Mahmoud Ibrahim, Aya Elgendy and Shaimaa
Fouad designed the study. Shereen A. Baioumy and Shaimaa Fouad Fouad
shared in sample collection. Shereen Mahmoud Ibrahim and Aya Elgendy
shared in sample collection and did the statistical analysis. Shaimaa
Hani Fouad wrote the draft. Shereen A. Baioumy and Aya Elgendy performed
the critical review of the manuscript. All authors reviewed and approved
the final version. All authors read and approved the final manuscript.

\textbf{Availability of data and materials}

All the data needed to support the current findings could be found in a
supporting sheet.

\textbf{References:}

\begin{enumerate}
\tightlist
\item
  \textbf{Zhu X, Cui J, Yi L, Qin J, et al.} The Role of T Cells and
  Macrophages in Asthma Pathogenesis: A New Perspective on Mutual
  Crosstalk. Mediators of Inflammation.2020; 7835284.
  https://doi.org/10.1155/2020/7835284
\item
  \textbf{Trompette A, Divanovic S, Visintin A, et al.} Allergenicity
  resulting from functional mimicry of a Toll-like receptor complex
  protein. Nature. 2009 ; 457:585-8.
\item
  \textbf{Taha A, Etewa S, Abdel-Rahman S, et al.} House dust mites
  among allergic patients at the Allergy and Immunology Unit, Zagazig
  University: an immunologic and serologic study. J Parasit Dis. 2018
  ;42(3):405--415 https://doi.org/10.1007/s12639-018-1016-9
\item
  \textbf{Breiteneder, H., Peng, Y.Q., Agache, I., et al.} Biomarkers
  for diagnosis and prediction of therapy responses in allergic diseases
  and asthma. Allergy.2020; 75(12), pp.3039-3068.
\item
  \textbf{Ohlsson B, Orho-Melander M, Nilsson PM.} Higher levels of
  serum zonulin may rather be associated with increased risk of obesity
  and hyperlipidemia, than with gastrointestinal symptoms or disease
  manifestations. International journal of molecular sciences. 2017
  ;18(3):582.
\item
  \textbf{Sheen YH, Jee HM, Kim DH, et al.} Serum zonulin is associated
  with presence and severity of atopic dermatitis in children,
  independent of total IgE and eosinophil. Clin Exp Allergy. 2018;
  48:1059--1062. DOI: 10.1111/cea.13158
\item
  National Institutes of Health. Global Initiative for Asthma. Global
  Strategy for Asthma Management and Prevention Revision, 2020.
  http://www.ginaasthma.org
\item
  \textbf{Bernstein IL, Li JT, Bernstein DI, et al.} Allergy diagnostic
  testing: An updated practice parameter. Ann Allergy Asthma Immunol.
  2008:100: S1--S148.
\item
  Australasian Society of Clinical Immunology and Allergy (ASCIA) Skin
  Prick Testing Guide for Diagnosis of Allergic Disease the ASCIA
  website (2020) www.allergy.org.au/members/committees\#wpim
\item
  \textbf{Bateman ED, Hurd SS, Barnes PJ, et al.} Global strategy for
  asthma management and prevention: GINA executive summary Eur Respir
  J.2008; 31 (1) : 143-178 10.1183/09031936.00138707
\item
  \textbf{Dhaliwal AK, Singh D, Randhawa RK, et al.} Sensitivity in
  allergic asthmatic subjects towards house dust mite allergens.
  Systematic and Applied Acarology. 2021;26(1):75-84.
\item
  Chu HT, Tran TN, Doyen V, et al. The protective effect of rural life
  on mite sensitization disappears among urban migrants in the South of
  Vietnam. World Allergy Organization Journal. 2019;12:100085
  http://doi.org/10.1016/j.waojou.2019.100085
\item
  \textbf{Walker J, Dieleman L, Mah D, et al.} High prevalence of
  abnormal gastrointestinal permeability in moderate-severe asthma.
  Clinical and Investigative Medicine. 2014; 1:E53-7.
\item
  \textbf{Ricciardolo FL, Levra S, Sprio AE, et al.} Asthma in the
  real-world: the relevance of gender. International archives of allergy
  and immunology. 2020;181(6):462-6. doi: 10.1159/000506808
\item
  \textbf{Sabry EY.} Relation of perimenstrual asthma with disease
  severity and other allergic co-morbidities (The first report of
  perimenstrual asthma prevalence in Saudi Arabia). Allergol
  Immunopathol. 2011; 39(1):23--36.
\item
  \textbf{Senna G., Latorre M., Bugiani M., et al.} Sex differences in
  severe asthma: results from severe asthma network in Italy-SANI.
  Allergy, Asthma \& Immunology Research. 2021; 13(2), 219.
\item
  \textbf{Holmquist L and Vesterberg O.} Immunochromatographic direct
  sampling on filter testing for aeroallergens. J Biochem Biophys
  Methods.2003; 57(3):183--190.
\item
  \textbf{Hijazi N, Abalkhail B, and Seaton A.} Diet and childhood
  asthma in a society in transition: a study in urban and rural Saudi
  ArabiaThorax 2000;55:775-779. https://doi.org/
  10.1136/thorax.55.9.775.
\item
  \selectlanguage{polish}\textbf{Henszel Ł, Kalisińska E, Kosik-Bogacka DI, et al.}\selectlanguage{english} Mites in
  Dust Samples Collected from Sleeping Places in Apartments. Polish
  Journal of Environmental Studies. 2010;19(4).
\item
  \textbf{Lawson JA, Rennie DC, Cockcroft DW, et al.} Childhood asthma,
  asthma severity indicators, and related conditions along an
  urban-rural gradient: a cross-sectional study. BMC pulmonary medicine.
  2017 Dec;17(1):1-9.
\item
  \textbf{Hassan AA and Hagrass SA.} Prevalence of bronchial asthma in
  primary school children. Am J Med Sci.2017;7(2):67--73.
\item
  \textbf{Antonios SN, Ashour DS, Elkholy MG, et al.} House dust mites
  as a risk factor in chest allergic patients in Gharbia Governorate,
  Egypt. J Egypt Med Microbiol.2012; 21(1):51--62
\item
  \textbf{Haggag MG, Aboelazm AA, Al-Gazzar AM, et al.} Immunoblotting
  technique versus skin prick test for detection of allergen specific
  immunoglobulin e in allergic conjunctivitis. Journal of ophthalmology
  and related sciences. 2017; 1(2):39-50.
\item
  \textbf{Kim EJ, Kwon JW, Lim YM, et al.} Assessment of total/specific
  IgE levels against 7 inhalant allergens in children aged 3to 6 years
  in Seoul, Korea. Allergy Asthma Immunol Res.2013; 5(3):162--169
\item
  \textbf{Fereidouni M, Hossini RF, Azad FJ, et al.} Skin prick test
  reactivity to common aeroallergens among allergic rhinitis patients in
  Iran. Allergologia et immunopathologia. 2009;37(2):73-9.
\item
  \textbf{Somani VK.} A study of allergen-specific IgE antibodies in
  Indian patients of atopic dermatitis. Indian J Dermatol Venereol
  Leprol. 2008;4(2):100--104.
\item
  \selectlanguage{polish}\textbf{Kovač K, Dodig S, Tješić-Drinković D, et al.}\selectlanguage{english} Correlation
  between asthma severity and serum IgE in asthmatic children sensitized
  to Dermatophagoides pteronyssinus. Archives of medical research. 2007
  Jan 1;38(1):99-105.
\item
  \textbf{Kenawy EM, El-Hafeez H, Mohamed NA., et al.} Interleukin
  (il)-17 as a biomarker in assessment of bronchial asthma severity.
  Al-Azhar Med. J.2017; 46(2), , 443-454. DOI: 10.12816/0038267
\item
  \textbf{Rotsides DZ, Goldstein IF, Canfield SM, et al.} Asthma,
  allergy, and IgE levels in NYC head start children. Respir Med.2010;
  104(3):345-55.
\item
  \textbf{Baldacci S, Modena P, Carrozzi L, et al.} Skin prick test
  reactivity to common aeroallergens in relation to total IgE,
  respiratory symptoms, and smoking in a general population sample of
  northern Italy. Allergy. 1996 Mar;51(3):149-56.
\item
  \textbf{Rose G, Arlian L, Bernstein D, et al.} Evaluation of household
  dust mite exposure and levels, of specific IgE and IgG antibodies in
  asthmatic patients enrolled in a trial of immunotherapy. J ALLERGY
  CLIN IMMUNOL.1996;97 (5): 1071-1078
\item
  \textbf{Yamaide F, Fikri B, Nakano T, et al.} Serum zonulin levels are
  higher in pediatric allergic patients than those in healthy children.
  Authorea Preprints. 2020 Jun 28.
\item
  \textbf{Djukanovic R, Roche WR, Wilson JW, et al.} Mucosal
  inflammation in asthma. Am Rev Respir Dis 1990;142:434-57.
\item
  \textbf{Hijazi Z, Molla A M, Al-Habashi H, et al.} Intestinal
  permeability is increased in bronchial asthma. Arch Dis
  Child.2004;89:227--229. doi: 10.1136/adc.2003.027680 Int. J. Mol. Sci.
  ,18, 582; doi:10.3390/ijms18030582 www.mdpi.com/journal/ijms
\item
  \textbf{Swindle EJ, Collins JE, and Davies DE.} Breakdown in
  epithelial barrier function in patients with asthma: Identification of
  novel therapeutic approaches. J Allergy Clin Immunol.
  2009;124:23-34.doi:10.1016/j.jaci.2009.05.037
\item
  \textbf{Paterson BM, Lammers KM, Arrieta MC, et al.} The safety,
  tolerance, pharmacokinetic and pharmacodynamic effects of single doses
  of AT-1001 in coeliac disease subjects: a proof of concept study.
  Aliment Pharmacol Ther 2007;26:757-66.
\item
  \textbf{Benard A, Desreumeaux P, Huglo D, et al.} Clinical aspects of
  allergic disease Increased intestinal permeability in bronchial asthma
  The Journal Of Allergy And Clinical Immunology.1996;97(6): 1173-1178
\item
  \selectlanguage{ngerman}\textbf{Cervantes-García D, Jiménez M, Rivas-Santiago CE, et
  al.}Lactococcus lactis NZ9000 prevents asthmatic airway inflammation
  and remodelling in rats through the improvement of intestinal barrier
  function and systemic TGF-\selectlanguage{greek}β \selectlanguage{english}production. International Archives of
  Allergy and Immunology. 2021;182(4):277-91.
\item
  \textbf{Fasano A.} Zonulin and its regulation of intestinal barrier
  function: the biological door to inflammation, autoimmunity, and
  cancer. Physiol Rev 2011; 91:151-75. PMID:21248165; http://dx.doi.
  org/10.1152/physrev.00003.2008
\item
  \textbf{Sturgeona C, and Fasanoa A} . Zonulin, a regulator of
  epithelial and endothelial barrier functions, and its involvement in
  chronic inflammatory diseases, review. Tissue barriers.2016; 4(4):
  e1251384 (19 pages) http://dx.doi.org/10.1080/21688370.2016.1251384
\item
  \textbf{Knutson TW, Bengtsson U, Dannaeus A, et al.} Intestinal
  reactivity in allergic and nonallergic patients: an approach to
  determine the complexity of the mucosal reaction. Journal of allergy
  and clinical immunology. 1993 Feb 1;91(2):553-9.
\item
  \textbf{Musken H, Fernandez-Caldas E, Maranon F, et al} .In vivo and
  in vitro sensitization to domestic mites in German urban and rural
  allergic patients. J Investig Allergol Clin Immunol.2002; 12:177--181
\end{enumerate}

\textbf{Table (1):} \textbf{Description of studied subjects: :}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Variable}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Control} \textbf{Group (n=48)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Percentage\%}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Asthmatic patients} \textbf{(n=48)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Percentage\%}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Sex:} Male Female\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
28 20\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
85.3\% 14.7\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
18 30\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
37.5\% 62.5\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Age (years):} X \selectlanguage{ngerman}± SD Median\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
32.7 \selectlanguage{ngerman}± 11.2 28\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
30.67\selectlanguage{ngerman}±15.609 30\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Residence:} Rural Urban\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
34 14\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
70.8\% 29.2\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
26 22\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
54.16\% 45.84\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Family history of atopy} \textbf{Positive:}
\textbf{Negative:}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11 37\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
22.9\% 77\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
39 9\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
81.3\% 18.8\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Skin prick:} \textbf{negative} \textbf{positive}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
48 0\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
100\% 0\%\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0 48\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0\% 100\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{House dust Mites} \textbf{D. pteronyssinus} \textbf{D. farina}
\textbf{Both}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\_\_\_\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\_\_\_\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
48 34 29 34\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
100\% 70.8\% 60.4\% 70.8\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\textbf{Asthma grade}\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 1\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
4\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8.3\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 2\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
24\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
50.0\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 3\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 3\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
12\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
25.0\%\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 4\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
asthma grade 4\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
16.7\%\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table (2): Comparison between Total IgE levels and zonulin
levels among both control and case groups:}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Variable}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Control} \textbf{Group 1 (n=48)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{Asthmatic patients} \textbf{Group 2 (n=48)}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{F}\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
\textbf{P}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{Total IgE level (IU/ml)} \selectlanguage{ngerman}\textbf{Mean ± SD}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
84.4 \selectlanguage{ngerman}± 18.6\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
223.3 \selectlanguage{ngerman}± 103.4\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{4.8}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{0.000HS}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{Zounlin (ng/ml)} \selectlanguage{ngerman}\textbf{Mean ± SD}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
80 \selectlanguage{ngerman}± 13\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
258.3 \selectlanguage{ngerman}± 153.01\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{6.12}\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\textbf{0.000HS}\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table (3) Factors affecting Total IgE level:}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Variables\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
Variables\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Total IgE}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Total IgE}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Test value}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{P value}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Mean}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{SD}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Sex}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{male}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
272.89\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
141.33\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.29*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.03 S}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{female}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
193.53\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
56.38\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Residence}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{urban}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
219.27\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
93.30\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.25*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.81 NS}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{rural}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
226.69\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
112.99\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Age}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{\textless{}30 Years}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
240.00\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
120.07\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.12*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.27 NS}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{\textgreater{}30 years}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
206.58\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
82.78\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Asthma grade}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 1}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
154.50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
.58\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5.77**\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.02 S}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
205.87\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
88.23\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 3}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
241.08\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
113.93\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 4}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
283.25\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
132.03\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Skin prick}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{negative}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
172.00\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
34.37\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3.33*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.002 HS}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{positive}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
244.41\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
114.85\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

*Student t test **One Way ANOVA test (post hoc test shows significant
difference between grade 1 vs grade 2)

\textbf{Table (4) Factors affecting Zonulin level:}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Zonulin level}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Zonulin level}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Test value}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{P value}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Mean}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{SD}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Sex}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{male}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
290.11\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
163.89\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.12*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.27 NS\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{female}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
239.27\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
145.59\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Residence}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{urban}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
315.55\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
157.34\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2.51*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.02 S\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{rural}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
209.92\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
133.81\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Age}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{\textless{}30 Years}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
261.67\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
152.41\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.15*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.88 NS\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{\textgreater{}30 years}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
255.00\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
156.83\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Asthma grade}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 1}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
127.50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8.66\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
64.54**\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textless{}0.001 HS\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 2}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
209.50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
103.91\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 3}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
218.50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
75.50\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{asthma grade 4}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
530.00\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
87.83\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Skin prick}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{negative}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
299.29\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
182.05\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
1.20*\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.24 NS\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{positive}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
241.47\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
138.88\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Zonulin level}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{Zonulin level}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Total IgE}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\textbf{Pearson Correlation}\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.14\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
0.14\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{P value}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.34NS\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.34NS\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

*Student t test **One Way ANOVA test (post hoc test shows significant
difference between grade 1 vs grade 2, grade 1 vs grade 3, grade 1 vs
grade 4, grade 2 vs grade 4 and grade 3 vs grade 4)

\textbf{Table 5: Linear regression analysis for factors affecting
Zonulin level:}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& Unstandardized Coefficients & Unstandardized Coefficients &
Standardized Coefficients & Sig. & 95.0\% Confidence Interval for B &
95.0\% Confidence Interval for B\tabularnewline
& B & Std. Error & Beta & & Lower Bound & Upper Bound\tabularnewline
Grade 3 or 4 & 167.317 & 29.962 & 0.838 & \textless{}0.001 HS & 107.007
& 227.628\tabularnewline
Total IgE & 0.075 & 0.183 & 0.062 & 0.684 NS & -.293 &
0.443\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table (6): Validity ofZonulin level for differentiation between
(grade 1 or 2) vs (grade 3 or 4)}\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
\textbf{Parameters} & \textbf{Cut off point} & \textbf{AUC} &
\textbf{Sig} & \textbf{Sensitivity} & \textbf{Specificity} &
\textbf{+PV} & \textbf{-PV} & \textbf{95\% Confidence Interval} &
\textbf{95\% Confidence Interval}\tabularnewline
& & & & & & & & \textbf{Lower Bound} & \textbf{Upper
Bound}\tabularnewline
Zonulin & \emph{\textgreater{}} 198 & 0.764 & .002 HS & 80\% & 71.4\% &
66.7\% & 83.3\% & 0.622 & 0.906\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure1.Correlation between asthma grade of severity and serum
zonulin level.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Correlation between serum zonulin level and total
serum IgE level.}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3. Roc curve of Zonulin level for differentiation between
(grade 1 or 2) vs (grade 3 or 4)}

\selectlanguage{english}
\FloatBarrier
\end{document}

