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의학석사 학위논문
Quantitative distribution of tissue eosinophils
in the gastrointestinal tract of children with
functional abdominal pain disorders
compared to normal references and
inflammatory bowel disease
소아청소년 기능성 복통 환자의 위장관
조직 내 호산구의 양적 분포 – 정상
대조군 및 염증성 장질환 환자와의 비교
2018 년 2 월
서울대학교 대학원
의학과 소아과학전공
이은혜
A thesis of the Master of Science in Medicine
소아청소년 기능성 복통 환자의 위장관
조직 내 호산구의 양적 분포 – 정상
대조군 및 염증성 장질환 환자와의 비교
Quantitative distribution of tissue eosinophils
in the gastrointestinal tract of children with
functional abdominal pain disorders
compared to normal references and
inflammatory bowel disease
February 2018
The Department of Medicine
Seoul National University Graduate School
Eun Hye Lee
Quantitative distribution of tissue eosinophils
in the gastrointestinal tract of children with
functional abdominal pain disorders
compared to normal references and
inflammatory bowel disease
by
Eun Hye Lee
A thesis submitted to the Department of Pediatrics
in partial fulfillment of the requirements for the
Degree of Master of Science in Medicine (Pediatrics)
at Seoul National University
October 2017
Professor ____________________ Chairman
Professor ____________________ Vice chairman
Professor ____________________
소아청소년 기능성 복통 환자의 위장관
조직 내 호산구의 양적 분포 – 정상
대조군 및 염증성 장질환 환자와의 비교
지도교수 양 혜 란
이 논문을 의학석사 학위논문으로 제출함
2017 년 10 월
서울대학교 대학원
의학과 소아과학전공
이 은 혜
이은혜의 의학석사 학위논문을 인준함
2017 년 12 월
위 원 장 김 나 영 (인)
부위원장 양 혜 란 (인)
위 원 이 현 주 (인)
i
ABSTRACT
Quantitative distribution of tissue eosinophils
in the gastrointestinal tract of children with
functional abdominal pain disorders
compared to normal references and
inflammatory bowel disease
Eun Hye Lee
Pediatrics, Medicine
The Graduate School
Seoul National University
Introduction: Functional abdominal pain disorders (FAPDs) are common in
children and adolescents. However, the accurate pathogenesis of FAPDs is
not yet known. Recently, micro-inflammation, particularly eosinophilia
associated with the gastrointestinal (GI) tract has been suggested as the
pathophysiology observed in several GI disorders. We aimed to evaluate
eosinophilic infiltration throughout the GI tract in children and adolescents
with FAPDs, compared to those diagnosed with inflammatory bowel
diseases (IBD) and to normal reference values.
ii
Methods: We included 56 children with FAPDs, 52 children with Crohn’s
disease, and 23 children with ulcerative colitis. All subjects underwent
esophagogastroduodenoscopic and colonoscopic examination with biopsies
performed on the same day. Tissue eosinophil counts were assessed in 10
regions throughout the GI tract between the stomach and the rectum. Tissue
eosinophil counts of children with FAPDs were compared to those obtained
from children with IBD or normal pathological references from each of the
10 regions assessed. To minimize the effects of regional inflammation of
IBD itself, eosinophil counts were further analyzed after eliminating
segments that endoscopically showed macroscopic involvement.
Results: Eosinophil counts of the stomach (gastric antrum), small bowel
(duodenum, terminal ileum), and colon (cecum, ascending colon) were
observed to be significantly higher in children with FAPDs compared to
normal reference values. Eosinophil counts of the stomach (gastric antrum
and body) and the entire colon between the cecum and the rectum were
observed to be significantly higher in children presenting with IBD than in
those presenting with FAPDs. After selecting histopathological slides of GI
segments that endoscopically did not show macroscopic involvement,
comparison of tissue eosinophil counts between children with IBDs and
those with FAPDs revealed that eosinophil counts of the stomach (gastric
body), and the colon (cecum, descending and sigmoid colon, and the rectum)
iii
were also significantly higher in children presenting with IBD than those
presenting with FAPDs. A comparison between children with FAPDs and
those with IBD before and after selecting the macroscopically unaffected GI
segments in the IBD groups showed that there were no significant
differences in eosinophil counts of the small bowel (duodenum, terminal
ileum).
Conclusions: Significantly high eosinophil counts of the stomach and colon
showed a definite order in terms of the disease entity IBD, followed by
FAPDs, and normal controls, even after selecting uninvolved GI segments
in patients with IBD. Therefore, eosinophilic infiltration of the GI tract
might be associated with the intrinsic pathogenesis of both FAPDs and IBD
in children.
Keywords: Abdominal pain, Child, Eosinophils, Functional
gastrointestinal disorder, Inflammatory bowel disease
Student number: 2016-21932
iv
CONTENTS
Abstract ................................................................................................ i
Contents .............................................................................................. iv
List of tables and figures ......................................................................v
List of abbreviations ........................................................................ viii
Introduction ......................................................................................... 1
Materials and Methods ........................................................................ 6
Results ............................................................................................... 13
1. Patients characteristics .............................................................. 13
2. Comparison of eosinophil counts in the gastrointestinal tract
between patients with functional abdominal pain disorders and
those with inflammatory bowel diseases…................................ 15
3. Comparison of tissue eosinophil counts between normal controls
and those with functional abdominal pain disorders and
inflammatory bowel diseases……………………………..........26
4. Comparison of tissue eosinophil counts according to types of
functional abdominal pain disorders, age, and sex of patients with
functional abdominal pain disorders ......................................... 35
Discussion ......................................................................................... 39
References ......................................................................................... 49
Abstract in Korean ............................................................................ 61
v
LIST OF TABLES AND FIGURES
Figure 1. Photomicrograph of hematoxylin and eosin staining for
eosinophils in the ascending colon. ..................................12
Table 1. Demographic characteristics of the subjects recruited......... 14
Table 2. Comparison of gastrointestinal tract mucosal eosinophil
counts between children with functional abdominal pain
disorders and inflammatory bowel diseases........................18
Figure 2. Eosinophil counts in each part of the gastrointestinal tract in
normal controls, children with functional abdominal pain
disorders, and pediatric inflammatory bowel diseases
patients........................................................................……20
Table 3. Comparison of the diagnostic accuracy of gastrointestinal
tract mucosal eosinophil counts between children with
functional abdominal pain disorders and those with
inflammatory bowel diseases……….……………………21
Table 4. Comparison of gastrointestinal tract mucosal eosinophil
counts between children with functional abdominal pain
vi
disorders and inflammatory bowel diseases except regional
inflammation site……………………………………….....23
Figure 3. Eosinophil counts in each part of the gastrointestinal tract in
normal control, children with functional abdominal pain
disorders, and pediatric inflammatory bowel diseases
patients after eliminating the gastrointestinal parts with
grossly affected regional inflammation of IBD on
endoscopy ….…………………………………................. 25
Table 5. Comparison of tissue eosinophil counts between normal
controls and children with functional abdominal pain and
those with inflammatory bowel diseases………..…......... 29
Table 6. Comparison of tissue eosinophil counts among normal
controls, children with functional abdominal pain, and those
with inflammatory bowel diseases except regional
inflammation site................................................................ 31
Figure 4. Eosinophils distribution of whole gastrointestinal tract in
normal controls (A), children with functional abdominal
pain disorders (B), those with inflammatory bowel diseases
vii
(C), and those with IBD without macroscopic lesions on
endoscopy (D)…................................................................ 33
Table 7. Comparison of tissue eosinophil counts according to subtypes
of functional abdominal pain disorders ............................... 36
Table 8. Comparison of tissue eosinophil counts according to age
group in children with functional abdominal pain
disorders…………………………………………………..37
Table 9. Comparison of tissue eosinophil counts according to sex of
children with functional abdominal pain disorders.............. 38
viii
LIST OF ABBREVIATIONS
FAPDs; Functional abdominal pain disorders
FAP; Functional abdominal pain
GI; Gastrointestinal
FD; Functional dyspepsia
IBS; Irritable bowel syndrome
AM; Abdominal migraine
FAP-NOS; Functional abdominal pain – not otherwise specified
FGID; Functional gastrointestinal disorder
IBD; Inflammatory bowel diseases
CD; Crohn’s disease
UC; Ulcerative colitis
SES-CD; Simple endoscopic score for Crohn’s disease
UCEIS; Ulcerative colitis endoscopic index of severity
T-ileum; Terminal ileum
ix
A-colon; Ascending colon
T-colon; Transverse colon
D-colon; Descending colon
S-colon; Sigmoid colon
1
INTRODUCTION
Functional abdominal pain disorders (FAPDs) are common in children.
Functional abdominal pain (FAP) constitutes more than 50% of the
consultations in pediatric gastroenterology practice and 2-4% of all general
pediatric clinic visits (1, 2). A recent meta-analysis has reported a global
pooled prevalence rate for FAPDs was 16.4% based on the Rome III criteria
(3). The prevalence of FAPDs was higher in South America (16.8%) and
Asia (16.5%) than in North America (13.4%) and Europe (10.5%) (3, 4). It
is well-known that FAPDs could be associated with long-term symptoms. A
systematic study has demonstrated the persistence of recurrent abdominal
pain in 29.1% of patients who were followed for a median duration of 5
years (2, 5). A prospective study has demonstrated that approximately 41%
of children presenting with FAP continued to report clinically significant
abdominal pain after a mean duration of a 9-year follow-up into adolescence
and young adulthood (2, 6). Children with FAPDs tend to have a lower
quality of life than healthy children, and FAPDs can lead to frequent school
absences (4, 7, 8).
The newly developed Rome IV criteria to diagnose functional
gastrointestinal (GI) disorders for children and adolescents were released in
2016 (9). Based on the Rome IV criteria, FAPDs comprise 4 subtypes
2
including functional dyspepsia (FD), irritable bowel syndrome (IBS),
abdominal migraine (AM), and functional abdominal pain – not otherwise
specified (FAP-NOS). Several studies have reported the coexistence of more
than 1 FAPD subtype in 13-33% of children (2, 9-11). This overlap of
FAPDs is more common in children than in adults. Reportedly, a
population-based study has shown that in adults, the overlap of GI symptom
complexes ranged between 4% and 9% of patients (12). Furthermore,
studies performed in both children and adults have reported that the
diagnosis was observed to have changed from one functional
gastrointestinal disorder (FGID) to another over time (2, 13, 14). These
characteristics of FGIDs suggest that FGIDs may include dynamic disease
entities, and that there may be an overlap in underlying pathophysiology of
FGIDs including FAPDs (2, 14).
The pathomechanism to explain FAPDs remains unclear and
multifactorial hypotheses have been suggested (4, 9) including impaired GI
motility, visceral hypersensitivity, immune dysregulation, gut dysbiosis,
genetic predisposition, abnormal gut-brain interaction, and psychosocial
distress (9, 15-21). Studies supporting these hypotheses have been
performed primarily in patients diagnosed with FD and/or IBS, and there is
increasing evidence suggesting that low-grade intestinal inflammation is
essentially involved in the underlying pathogenesis of FGIDs, particularly in
3
patients presenting with FD and IBS (22-29). Many studies have reported
that increased infiltration of immune cells such as eosinophils, mast cells,
and lymphocytes and the subsequent degranulation of these activated
immune cells reflect low-grade mucosal inflammation in the GI tract that is
observed in patients with FD and/or IBS. Studies performed in adults
presenting with FGIDs have demonstrated that duodenal inflammation with
eosinophils and mast cells was primarily associated with FD (19, 26, 27).
This evidence supports the hypothesis that low-grade duodenal
inflammation is a driver of altered neural function associated with FD and
may explain the gastroduodenal motor and sensory abnormalities observed
in adults presenting with FD (19). However, in adults diagnosed with IBS,
most studies have shown increased small intestinal and colonic infiltration
of mast cells and T lymphocytes (28). However, the types of immune cells
that were increased, the degree of inflammation, and the segments of the GI
tract that were investigated have not been consistent across all these studies
performed in adults. Additionally, histopathological studies pertaining to
childhood FAPDs are relatively few, and studies involving children are
primarily limited to studies pertaining to FD (20, 30-34). Furthermore, the
results of some studies performed in children emphasize the role of gastric
immune cells, which are contrary to the results demonstrated by studies
performed in adults (4, 32, 33, 35).
4
In our previous study published in 2016, we demonstrated that
eosinophil counts in the gastric antrum and body mucosa were significantly
high in a definite order of in terms of the disease entity normal reference
values, followed by children with FAPDs, and then those with H. pylori
gastritis (4). Eosinophil counts in the duodenal mucosa also were high in the
same order, although this was not a significant change. There were no
significant differences observed in the eosinophil counts of the stomach or
duodenum among the 4 subtypes of FAPDs based on the Rome III criteria
(4). Based on these results, we concluded that gastric, rather than duodenal
eosinophilia, might have been associated with pediatric FAPDs observed in
our previous study (4).
In the present study, we extended our study to investigate the entire
GI tract. Unlike the esophagus, eosinophils are resident cells of the stomach,
small and large intestine. However, to date, the normal range of eosinophils
in the GI tract tissue is not well-defined; thus, interpretation of pathological
intestinal eosinophilia is difficult (36-38). It is also known that eosinophils
are not uniformly distributed throughout the length of the intestine (38-42).
Several studies have reported that eosinophils were more numerous in the
cecum and ascending colon than they were elsewhere (38-40, 43, 44).
Therefore, eosinophils of the GI tract should be analyzed and compared in
each specific region, even within the colon. To our knowledge, no studies
5
have investigated the entire GI tract and compared tissue eosinophilic
infiltration in patients presenting with FGIDs.
Therefore, we aimed to evaluate tissue eosinophilic infiltration
between the stomach and the rectum in children with FAPDs compared to
the normal reference values and disease controls with inflammatory bowel
diseases (IBD) in each segment throughout the bowel. In comparing with
the IBD group, additional analysis was performed after selecting the
segments of the GI tract, which endoscopically did not show macroscopic
involvement of IBD inflammation, in addition to a comparison with all
tissues showing IBD.
6
MATERIALS AND METHODS
Subjects
Our retrospective study included children and adolescents who visited the
Department of Pediatric Gastroenterology at the Seoul National University
Bundang Hospital with complaints of chronic recurrent abdominal pain and
accompanying GI symptoms including constipation, diarrhea, and bloating
et al. These children had been diagnosed with FAPDs between January 2009
and October 2015 and had undergone an upper endoscopic and colonoscopic
examination. Children excluded from this study were those with a
significant allergy such as allergic rhinitis, atopic dermatitis, asthma, and
food allergy, and those with underlying liver, kidney or heart disease that
could cause GI symptoms.
All children underwent laboratory tests including complete blood cell
counts, erythrocyte sedimentation rate (ESR), highly sensitive C-reactive
protein (CRP), liver function tests, serum amylase, lipase, serum electrolytes,
urinalysis, and stool examination for parasite, stool occult blood check, and
stool cultures for bacteria, as well as abdominal X-ray and ultrasonography.
Those showing abnormalities on laboratory tests including leukocytosis,
anemia, peripheral blood eosinophilia (>500/㎕), elevated liver or
7
pancreatic enzyme, electrolyte imbalance, high ESR or CRP, pyuria,
hematuria, detection of parasite or bacteria or occult blood in stool sample
were excluded. Those showing any abnormal imaging studies were likewise
excluded from the study. All enrolled children also underwent
esophagogastroduodenoscopic and colonoscopic examination with biopsies
obtained from all segments of the GI tract between the esophagus and
rectum. Children and adolescents who demonstrated any macroscopic
abnormality including mucosal nodularity, erosions, and/or ulcerations on
endoscopic examination were also excluded from the study. Eventually, 56
children with FAPDs were included and classified into subtypes of FAPDs
including FD, IBS, AM, and FAP-NOS based on the Rome IV criteria.
Among the children and adolescents who had been diagnosed with
IBD and had been treated in our tertiary medical center, those who
underwent both esophagogastroduodenoscopic and colonoscopic
examination with biopsies during the same period were also recruited
retrospectively as disease controls. Eventually, 52 children with Crohn’s
disease (CD) and 23 children with ulcerative colitis (UC) were enrolled.
Although the comparison with age- and sex-matched healthy children
as normal controls would be ideal, this was not performed because of ethical
considerations regarding subjecting healthy children without any GI
8
symptoms to endoscopic examination. Instead, we used previously
published normal pediatric pathology references as substitution for the data
of normal controls (4, 39, 40). Because the study reported by DeBrosse et al.
(39) does not provide information regarding the cecum and the descending
and sigmoid colon, we used another normal reference to compare data
pertaining to the colonic area. Thus, we used the study reported by
DeBrosse, et al. (39) as a reference to compare eosinophil infiltration in the
upper GI tract between the stomach and ileum and another study reported by
Saad et al. (40) to compare colonic tissue eosinophil counts.
Our retrospective study was approved by the Institutional Review
Board of Seoul National University Bundang Hospital (IRB No. B-1609-
363-102).
Endoscopic biopsy and histopathology
Esophagogastroduodenoscopy was performed using a GIF-Q260 or GIF-
XP260 scope (Olympus, Tokyo, Japan) and a colonoscopy was performed
using a PCF-Q260AL, GIF-Q260, or GIF-XP260 scope (Olympus, Tokyo,
Japan) based on the child’s weight, and age in most enrolled children.
However, in some children with CD, the presence/absence of anal stenosis
determined the choice of the colonoscope used. Endoscopic mucosal
9
biopsies were obtained from the esophagus, gastric antrum and body,
duodenum, terminal ileum, cecum, ascending, transverse, descending, and
sigmoid colon, as well as the rectum.
Biopsy tissues were immediately fixed using formalin and processed
with embedding in paraffin wax. Sections of size 3 ㎛ were cut from the
paraffin block, and stained using hematoxylin and eosin stains. The
eosinophil count was obtained from 5 randomly selected high-power fields
(HPF). Quantification of eosinophils was performed using an Axioskope40
microscope (Mirax-Carl Zeiss, Oberkochen, Germany) at X 400
magnification. Cell counts were performed by 2 pathologists who were
blinded to the status of the children, and a mean value of the 5 HPF counts
obtained was calculated for each child (Fig. 1).
We ensured that all recruited children with FAPDs demonstrated no
eosinophils in their esophagus and all children including those with FAPDs
and IBD demonstrated no H.pylori infection in the stomach biopsies.
Because it is known that eosinophils are not usually observed in the
esophageal mucosa in a non-pathological state, we excluded the esophagus
from the areas that were compared. Tissue eosinophil counts in each of the
10 regions of the GI tract of children with FAPDs were compared with those
in normal pathology references (normal controls) and those from children
10
diagnosed with IBD (disease controls), respectively.
To minimize the confounding effects of IBD-induced inflammation,
after selecting the histopathological specimens of the GI tract segments that
endoscopically did not show macroscopic IBD involvement, we analyzed
eosinophil counts as well. Local segments of macroscopically uninvolved
GI tract were selected based on use of 4 variables relating to the simple
endoscopic score for CD (SES-CD) (45) and 3 descriptors of ulcerative
colitis endoscopic index of severity (UCEIS) (46, 47). Variables considered
for SES-CD were ulcer size, ulcerated surface, affected surface, and the
presence of narrowing. Descriptors of UCEIS were vascular pattern,
bleeding, and erosions and ulcers. The segments in which all scores of all 4
variables pertaining to SES-CD and of all 3 descriptors pertaining to UCEIS
were 0 were selected for analysis after eliminating IBD-induced regional
inflammation.
Statistical Methods
Data for continuous variables are presented as mean ± SD for parametric or
median values along with range (minimum-maximum) for nonparametric
variables. Categorical variables are presented as a percentage of the total
number. Continuous data were analyzed using the Student’s t-test for
11
parametric variables and the Mann-Whitney U test for nonparametric
variables. To distinguish between FAPDs and IBD, we analyzed tissue
eosinophil counts as dichotomous variables after computing a receiver-
operating characteristic (ROC) curve and identifying the cut-off with
maximal sensitivity and specificity. For all statistical analyses, a two-sided
P value < 0.05 was considered statistically significant. All statistical
analyses were performed using PASW Statistics (version 20.0, SPSS Inc.,
Chicago, IL, USA) and MedCalc software (MedCalc 12.7.0, MedCalc
software, Mariakerke, Belgium).
12
Figure 1. Photomicrograph of hematoxylin and eosin staining for eosinophils in the
ascending colon. (magnification, x 400) (A) specimen of normal controls, (B) specimen of
functional abdominal pain disorders, (C) specimen of Crohn’s disease, (D) specimen of
ulcerative colitis
13
RESULTS
Patients Characteristics
Our study included 56 children fulfilling the Rome IV criteria for FAPDs
(39 boys and 17 girls, median age 14.1 years, range 2.8-18.9) who were
classified as IBS (n = 37), IBS + FD (n = 5), IBS + FAP-NOS (n = 3), IBS +
AM (n = 1), FAP-NOS (n = 6), FD (n = 3), and AM (n = 1).
Normal controls for the upper GI tract included 19 children based on
the reference cases: 8 boys and 11 girls, median age 12.0 years (range 2.0-
17.0). Normal controls for the lower GI tract included 41 children based on
the reference cases: 21 boys and 20 girls, median age 12.2 years (range 3.3-
17.9).
Disease controls with IBD included a total of 75 children: 55 boys
and 20 girls, median age 14.7 years (range 2.5-19.5). Disease controls with
IBD comprised 52 children with CD and 23 children with UC (Table 1).
14
Table 1. Demographic characteristics of the subjects recruited
Variable FAPDs Normal reference Normal reference IBD [CD+UC]
(n = 56) (upper GI, n = 19)
(lower GI, n = 41) (n = 75)
Sex
(boys : girls)
39:17 8:11
21:20
55:20
Age, yr
(mean ± SD)
13.3 ± 3.5 10.6 ± 4.2 11.7 ± 4.3 13.9 ± 3.6
Age is shown as mean ± SD.
FAPDs, functional abdominal pain disorders; upper GI, upper gastrointestinal region including gastric antrum, stomach body, duodenum and
terminal ileum; lower GI, lower gastrointestinal region including cecum, ascending colon, transverse colon, descending colon and
rectosigmoid colon; IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis
15
Comparison of eosinophil counts in the gastrointestinal tract
between patients with functional abdominal pain disorders
and those with inflammatory bowel diseases
Eosinophil counts of the stomach and the entire colon between cecum and
the rectum were significantly higher in children with IBD than in those with
FAPDs (Table 2, Fig. 2). Eosinophil counts of the duodenum and the
terminal ileum (T-ileum) were also higher in children with IBD, but this was
not a significant difference when compared with children diagnosed with
FAPDs (Table 2, Fig. 2).
A comparison between children diagnosed with FAPDs and those
with CD showed that the eosinophil counts of the stomach (both gastric
antrum and body) and the left colon (transverse [T] colon, descending [D]
colon, sigmoid [S] colon, and rectum) in children with CD were
significantly higher than those observed in children with FAPDs (Table 2).
Eosinophil counts of the duodenum, T-ileum, cecum, and the ascending
colon (A-colon) were also higher in children with CD than those observed
in children with FAPDs, although this difference was not statistically
significant. A comparison between children diagnosed with FAPDs and
those with UC showed that eosinophils throughout the colon between the
cecum and rectum were significantly higher in children with UC than in
16
children with FAPDs (Table 2). Eosinophil counts of the stomach and small
bowel (duodenum and T-ileum) were not significantly different between
children with FAPDs and those with UC (Table 2). Cut-off values of
eosinophil counts in the stomach and the entire colon to distinguish between
FAPDs and IBD are shown in Table 3.
To minimize the confounding effects of IBD-induced inflammation,
after histopathology slides of macroscopically uninvolved GI segments
without IBD mucosal lesions on endoscopy were selected, eosinophil counts
were analyzed between children with FAPDs and those with IBD as well.
Eosinophil counts in children with IBD were significantly higher than those
observed in children with FAPDs in the gastric body, cecum, D-colon, S-
colon, and rectum (Table 4, Figure 3). Eosinophil counts of the A-colon and
T-colon were also higher in children with IBD, but this difference was not
significant when compared to those in children diagnosed with FAPDs
(Table 4, Figure 3). Eosinophil counts of the small bowel (duodenum and
terminal ileum) were not significantly different between children with
FAPDs and those with IBD (Table 4, Figure 3). A comparison between
children with FAPDs and those with CD after eliminating the specimens of
the GI segments with macroscopic lesions, showed that the eosinophil
counts of the stomach (both gastric antrum and body) and 2 segments of the
large intestine (T-colon and rectum) in children with CD were significantly
17
higher than those observed in children with FAPDs (Table 4). A comparison
between children with FAPDs and those with UC without macroscopic
lesions, showed that the eosinophils in a section of the colon (A-colon, D-
colon, S-colon, and rectum) were significantly higher in children with UC
than those observed in children with FAPDs (Table 4).
A comparison between children with CD and those with UC showed
that children with UC demonstrated significantly higher numbers of
eosinophils in the A-colon, D-colon, S-colon, and rectum than those
observed in children with CD. However, after excluding the tissue
specimens with macroscopically active inflammatory lesions, there were no
significant differences observed in tissue eosinophil counts except in terms
of the A-colon (Data are not shown).
18
Table 2. Comparison of gastrointestinal tract mucosal eosinophil counts between children with functional abdominal
pain disorders and inflammatory bowel diseases.
Data indicate the mean number of eosinophils/high-power field for each anatomical region of the gastrointestinal tract. Data are shown as
mean ± SD.
*P value between FAPDs and CD was calculated by independent t- test.
Anatomical region FAPDs
(n = 56)
CD
(n = 52)
UC
(n = 23)
IBD [CD + UC]
(n = 75)
*P value
[FAPDs-CD]
†P value
[FAPDs-UC]
‡P value
[FAPDs-IBD]
Gastric antrum 4.1 ± 6.1 11.2 ± 13.0 5.4 ± 5.1 9.5 ± 11.5 0.001 0.207 0.001
Gastric body 2.6 ± 2.5 7.0 ± 9.7 5.0 ± 6.6 6.4 ± 8.9 0.004 0.104 0.001
Duodenum 13.0 ± 8.5 18.5 ± 19.4 15.0 ± 9.3 17.4 ± 17.1 0.078 0.389 0.068
Terminal ileum 22.3 ± 17.6 27.8 ± 17.7 19.1 ± 9.6 25.5 ± 16.3 0.291 0.983 0.488
Cecum 23.0 ± 19.2 27.0 ± 17.3 34.3 ± 18.6 29.4 ± 17.9 0.382 0.033 0.047§
Ascending colon 15.7 ± 9.5 20.4 ± 17.8 27.3 ± 15.9 22.7 ± 17.4 0.140 0.002 0.011
Transverse colon 13.1 ± 9.1 23.4 ± 20.8 27.3 ± 20.1 24.6 ± 20.5 0.004 0.005 < 0.001
Descending colon 12.7 ± 8.8 19.5 ± 15.3 31.8 ± 17.8 23.4 ± 17.0 0.014 <0.001 < 0.001
Sigmoid colon 11.9 ± 12.8 18.8 ± 16.7 29.3 ± 20.9 22.0 ± 18.6 0.021 <0.001 0.001
Rectum 3.3 ± 3.0 7.8 ± 9.8 25.9 ± 26.3 13.1 ± 18.1 0.003 <0.001 < 0.001
19
†P value between FAPDs and UC was calculated by independent t- test.
‡P value between FAPDs and IBD (CD+UC) was calculated by independent t- test except for
§P value by Mann-Whitney U test.
FAPDs, functional abdominal pain disorders; IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis
20
Figure 2. Eosinophil counts in each part of the gastrointestinal tract in normal controls,
children with functional abdominal pain disorders (FAPDs), and pediatric inflammatory
bowel diseases (IBD) patients. Eosinophil counts were expressed as mean ± SE. The order
of bar graphs is normal controls, FAPDs, and IBD. In the rectum, eosinophil counts
according to both upper GI reference and lower GI reference were presented because lower
GI tract pathology references presented the ‘rectosigmoid area’, with no clear division
between the sigmoid and the rectum. The ‘*’ mark means that P value is less than 0.05. The
reference used to compare eosinophil infiltration in the upper GI tract between the stomach
and ileum is the study reported by DeBrosse, et al.(39) The reference used to compare
colonic tissue eosinophil counts is the study reported by Saad et al.(40)
21
Table 3. Comparison of the diagnostic accuracy of gastrointestinal tract mucosal eosinophil counts between children with
functional abdominal pain disorders and those with inflammatory bowel diseases.
Data indicate the mean number of eosinophils/high-power field for each anatomical region of the upper gastrointestinal tract. Data are shown
as mean ± SD.
Anatomical region FAPDs
(n = 56)
IBD [CD+UC]
(n = 75)
*P value
[FAPD- IBD]
Cut-off value AUC sensitivity specificity †P value
Gastric antrum 4.1 ± 6.1 9.5 ± 11.5 0.001 4 0.697 63.2 74.5 <0.0001
Gastric body 2.6 ± 2.5 6.4 ± 8.9 0.001 4 0.672 50.7 81.1 0.0004
Duodenum 13.0 ± 8.5 17.4 ± 17.1 0.068
Terminal ileum 22.3 ± 17.6 25.5 ± 16.3 0.488
Cecum 23.0 ± 19.2 29.4 ± 17.9 0.047§ 25 0.631 55.1 71.9 0.0433
Ascending colon 15.7 ± 9.5 22.7 ± 17.4 0.011 23 0.617 39.7 83.7 0.0356
Transverse colon 13.1 ± 9.1 24.6 ± 20.5 < 0.001 10 0.703 79.7 52.3 <0.0001
Descending colon 12.7 ± 8.8 23.4 ± 17.0 < 0.001 19 0.729 51.6 86.0 <0.0001
Sigmoid colon 11.9 ± 12.8 22.0 ± 18.6 0.001 11 0.711 69.6 67.9 <0.0001
Rectum 3.3 ± 3.0 13.1 ± 18.1 < 0.001 8 0.754 44.9 94.3 <0.0001
22
*P value between FAPDs and IBD (CD+UC) was calculated by independent t- test except
§P value by Mann-Whitney U test.
†P value of cut-off value between FAPDs and IBD (CD+UC) was calculated by ROC curve.
CD, Crohn’s disease; UC, ulcerative colitis; AUC, area under the curve
23
Table 4. Comparison of gastrointestinal tract mucosal eosinophil counts between children with functional abdominal
pain disorders and inflammatory bowel diseases except regional inflammation site.
Data indicate the mean number of eosinophils/high-power field for each anatomical region of the upper gastrointestinal tract. Data are shown
as mean ± SD.
*P value was analyzed between FAPDs and CD except regional inflammatory sites.
Anatomical
region
FAPDs
(n = 56)
CD except
regional
inflammation
(n = 52)
UC except
regional
inflammation
(n = 23)
IBD except
regional
inflammation
(n = 75)
*P value
[FAPDs-CD]
†P value
[FAPDs-UC]
‡P value
[FAPDs-IBD]
Gastric antrum 4.1 ± 6.1 5.8 ± 4.1 5.6 ± 5.1 5.8 ± 4.4 0.002 b 0.141 0.096
a
Gastric body 2.6 ± 2.5 5.3 ± 5.7 5.2 ± 6.8 5.3 ± 6.0 0.007 b 0.088 0.002
a
Duodenum 13.0 ± 8.5 11.4 ± 6.9 13.7 ± 8.6 12.2 ± 7.5 0.365 0.686 0.614a
Terminal ileum 22.3 ± 17.6 25.5 ± 10.1 17.6 ± 8.7 22.2 ± 10.1 0.562 0.813 0.496b
Cecum 23.0 ± 19.2 29.9 ± 18.7 35.9 ± 16.7 31.6 ± 18.0 0.228 0.050 0.032b
Ascending colon 15.7 ± 9.5 17.1 ± 12.6 28.1 ± 16.4 20.1 ± 14.3 0.597 0.020 0.117a
Transverse colon 13.1 ± 9.1 18.3 ± 11.4 13.6 ± 7.5 17.2 ± 10.7 0.044 a 0.671 0.076
a
Descending colon 12.7 ± 8.8 18.8 ± 18.1 21.1 ± 10.7 19.4 ± 16.6 0.053 0.021 0.021a
Sigmoid colon 11.9 ± 12.8 15.7 ± 10.6 23.4 ± 16.4 17.4 ± 12.3 0.178 0.020 0.046a
Rectum 3.3 ± 3.0 5.8 ± 4.4 29.0 ± 37.9 9.2 ± 16.3 0.002 a 0.021 0.027
a
24
†P value between FAPDs and UC was analyzed by Mann-Whitney U test.
‡P value was analyzed between FAPDs and IBD.
a: P value was calculated by independent t- test. b: P value was calculated by Mann-Whitney U test.
FAPDs, functional abdominal pain disorders; IBD, inflammatory bowel disease; CD, Crohn’s disease; UC, ulcerative colitis
25
Figure 3. Eosinophil counts in each part of the gastrointestinal (GI) tract in normal controls,
children with functional abdominal pain disorders (FAPDs), and pediatric inflammatory
bowel diseases (IBD) patients after eliminating the GI segments with grossly affected
regional inflammation of IBD on endoscopy. Eosinophil counts were expressed as mean ±
SE. The order of bar graphs is normal controls, FAPDs, and IBD. In the rectum, eosinophil
counts according to both upper GI reference and lower GI reference were presented because
lower GI tract pathology references presented the ‘rectosigmoid area’, with no clear
division between the sigmoid and the rectum. The ‘*’ mark means that P value is less than
0.05.
The reference used to compare eosinophil infiltration in the upper GI tract between the
stomach and ileum is the study reported by DeBrosse, et al.(39) The reference used to
compare colonic tissue eosinophil counts is the study reported by Saad et al.(40)
26
Comparison of tissue eosinophil counts between normal
controls and those with functional abdominal pain disorders
and inflammatory bowel diseases
A comparison between children with FAPDs and normal controls showed
that eosinophil counts of children with FAPDs were significantly higher in
the stomach (gastric antrum), small bowel (duodenum, T-ileum), cecum, and
the A-colon than those observed in normal controls (Table 5, Fig. 2).
A comparison between children with IBD and normal controls
showed that eosinophil counts in all segments between the stomach and the
rectum were significantly higher in children with IBD than those observed
in normal controls (Table 5, Fig. 2). Because the pediatric lower GI tract
pathology references which we used as a substitute for normal controls
presented a ‘rectosigmoid area’ and the region was not definitively divided
into the sigmoid and rectum, it was difficult to accurately compare the
eosinophil counts in the rectum. Eosinophil counts of the rectum were
significantly higher in children with IBD than in those of normal controls
when the rectal eosinophils were compared using the values noted in the
pediatric upper GI tract pathology references (Table 5, Fig. 2).
A comparison between children with UC and those studied as normal
controls showed that the eosinophil counts throughout the GI tract between
27
the stomach and rectum were significantly higher in children with UC than
in those studied as normal controls (Table 5). A comparison between
children with CD and those studied as normal controls showed that the
eosinophil counts between the stomach and the sigmoid colon were
significantly higher in children with CD than in those studied as normal
controls (Table 5). Eosinophil counts of the rectum were not significantly
different between children with CD and those studied as normal controls
even when the rectal eosinophils were compared using the values obtained
from the pediatric upper GI tract pathology references (Table 5).
After selecting the histopathological specimens of the GI tract
segments that endoscopically did not show macroscopic IBD involvement,
we analyzed eosinophil counts between normal controls and children with
IBD, in these segments to minimize the effects of IBD-induced local
inflammation. Eosinophil counts in children with IBD showing
histopathology slides without macroscopic lesions were significantly higher
than in those studied as normal controls in the gastric antrum, gastric body,
T-ileum, and throughout the colon except the rectum (Table 6, Fig. 3).
Eosinophil counts in the duodenum were also higher in children with IBD
without macroscopic lesions, but not significantly different when compared
to normal controls (Table 6, Fig. 3).
28
A comparison between CD pathology without macroscopic lesions
and those studied as normal control showed that the eosinophil counts of the
stomach (gastric antrum and body), T-ileum, cecum, A-colon, T-colon, and
D-colon were significantly higher in those with CD without macroscopic
lesions than in those studied as normal controls (Table 6). A comparison
between children with UC and normal controls after excluding the tissue
specimens with macroscopically observed active inflammatory lesions
showed that the eosinophil counts of the stomach (gastric antrum and body),
duodenum, cecum, A-colon, D-colon, and S-colon were significantly higher
in those with UC than those observed in normal controls (Table 6).
Eosinophil counts of rectum in children with UC without macroscopic
lesions were much higher than in those studied as normal controls but this
difference was not statistically significant because the number of
eliminating slides was too large to show statistical power (Table 6).
Figure 4 shows eosinophilic distribution of whole GI tract in normal
controls, children with FAPDs, those with IBD, and those with IBD without
macroscopic lesions on endoscopy.
29
Table 5. Comparison of tissue eosinophil counts between normal controls and children with functional abdominal
pain and those with inflammatory bowel diseases.
*P value between normal control and FAPDs was calculated by independent t-test.
†P value between normal control and CD was calculated by independent t-test.
Anatomical
region
Normal control
(upper, n = 19)
(lower, n = 41)
FAPDs
(n = 56)
CD
(n = 52)
UC
(n = 23)
IBD
[CD+UC]
(n = 75)
*P value
[Control-
FAPDs]
†P value
[Control-
CD]
‡P value
[Control-
UC]
‡P value
[FAPDs
-IBD]
Gastric antrum 1.9 ± 1.3 4.1 ± 6.1 11.2 ± 13.0 5.4 ± 5.1 9.5 ± 11.5 0.006 <0.001 0.001 <0.001
Gastric body 2.1 ± 2.4 2.6 ± 2.5 7.0 ± 9.7 5.0 ± 6.6 6.4 ± 8.9 0.235 0.001 0.037 <0.001
Duodenum 9.6 ± 5.3 13.0 ± 8.5 18.5 ± 19.4 15.0 ± 9.3 17.4 ± 17.1 0.034 0.002 0.016 0.001
Terminal ileum 12.4 ± 5.4 22.3 ± 17.6 27.8 ± 17.7 19.1 ± 9.6 25.5 ± 16.3 0.009 <0.001 0.024 <0.001
Cecum 14.2 ± 6.1 23.0 ± 19.2 27.0 ± 17.3 34.3 ± 18.6 29.4 ± 17.9 0.006 <0.001 <0.001 <0.001
Ascending colon 12.0 ± 5.3 15.7 ± 9.5 20.4 ± 17.8 27.3 ± 15.9 22.7 ± 17.4 0.013 0.002 <0.001 <0.001
Transverse colon 11.9 ± 4.6 13.1 ± 9.1 23.4 ± 20.8 27.3 ± 20.1 24.6 ± 20.5 0.219 <0.001 <0.001 <0.001
Descending colon 10.7 ± 5.6 12.7 ± 8.8 19.5 ± 15.3 31.8 ± 17.8 23.4 ± 17.0 0.094 <0.001 <0.001 <0.001
Sigmoid colon 12.4 ± 6.1a 11.9 ± 12.8 18.8 ± 16.7 29.3 ± 20.9 22.0 ± 18.6 0.401 0.007 <0.001 <0.001
Rectum 12.4 ± 6.1a 3.3 ± 3.0 7.8 ± 9.8 25.9 ± 26.3 13.1 ± 18.1 <0.001 0.003 0.012 0.384
8.3 ± 5.9b 0.001 0.406 0.002 0.037
30
‡P value between normal control and was calculated by independent t-test.
§P value between normal control and IBD (CD+UC) was calculated by independent t-test.
a: Tissue eosinophil counts in the rectosigmoid according to normal pediatric lower gastrointestinal tract pathology references which we
adopted as substitution for the data of normal controls. This reference presented rectosigmoid area, not divided into sigmoid and rectum.
b: Tissue eosinophil counts in the rectum according to normal pediatric gastrointestinal tract pathology references which we adopted as
substitution for upper gastrointestinal tract data of normal controls.
The reference to compare eosinophil infiltration in the upper GI tract between the stomach and ileum is the study reported by DeBrosse, et al.
(39)
The reference to compare colonic tissue eosinophil counts is the study reported by Saad et al. (40)
FAPD, functional abdominal pain disorders; CD, Crohn’s disease; UC, ulcerative colitis; upper, upper gastrointestinal region including
gastric antrum, stomach body, duodenum and terminal ileum; lower, lower gastrointestinal region including cecum, ascending colon,
transverse colon, descending colon and rectosigmoid colon
31
Table 6. Comparison of tissue eosinophil counts among normal controls, children with functional abdominal pain,
and those with inflammatory bowel diseases except regional inflammation site.
*P value between normal control and FAPDs was calculated by independent t-test.
Anatomical
region
Normal control
(upper, n = 19)
(lower, n = 41)
FAPDs
(n = 56)
CD except
regional
inflammation
(n = 52)
UC except
regional
inflammation
(n = 23)
IBD except
regional
inflammation
(n = 75)
*P value
[Control-
FAPDs]
†P value
[Control
-CD]
‡P value
[Control
-UC]
§P value
[Control
-IBD]
Gastric antrum 1.9 ± 1.3 4.1 ± 6.1 5.8 ± 4.1 5.6 ± 5.1 5.8 ± 4.4 0.006 <0.001 0.001 <0.001
Gastric body 2.1 ± 2.4 2.6 ± 2.5 5.3 ± 5.7 5.2 ± 6.8 5.3 ± 6.0 0.235 0.001 0.035 0.001
Duodenum 9.6 ± 5.3 13.0 ± 8.5 11.4 ± 6.9 13.7 ± 8.6 12.2 ± 7.5 0.034 0.165 0.049 0.069
Terminal ileum 12.4 ± 5.4 22.3 ± 17.6 25.5 ± 10.1 17.6 ± 8.7 22.2 ± 10.1 0.009 <0.001 0.056 <0.001
Cecum 14.2 ± 6.1 23.0 ± 19.2 29.9 ± 18.7 35.9 ± 16.7 31.6 ± 18.0 0.006 <0.001 <0.001 <0.001
Ascending colon 12.0 ± 5.3 15.7 ± 9.5 17.1 ± 12.6 28.1 ± 16.4 20.1 ± 14.3 0.013 0.035 0.003 0.002
Transverse colon 11.9 ± 4.6 13.1 ± 9.1 18.3 ± 11.4 13.6 ± 7.5 17.2 ± 10.7 0.219 0.004 0.281 0.005
Descending colon 10.7 ± 5.6 12.7 ± 8.8 18.8 ± 18.1 21.1 ± 10.7 19.4 ± 16.6 0.094 0.016 0.006 0.003
Sigmoid colon 12.4 ± 6.1a 11.9 ± 12.8 15.7 ± 10.6 23.4 ± 16.4 17.4 ± 12.3 0.401 0.066 0.031 0.013
Rectum 12.4 ± 6.1a 3.3 ± 3.0 5.8 ± 4.4 29.0 ± 37.9 9.2 ± 16.3 <0.001 <0.001 0.142 0.120
8.3 ± 5.9b 0.001 0.076 0.092 0.382
32
†P value between normal control and CD except regional inflammation was calculated by independent t-test.
‡P value between normal control and UC except regional inflammation was calculated by independent t-test.
§P value between normal control and IBD (CD+UC) except regional inflammation was calculated by independent t-test.
a: Tissue eosinophil counts in the rectosigmoid according to normal pediatric lower gastrointestinal tract pathology references which we
adopted as substitution for the data of normal controls. This reference presented rectosigmoid area, not divided into sigmoid and rectum.
b: Tissue eosinophil counts in the rectum according to normal pediatric gastrointestinal tract pathology references which we adopted as
substitution for upper gastrointestinal tract data of normal controls.
The reference to compare eosinophil infiltration in the upper GI tract between the stomach and ileum is the study reported by DeBrosse, et al.
(39)
The reference to compare colonic tissue eosinophil counts is the study reported by Saad et al. (40)
FAPDs, functional abdominal pain disorders; CD, Crohn’s disease; UC, ulcerative colitis; upper, upper gastrointestinal region including
gastric antrum, stomach body, duodenum and terminal ileum; lower, lower gastrointestinal region including cecum, ascending colon,
transverse colon, descending colon and rectosigmoid colon
33
Figure 4. Eosinophils distribution of whole gastrointestinal (GI) tract in normal controls (A),
functional abdominal pain disorders (FAPDs) group (B), inflammatory bowel diseases (IBD)
34
group (C), and IBD group after selecting the histopathological specimens of the GI tract
segments that endoscopically did not show macroscopic IBD involvement (D).
Eosinophil counts were expressed as mean ± SE. In the rectum, eosinophil counts according
to both upper GI reference and lower GI reference were presented because lower GI tract
pathology references presented the ‘rectosigmoid area’, with no clear division between the
sigmoid and the rectum. The ‘*’ mark means normal reference of rectum by the upper GI
tract pathology references. The reference used to compare eosinophil infiltration in the
upper GI tract between the stomach and ileum is the study reported by DeBrosse, et al.(39)
The reference used to compare colonic tissue eosinophil counts is the study reported by
Saad et al.(40)
35
Comparison of tissue eosinophil counts according to types of
functional abdominal pain disorders, age, and sex of patients
with functional abdominal pain disorders
We compared tissue eosinophil counts in each specific region according to
types of FAPDs. Total of 56 children with FAPDs were classified as IBS (n
= 37), IBS + FD (n = 5), IBS + FAP-NOS (n = 3), IBS + AM (n = 1), FAP-
NOS (n = 6), FD (n = 3), and AM (n = 1). We divided into 3 groups
including pure IBS (n = 37), overlap (IBS + FD, IBS + FAP-NOS, IBS +
AM, n = 9), and non-IBS (n = 10). Eosinophil counts of the each region
were not significantly different among 3 groups (Table 7).
We compared tissue eosinophil counts in each specific region
according to age of children with FAPDs. We divided children with FAPDs
into 2 groups with age 13 to separate childhood and adolescence. Eosinophil
counts of the each region were not significantly different on the basis of age
(Table 8).
We compared tissue eosinophil counts in each region based on sex of
children with FAPDs. Eosinophil counts of the each region were not
significantly different on the basis of sex (Table 9).
36
Table 7. Comparison of tissue eosinophil counts according to subtypes of functional abdominal pain disorders.
Anatomical region IBS only
(n=37)
Overlap
(n = 9)
Non – IBS
(n = 10)
*P value
Gastric antrum 3.6 ± 6.6 5.4 ± 6.4 4.7 ± 3.6 0.249
Gastric body 2.1 ± 2.2 3.9 ± 3.8 3.3 ± 2.3 0.204
Duodenum 12.8 ± 8.5 21.4 ± 22.8 12.9 ± 3.9 0.716
Terminal ileum 24.1 ± 19.4 23.0 ± 8.0 7.5 ± 3.5 0.242
Cecum 22.5 ± 20.7 24.4 ± 20.7 24.0 ± 13.3 0.800
Ascending colon 17.2 ± 10.3 13.0 ± 6.4 11.7 ± 8.2 0.518
Transverse colon 14.0 ± 10.3 12.7 ± 7.5 9.3 ± 2.4 0.853
Descending colon 13.3 ± 8.5 9.0 ± 3.8 14.2 ± 12.9 0.411
Sigmoid colon 12.8 ± 15.2 10.1 ± 7.5 10.5 ± 6.9 0.916
Rectum 3.3 ± 3.1 3.7 ± 2.5 2.8 ± 3.2 0.606
*P value between normal control and FAPDs was calculated by Kruskal-Wallis test.
IBS; irritable bowel syndrome, Non – IBS; non irritable bowel syndrome
Overlap included IBS + FD (n = 5), IBS + FAP-NOS (n = 3), and IBS + AM (n = 1).
Non – IBS included FAP-NOS (n = 6), FD (n = 3), and AM (n = 1).
37
Table 8. Comparison of tissue eosinophil counts according to age group in children with functional abdominal pain
disorders.
Anatomical region Age < 13 years
(n = 23)
Age ≥ 13 years
(n = 33)
*P value
Gastric antrum 3.6 ± 4.8 4.3 ± 6.9 0.358
Gastric body 2.8 ± 3.0 2.5 ± 2.2 1.000
Duodenum 13.6 ± 15.4 12.0 ± 8.8 0.802
Terminal ileum 21.9 ± 12.8 22.5 ± 20.2 0.661
Cecum 26.8 ± 23.4 20.1 ± 15.4 0.459
Ascending colon 10.3 ± 9.4 13.2 ± 11.5 0.480
Transverse colon 12.6 ± 7.2 13.3 ± 10.2 0.845
Descending colon 11.5 ± 8.5 11.2 ± 9.8 0.738
Sigmoid colon 10.2 ± 7.0 13.2 ± 15.7 0.285
Rectum 3.4 ± 3.8 3.2 ± 2.2 0.523
*P value between normal control and FAPDs was analyzed by Mann-Whitney U test.
38
Table 9. Comparison of tissue eosinophil counts according to sex of children with functional abdominal pain
disorders.
Anatomical region Boys (n = 38) Girls (n = 17)
*P value
Gastric antrum 4.0 ± 6.7 4.2 ± 4.7 0.502
Gastric body 2.5 ± 2.6 2.9 ± 2.5 0.444
Duodenum 13.5 ± 12.0 16.1 ± 10.8 0.273
Terminal ileum 26.1 ± 19.3 13.5 ± 8.3 0.136
Cecum 25.0 ± 21.0 16.1 ± 8.5 0.412
Ascending colon 16.4 ± 10.6 13.8 ± 5.8 0.776
Transverse colon 13.6 ± 9.5 11.7 ± 8.5 0.463
Descending colon 11.4 ± 5.8 15.7 ± 13.3 0.641
Sigmoid colon 9.9 ± 5.7 16.6 ± 21.4 0.224
Rectum 3.1 ± 2.9 3.6 ± 3.2 0.597
*P value between normal control and FAPDs was analyzed by Mann-Whitney U test.
39
DISCUSSION
We aimed to evaluate the relationship between the GI tract eosinophilia and
pediatric FAPDs to better understand its underlying pathomechanism
because the exact pathogenesis of FAPDs in children remains unclear.
Recent studies suggest that micro-inflammation of the GI tract could play a
key role in the pathophysiology of FGIDs (4, 27). Although the mechanism
to explain the emergence of low-grade inflammation of the GI tract is
unclear, it has been suggested that a dysfunction of the intestinal barrier
facilitates the mucosal penetration of antigens in IBS and IBD, precipitating
an immune reaction (4, 48, 49). It has been hypothesized that an abnormally
increased intestinal permeability facilitates the entry of dietary antigens or
GI pathogens into the GI lumen and accelerates an inappropriate stimulation
of the host immune reaction in patients with IBS or FD (4, 23, 26, 50).
Several studies have reported that increased infiltration of immune cells
including eosinophils, mast cells, and lymphocytes and degranulation of
these activated immune cells leads to low-grade mucosal inflammation in
the GI tract in those presenting with FD and/or IBS. Studies performed in
adults with FGIDs have demonstrated that duodenal inflammation with
eosinophils and mast cells is primarily associated with FD (19, 26, 27).
However, in adults with IBS, most studies have shown increased infiltration
40
of mast cells and T lymphocytes in the small intestine and colon (28).
A few reports mention that, to date, studies have not demonstrated a
significant difference in the tissue eosinophil counts between patients with
IBS (23, 25, 51, 52). However, our study showed that eosinophil counts of
the stomach (gastric antrum), small bowel (duodenum, T-ileum), and colon
(cecum, A-colon) were significantly higher in children with FAPDs than in
normal controls based on an assessment of tissue eosinophils throughout the
GI tract. In the present study, 56 children with FAPDs were classified as
having IBS (n = 37), IBS + FD (n = 5), IBS + FAP-NOS (n = 3), IBS + AM
(n = 1), FAP-NOS (n = 6), FD (n = 3), and AM (n = 1). Among these 56
children with FAPDs, a majority (46 [82.1%]) were those with IBS. Our
results suggest that eosinophils of the GI tract might be associated with
FAPDs including IBS in children and might contribute to its pathogenesis.
Eosinophils are known to be involved with both the innate and
adaptive immune response and affect tissue damage and repair processes
(53). Because eosinophils contribute to multiple phases of the immune
response, they can significantly influence disease processes through a
variety of mechanisms (53). Eosinophils are activated by an inflammatory
reaction initiated by internal and external triggers (4, 27, 54). Internal
triggers such as anxiety and stress affect the gut physiology acting via brain-
41
gut axis to induce an inflammatory response, whereas external triggers such
as microbes and allergens stimulate inflammation via highly conserved
pathogen-associated molecular patterns (27, 55). Following activation,
eosinophils release cytotoxic charged proteins such as major basic protein,
eosinophil peroxidase, eosinophil-derived neurotoxin, and eosinophil
cationic protein (53). The accumulation and degradation of eosinophils
causes neural stimulation and smooth muscle contraction, which
consequently produces GI symptoms including abdominal pain, bloating,
and flatulence in those presenting with FGIDs (4, 27, 56).
Early studies describing mucosal biopsies obtained from IBD patients
have shown mucosal eosinophilia compared to those obtained from healthy
controls (36). Additionally, mucosal eosinophilia was more prominent in
IBD patients compared to those with IBS (36). Furthermore, eosinophil
counts or eosinophil cationic protein levels positively correlated with
disease activity and negatively correlated with treatment response in IBD
patients (53, 57, 58). Therefore, eosinophils are known to play a role in the
pathogenesis of IBD, even though their exact relationship with IBD remains
unclear (36, 37, 42, 59).
We hypothesized that micro-inflammation might be the primary
pathogenesis that causes GI symptoms in FAPDs. Therefore, we expected
42
the density of inflammatory cells in the GI tract of the FAPDs group to be
higher than that observed in normal controls without any inflammation and
lower than that observed in the IBD group with definite inflammation. In the
present study, we found that significantly high eosinophil counts of the
stomach and colon showed a definite order in terms of the disease entity
IBD, followed by FAPDs, and normal controls regardless of endoscopically
detected macroscopic IBD lesions.
Our study results are in agreement with those of previous studies
reported by Bass et al. (60), Pensabene et al. (38), and Flores et al. (61) in
that eosinophil counts of the colonic area were significantly higher in
children with IBD than in those with FAPDs or IBS or normal controls.
However, a study reported by Bass et al. (60) also has described that tissue
eosinophil counts of the stomach and the duodenum were not significantly
different between children with IBD and those with FAPDs, unlike the
results obtained in our study. Additionally, a study reported by Pensabene et
al. differs from ours in that their study showed that eosinophil counts were
not observed to be significantly different between children with CD and
those with UC. However, a study reported by Carvalho et al. (62) has shown
results consistent with those of our study in that high eosinophil counts of
the colon showed a definite order in terms of the disease entity UC,
followed by CD, non-inflamed CD, and IBS. Their results suggested that the
43
uninvolved mucosa may not be entirely normal in patients with IBD and
that eosinophils could play some role in the pathogenesis of the early lesions
in IBD (62).
To date, several trials have been performed to determine cut-off
values of tissue eosinophil counts to distinguish between FAPDs and
controls or between CD and UC (20, 61). Wauters et al. (20) showed that
duodenal eosinophils > 112/mm2 could predict the presence of FD between
FD and controls. Flores et al. (61) showed that colonic eosinophil counts ≥
70/HPF was a cut-off value to predict UC when comparing between CD and
UC. In the present study, we analyzed the cut-off value of tissue eosinophils
differentiating between FAPDs and IBD in each region of the GI tract that
we evaluated, as presented in Table 3. However, the use of a cut-off value of
tissue eosinophil counts may not be effective because of the relative low
sensitivity and the specificity.
To our knowledge, the present study is the first comparative study
completed wherein the entire GI tract was evaluated to determine
differences in tissue eosinophil infiltration in patients with FGIDs in studies
performed on both, adults and children. Histopathological studies in
children with FAPDs are relatively rare. Moreover, pediatric studies have
primarily been limited to studies investigating FD (20, 30-34), and have
44
focused on the stomach and duodenum. Therefore, the histopathological
features of the GI tract beyond the duodenum in children with FAPDs
remain unclear. Our study showed that eosinophil counts of children with
FAPDs were significantly higher in the small bowel (duodenum, T-ileum),
cecum, and the A-colon as well as stomach (gastric antrum) than those
observed in normal controls. Our study suggests that eosinophils in the
stomach, small bowel, and proximal colon contribute to development of
FAPDs.
A gradient of eosinophil density is known to exist from the proximal
to distal colon (41, 42); therefore, it is important that GI tract eosinophils
should be analyzed through comparisons performed in each specific region
even in the colonic area. In several studies performed on adults with IBS,
the biopsy specimens studied were primarily from the D-colon or
rectosigmoid area (28). Several recent comparative studies involving
patients with IBS and/or IBD, have not clearly described which part of the
colon was analyzed or were performed with tissue obtained only from the
rectosigmoid/rectal area (38, 57, 60-62). An inappropriate comparison of
eosinophils within different sections of the colon can lead to inaccurate
results. Furthermore, symptoms of FAPDs are not related to the exclusive
involvement of localized areas of the GI tract such as the rectosigmoid area.
A close correlation exists between each segment of the GI tract and each
45
segment affects the other. It is proposed that the interaction of multifactorial
pathophysiological factors involve on the generation of FGIDs explained by
the disorders of gut-brain interaction (63). In this regard, our study involved
assessment of the entire GI tract and compared eosinophils in each segment
of the gut, which is strength of our study.
We also investigated whether GI tract mucosal inflammation of
children with FAPDs was different depending on subtypes of FAPDs or age
or sex. According to adult studies that reported burdens of FGIDs were
heavier when one disorder overlaps with one or more disorders (64), we
expected that overlap group has more severe inflammation than other groups
in FAPDs and shows higher eosinophils than other groups. However, when
we divided into 3 groups including IBS (n = 37), overlap (IBS + FD, IBS +
FAP-NOS, IBS + AM, n = 9), and non-IBS group (n = 10), eosinophil
counts of the each region were not significantly different among 3 groups.
Eosinophil counts of the each region were also not significantly according to
sex and age as well.
The present study has some limitations. First, this study did not recruit
healthy individuals as normal controls because ethical considerations do not
justify endoscopic examination with biopsy sampling for the evaluation of
eosinophilic infiltration in otherwise healthy children. Instead, as a
46
substitute for enrolling a normal control, we adopted normal reference
values based on a previously published study, which evaluated endoscopic
biopsies from each segment of the GI tract for children with no identified
organic findings, and performed final clinical assessment. Another issue in
terms of the use of normal references was that the 1st references we used did
not include an evaluation of the cecum, descending and sigmoid colon, and
thus we had to select a separate normal reference for comparison of the
colonic area. Consequently, we used two references as the normal control,
and an upper GI tract reference used for comparison of eosinophil
infiltration between the stomach and the ileum was different from the lower
GI tract reference used for comparison of colonic tissue eosinophil counts.
These reference groups did not comprise Korean children; therefore, they
might not accurately represent normal controls. The upper GI tract reference
group also included a few children with FAPDs and a few with allergies. As
a result, the normal reference values of the upper GI tract used in this study
are likely to reflect elevated tissue eosinophil counts compared to those
expected in normal individuals. The pediatric lower GI tract pathology
references, which we used, presented the ‘rectosigmoid area’, with no clear
division between the sigmoid and the rectum. Thus, an accurate comparison
of eosinophil counts in the rectum was difficult. Second, this was a
retrospective study with a relatively small sample size and evaluated only
47
eosinophils without other inflammatory cells such as mast cells or T
lymphocytes. Third, majority (46 [82.1%]) of children with FAPDs were
patients with IBS. This is because children with IBS mostly undergo a
colonoscopy.
Therefore, we recommend that further prospective studies including
a larger number of children, balanced recruitment of other subtypes of
FAPDs and additional cell types such as mast cells or lymphocytes are
required to confirm low-grade inflammation in children with FAPDs.
In summary, eosinophil counts of the stomach (gastric antrum), small
bowel (duodenum, T-ileum), and colon (cecum, A-colon) were observed to
be significantly higher in children with FAPDs compared to normal
reference values. Eosinophil counts of the stomach (gastric antrum and body)
and the entire colon between the cecum and the rectum were significantly
higher in children with IBD than in those with FAPDs. After eliminating the
GI segments that showed macroscopic IBD-associated regional
inflammation on endoscopic examination, comparison of tissue eosinophil
counts between IBD and FAPDs showed similar results in that the
eosinophil counts of the stomach (gastric body) and colon (cecum, D-colon,
S-colon, rectum) were significantly higher in children with IBD than in
those with FAPDs. In a comparison of FAPDs with IBD both before and
after selecting GI segments that macroscopically were unaffected by IBD,
48
there were no statistically significant differences observed in duodenal and
terminal ileal eosinophil counts.
In conclusion, the present study showed that eosinophils of the GI
tract including the stomach and colon might be associated with FAPDs
including IBS in children. Therefore, the results of our study suggest that GI
tract eosinophilia might contribute to the development of pediatric FAPDs.
GI tract eosinophilia could to some extent be a possible cause of FAPDs or
could represent a simple phenomenon secondary to inflammation of the GI
tract. Assessment of eosinophil counts alone cannot explain the potential
role of GI tract eosinophils in the pathophysiology of FAPDs. Further
studies on eosinophil recruitment, activation, and degradation in association
with GI motility tests along with studies on other immune cells are needed
to better understand the underlying pathomechanisms of FAPDs.
49
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61
국문 초록
서론: 기능성 복통 질환은 소아청소년에서 매우 흔한 질환이지만,
그 기전은 아직 명확하게 밝혀지지 않았다. 최근 장관 내 미세염
증, 특히 위장관에 침착된 호산구가 기능성 복통 질환을 일으키는
병태생리로서 제시되었다. 본 연구에서는, 기능성 복통이 있는 소
아청소년 환자의 위장관 점막에 침윤된 호산구를 평가하기 위해
질병 대조군인 소아 염증성 장질환 환자와 정상 대조군에서 위장
관 점막에 침윤된 호산구 수를 기능성 복통 질환 소아 환자의 위
장관 점막 내 호산구 수와 비교 분석하였다.
방법: 기능성 복통 질환이 있는 소아 환자 56명, 소아 크론병 환자
52명, 소아 궤양성대장염 환자 23명이 연구대상으로 모집되었다.
대상 환자들은 모두 위내시경과 대장내시경을 통한 조직 검사를
같은 날에 시행 받았다. 위에서 직장까지 총 10곳의 위장관 부위
에서 채취한 조직에서 위장관 조직 내 호산구 수를 측정하였다.
소아의 정상 대조군에서의 조직 내 호산구 수는 기존에 보고된 정
상인에서의 정상 참고치로 정하였다. 위장관 내 10곳의 부위에 대
해 소아청소년 기능성 복통 환자의 조직 내 호산구 수와 소아 염
62
증성 장질환 환자의 조직 내 호산구 수, 그리고 정상 참고치를 각
각 부위별로 서로 비교하였다. 또한, 염증성 장질환 자체의 국소적
인 염증의 영향을 최소화하기 위하여 내시경 검사에서 육안적 장
관 점막 병변이 있는 부위를 제외한 후 각각 부위별로 추가 분석
을 시행하였다.
결과: 기능성 복통이 있는 소아청소년 환자를 정상 참고치와 비교
했을 때, 위 전정부 (P = 0.006), 십이지장 (P = 0.034), 말단 회
장 (P = 0.009), 맹장 (P = 0.006), 상행 결장 (P = 0.013)에서 침
윤된 호산구의 수가 의미있게 높았다. 소아 염증성 장질환 환자
를 소아 기능성 복통 환자와 비교했을 때, 위 전정부 (P =
0.001), 위 체부 (P = 0.001), 그리고 맹장부터 직장에 이르는 대
장 전체 (맹장 [P = 0.047], 상행 결장 [P = 0.011], , 횡행 결장과
하행 결장 및 직장 [P < 0.001), 구불 결장 [P = 0.001] 에서
침윤된 호산구의 수가 의미있게 높았다. 염증성 장질환 환자에
서 육안적 병변이 없는 장관 부위만을 대상으로 재분석을 하였
을 때, 소아 염증성 장질환 환자는 소아 기능성 복통 환자에 비
해서 위 체부 (P = 0.002), 맹장 (P = 0.032), 하행 결장 (P =
63
0.021), 구불 결장 (P = 0.046), 직장 (P = 0.027)에서 침윤된 호산
구의 수가 의미 있게 높았다. 육안적 병변 부위의 포함 여부와
상관 없이, 십이지장과 회장 말단의 조직 내 호산구 수는 각 군
간에 유의한 차이를 보이지 않았다.
결론: 염증성 장질환 자체가 갖는 염증의 영향을 감안하더라도, 위
와 대장의 조직 내 침윤된 호산구 수는 염증성 장질환, 기능성 복
통 질환, 정상 참고치의 순서로 높게 나타났다. 즉, 위장관 조직,
특히 위와 대장 조직에 침윤되는 호산구는 소아청소년에서 기능성
복통 질환 및 염증성 장질환의 발병에 있어 주요 병태생리와 연관
되어 있음을 시사한다.
주요어: 복통, 소아, 호산구, 염증, 기능성 위장관 질환, 염증성 장질
환
학 번: 2016-21932
