J Neurogastroenterol Motil, Vol. 24 No. 4 October, 2018

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1 JNM J Neurogstroenterol Motil, Vol. 24 No. 4 October, 18 pissn: eissn: Journl of Neurogstroenterology nd Motility Originl Article Quntittive Anlysis of Distribution of the Gstrointestinl Trct Eosinophils in Childhood Functionl Abdominl Pin Disorders Eun Hye Lee, 1,2 Hye Rn Yng, 3,4 nd Hye Seung Lee 5,6 1 Deprtment of Peditrics, Nowon Eulji Medicl Center, Seoul, Kore; 2 Deprtment of Peditrics, Eulji University College of Medicine, Dejeon, Kore; 3 Deprtment of Peditrics, Seoul Ntionl University Bundng Hospitl, Seongnm, Gyeonggi-do, Kore; 4 Deprtment of Peditrics, Seoul Ntionl University College of Medicine, Seoul, Kore; 5 Deprtment of Pthology, Seoul Ntionl University Bundng Hospitl, Seongnm, Gyeonggi-do, Kore; nd 6 Deprtment of Pthology, Seoul Ntionl University College of Medicine, Seoul, Kore Bckground/Aims Although functionl bdominl pin disorders (FAPDs) re common in children, the ccurte pthogenesis of FAPDs is not known yet. Micro-inflmmtion, prticulrly tissue eosinophili of gstrointestinl (GI) trct, hs been suggested s the pthophysiology observed in severl GI disorders. We imed to evlute eosinophilic infiltrtion throughout the entire GI trct in children with FAPDs, compred to those with inflmmtory bowel diseses (IBD) nd to norml reference vlues. Methods We included 56 children with FAPDs, 52 children with Crohn s disese, nd 23 children with ulcertive colitis. All subjects underwent esophgogstroduodenoscopic nd colonoscopic exmintion with biopsies. Tissue eosinophil counts were ssessed in 1 regions throughout the GI trct. Results Eosinophil counts of the gstric ntrum, duodenum, terminl ileum, cecum, nd scending colon were significntly higher in children with FAPDs compred to norml reference vlues. Eosinophil counts of the stomch nd the entire colon were observed to be significntly higher in children with IBD thn in those with FAPDs. Even fter selecting mcroscopiclly uninvolved GI segments on endoscopy in children with IBD, eosinophil counts of the gstric body, cecum, descending colon, sigmoid colon, nd the rectum were lso significntly higher in children with IBD thn those with FAPDs. Conclusions Significntly high eosinophil counts of the stomch nd colon were observed in the order of IBD, followed by FAPDs, nd norml controls, regrdless of endoscopiclly detected mcroscopic IBD lesions in children. This suggests some contribution of GI trct eosinophils in the intrinsic pthogenesis of FAPDs in children. (J Neurogstroenterol Motil 18;24: ) Key Words Abdominl pin; Child; Eosinophils; Functionl gstrointestinl disorder; Inflmmtory bowel diseses Received: Mrch 3, 18 Revised: July 13, 18 Accepted: July 31, 18 This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( org/licenses/by-nc/4.) which permits unrestricted non-commercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. Correspondence: Hye Rn Yng, MD, PhD Deprtment of Peditrics, Seoul Ntionl University Bundng Hospitl, Seoul Ntionl University, 82, Gumi-ro 173 Beon-gil, Bundng-gu, Seongnm, Gyeonggi-do 136, Kore Tel: , Fx: , E-mil: hryng@snubh.org 614 c 18 The Koren Society of Neurogstroenterology nd Motility J Neurogstroenterol Motil, Vol. 24 No. 4 October, 18

2 GI Eosinophils in Childhood FAPDs Introduction Functionl bdominl pin disorders (FAPDs) re common in children. A recent met-nlysis hs reported globl pooled prevlence rte for FAPDs ws 16.5% bsed on the Rome III criteri. 1 It is well-known tht FAPDs could be ssocited with long-term symptoms. A prospective study hs demonstrted tht pproximtely 41.% of children with functionl bdominl pin (FAP) continued to report cliniclly significnt bdominl pin fter men durtion of 9-yer follow-up into dolescence nd young dulthood. 2,3 The pthomechnism to explin FAPDs remins uncler nd multifctoril hypotheses hve been suggested. 4,5 Recently mny studies hve suggested tht low-grde intestinl inflmmtion is essentilly involved in the underlying pthogenesis of functionl gstrointestinl disorders (FGIDs). 6,7 These studies hve reported tht incresed infiltrtion of immune cells such s eosinophils, mst cells, nd lymphocytes nd the subsequent degrnultion of these ctivted immune cells produce low-grde mucosl inflmmtion in the gstrointestinl (GI) trct of dults with functionl dyspepsi (FD) nd/or irritble bowel syndrome (IBS). However, the types of immune cells tht were incresed, the degree of inflmmtion, nd the segments of the GI trct tht were investigted hve not been consistent cross ll these studies performed in dults. Furthermore, histopthologicl studies pertining to childhood FAPDs re reltively few, nd studies involving children re primrily limited to studies regrding FD. 8,9 In our previous study published in 16, we demonstrted tht eosinophil counts in the gstric ntrum nd body mucos were significntly high in definite order of in terms of the disese entity norml reference vlues, followed by children with FAPDs, nd then those with Helicobcter pylori gstritis. 1 In the present study, we extended the scope of our study to investigte the entire GI trct. Unlike the esophgus, eosinophils re resident cells of the stomch, smll nd lrge intestine. However, to dte, the norml rnge of eosinophils in the GI trct mucos is not well-defined; thus, interprettion of pthologicl intestinl eosinophili is difficult. 11 It is lso known tht eosinophils re not uniformly distributed throughout the length of the intestine. 12 Therefore, eosinophils of the GI trct should be nlyzed nd compred in ech specific region, even within the colon. We im to evlute tissue eosinophilic infiltrtion of the entire GI trct from the stomch through the rectum in children with FAPDs, compred to the norml reference vlues nd disese controls with inflmmtory bowel diseses (IBD) in ech segment throughout the bowel. Mterils nd Methods Subjects Our retrospective study included children nd dolescents who visited the Deprtment of Peditric Gstroenterology t the Seoul Ntionl University Bundng Hospitl with complints of chronic recurrent bdominl pin, nd who hd undergone both esophgogstroduodenoscopic nd colonoscopic exmintion with biopsies due to ccompnied red flg signs, nd finlly hd been dignosed with FAPDs between Jnury 9 nd October 15. Children excluded from this study were those with significnt llergy such s llergic rhinitis, topic dermtitis, sthm, nd food llergy tht my ffect tissue eosinophils in the GI trct, nd those with underlying liver, kidney or hert disese tht could cuse GI symptoms. All children underwent lbortory tests including complete blood cell counts, erythrocyte sedimenttion rte, C-rective protein, liver function tests, serum mylse nd lipse, serum electrolytes, urinlysis, stool exmintion for prsite, fecl occult blood, nd stool cultures for bcteri, s well s bdominl X-ry nd ultrsonogrphy. Those showing bnormlities on lbortory tests nd imging studies were excluded. Children who demonstrted endoscopiclly ny mcroscopic bnormlity were lso excluded from the study. Eventully, 56 children with FAPDs were included nd clssified into 4 subtypes of FAPDs including FD, IBS, bdominl migrine (AM), nd FAP not otherwise specified (FAP-NOS) bsed on the Rome IV criteri. 4 Among the children who hd been dignosed with IBD nd hd been treted in our tertiry medicl center, those who underwent both esophgogstroduodenoscopic nd colonoscopic exmintion with biopsies during the sme period were lso recruited retrospectively s disese controls. Eventully, 52 children with Crohn s disese (CD) nd 23 children with ulcertive colitis (UC) were enrolled. We used the study reported by DeBrosse et l 12 s n upper GI reference to compre eosinophil infiltrtion between the stomch nd ileum nd nother study reported by Sd 13 s lower GI reference to compre colonic eosinophil counts. Our retrospective study ws pproved by the Institutionl Review Bord of Seoul Ntionl University Bundng Hospitl (IRB No. B ). Vol. 24, No. 4 October, 18 ( ) 615

3 Eun Hye Lee, et l Endoscopic Biopsy nd Histopthology Esophgogstroduodenoscopy ws performed using GIF- Q26 or GIF-XP26 scope (Olympus, Tokyo, Jpn) nd colonoscopy ws performed using PCF-Q26AL, GIF-Q26, or GIF-XP26 scope (Olympus). Endoscopic mucosl biopsies were obtined from the esophgus, gstric ntrum nd body, duodenum, terminl ileum, cecum, scending, trnsverse, descending, nd sigmoid colon, nd the rectum. Biopsy tissues were immeditely fixed using formlin nd processed with embedding in prffin wx. Sections of size 3 μm were cut from the prffin block, nd stined using hemtoxylin nd eosin stins. The eosinophil count ws obtined from 5 rndomly selected high-power fields. Quntifiction of eosinophils ws performed using n Axioskope microscope (Mirx-Crl Zeiss, Oberkochen, Germny) t mgnifiction. Cell counts were performed by 2 pthologists who were blinded to the sttus of the children, nd men vlue of the 5 high-power fields counts obtined ws clculted for ech child (Fig. 1). Becuse it is known tht eosinophils re not usully observed in the esophgel mucos in non-pthologicl stte, we excluded the esophgus from the res tht were compred. Mucosl eosinophil counts in ech of the 1 regions of the GI trct of children with FAPDs were compred with those in norml pthology references (norml controls) nd those from children with IBD (disese controls), respectively. To minimize the confounding effects of IBD-induced inflmmtion, fter selecting histopthologicl specimens of the GI trct segments tht endoscopiclly did not show mcroscopic IBD involvement, we nlyzed eosinophil counts s well. Mcroscopiclly uninvolved segments of GI trct were selected bsed on use of 4 vribles of the simple endoscopic score for CD (SES-CD) 14 nd 3 descriptors of UC endoscopic index of severity (UCEIS). 15,16 The segments in which ll scores of ll 4 vribles pertining to SES- CD nd of ll 3 descriptors pertining to UCEIS were were selected for nlysis fter eliminting IBD-induced regionl inflmmtion. Sttisticl Methods Dt for continuous vribles re presented s men ± SD for prmetric or medin vlues long with rnge (minimummximum) for nonprmetric vribles. Ctegoricl vribles re presented s percentge of the totl number. Continuous dt were nlyzed using the independent t test for prmetric vribles, nd the Mnn-Whitney U test for nonprmetric vribles. For ll sttisticl nlyses, two-sided P-vlue <.5 ws considered sttisticlly significnt. All sttisticl nlyses were performed using PASW Sttistics (version.; IBM Corp, Armonk, NY, USA). A B C D Figure 1. Photomicrogrph of hemtoxylin nd eosin stining for eosinophils in the scending colon. Infiltrtion of tissue eosinophils incresed in definite order of specimen of norml controls (A), functionl bdominl pin disorders (B), Crohn s disese (C), nd ulcertive colitis (D) (mgnifiction, ). 616 Journl of Neurogstroenterology nd Motility

4 GI Eosinophils in Childhood FAPDs Results Ptients Chrcteristics Tble 1 shows the demogrphic dt of the subjects. Our study included 56 children fulfilling the Rome IV criteri for FAPDs who were clssified s IBS (n = 37), IBS + FD (n = 5), IBS + FAP-NOS (n = 3), IBS + AM (n = 1), FAP-NOS (n = 6), FD (n = 3), nd AM (n = 1). Norml controls for the upper GI trct were 19 children nd those for the lower GI trct were 41 children. Disese controls with IBD comprised 52 children with CD nd 23 children with UC. Comprison of Eosinophil Counts in the Gstrointestinl Trct Between Ptients With Functionl Abdominl Pin Disorders nd Those With Inflmmtory Bowel Diseses Eosinophil counts of the stomch nd the entire colon between cecum nd the rectum were significntly higher in children with IBD thn in those with FAPDs (Tble 2 nd Fig. 2). Eosinophil counts of the duodenum nd the terminl ileum were lso higher in children with IBD, but this ws not significnt difference (Tble 2 nd Fig. 2). Comprisons between children with FAPDs nd those with CD s well s those with UC re lso shown in Tble 2. A comprison between children dignosed with FAPDs nd those with CD showed tht the eosinophil counts of the stomch (both gstric ntrum nd body) nd the left colon (trnsverse colon, descending colon, sigmoid colon, nd rectum) in children with CD were Tble 1. Demogrphic Chrcteristics of the Subjects Recruited Vrible FAPDs (n = 56) Norml reference (upper GI, n = 19) Norml reference (lower GI, n = 41) IBD (CD + UC) (n = 75) Sex (boys:girls) 39:17 8:11 21: 55: Age (medin [rnge], yr) 14.1 ( ) 12. (2.-17.) 12.2 ( ) 14.7 ( ) FAPDs, functionl bdominl pin disorders; upper GI, upper gstrointestinl region including gstric ntrum, gstric body, duodenum, nd terminl ileum; lower GI, lower gstrointestinl region including cecum, scending colon, trnsverse colon, descending colon, nd rectosigmoid colon; IBD, inflmmtory bowel disese; CD, Crohn s disese; UC, ulcertive colitis. Age is shown s medin vlue long with rnge (minimum-mximum). Tble 2. Comprison of Tissue Eosinophil Counts of the Gstrointestinl Trct Between Children With Functionl Abdominl Pin Disorders nd Those With Inflmmtory Bowel Diseses Antomicl region FAPDs (n = 56) CD (n = 52) UC (n = 23) IBD (CD + UC) (n = 75) P-vlue (FAPDs-CD) b P-vlue (FAPDs-UC) c P-vlue (FAPDs-IBD) Gstric ntrum 4.1 ± ± ± ± Gstric body 2.6 ± ± ± ± Duodenum 13. ± ± ± ± Terminl ileum 22.3 ± ± ± ± Cecum 23. ± ± ± ± d Ascending colon 15.7 ± ± ± ± Trnsverse colon 13.1 ± ± ± ± <.1 Descending colon 12.7 ± ± ± ± <.1 <.1 Sigmoid colon 11.9 ± ± ± ± <.1.1 Rectum 3.3 ± ± ± ± <.1 <.1 P-vlue between functionl bdominl pin disorders (FAPDs) nd Crohn s disese (CD) ws clculted by independent t test. b P-vlue between FAPDs nd ulcertive colitis (UC) ws clculted by independent t test. c P-vlue between FAPDs nd inflmmtory bowel disese (IBD) (CD + UC) ws clculted by independent t test except for d P-vlue by Mnn-Whitney U test. Dt indicte the men number of eosinophils/high-power field for ech ntomicl region of the gstrointestinl trct. Dt re shown s men ± SD. Vol. 24, No. 4 October, 18 ( ) 617

5 Eun Hye Lee, et l Eosinophil counts/hpf (men + SE) 1 Control FAPDs IBD Control FAPDs IBD Control FAPDs IBD Control FAPDs IBD Gstric ntrum Gstric body Duodenum Terminl ileum Eosinophil counts/hpf (men + SE) 1 Cecum Control FAPDs IBD Control FAPDs IBD Control FAPDs IBD A T Control FAPDs IBD D Control FAPDs IBD S Control FAPDs IBD Rectum Figure 2. Eosinophil counts in ech prt of the gstrointestinl trct in norml controls, children with functionl bdominl pin disorders (FAPDs), nd children with inflmmtory bowel diseses (IBD). The order of br grphs is norml controls, FAPDs, nd IBD. In the rectum, eosinophil counts ccording to both upper gstrointestinl (GI) reference nd lower GI reference were presented becuse lower GI trct pthology reference presented the rectosigmoid re, with no cler division between the sigmoid nd the rectum. P-vlue is less thn.5. HPF, high-power field; A, scending colon; T, trnsverse colon; D, descending colon; S, sigmoid colon. significntly higher thn those observed in children with FAPDs (Tble 2). Eosinophil counts of the duodenum, terminl ileum, cecum, nd the scending colon were lso higher in children with CD thn those observed in children with FAPDs, lthough this difference ws not sttisticlly significnt. A comprison between children dignosed with FAPDs nd those with UC showed tht eosinophils throughout the colon between the cecum nd rectum were significntly higher in children with UC thn in children with FAPDs (Tble 2). Eosinophil counts of the stomch nd smll bowel (duodenum nd terminl ileum) were not significntly different between children with FAPDs nd those with UC (Tble 2). To minimize the confounding effects of IBD-induced inflmmtion, fter histopthologicl slides of mcroscopiclly uninvolved GI segments on endoscopy were selected, eosinophil counts were nlyzed between children with FAPDs nd those with IBD s well. Eosinophil counts in children with IBD were significntly higher thn those observed in children with FAPDs in the gstric body, cecum, descending colon, sigmoid colon, nd rectum (Tble 3 nd Fig. 3). Eosinophil counts of the scending colon nd trnsverse colon were lso higher in children with IBD, but this difference ws not sttisticlly significnt. Eosinophil counts of the smll bowel (duodenum nd terminl ileum) were not significntly different between children with FAPDs nd those with IBD. Comprisons between children with FAPDs nd those with CD s well s those with UC fter eliminting the specimens of the GI segments with IBD lesions on endoscopy re shown in Tble 3. A comprison 618 Journl of Neurogstroenterology nd Motility

6 GI Eosinophils in Childhood FAPDs Tble 3. Comprison of Gstrointestinl Trct Mucosl Eosinophil Counts Between Children With Functionl Abdominl Pin Disorders nd Inflmmtory Bowel Diseses Except Regionl Inflmmtion Site Antomicl region FAPDs (n = 56) CD except regionl inflmmtion (n = 52) UC except regionl inflmmtion (n = 23) IBD except regionl inflmmtion (n = 75) P-vlue (FAPDs- CD) b P-vlue (FAPDs- UC) c P-vlue (FAPDs- IBD) Gstric ntrum 4.1 ± ± ± ± d Gstric body 2.6 ± ± ± ± 6..7 d.88.2 Duodenum 13. ± ± ± ± Terminl ileum 22.3 ± ± ± ± d Cecum 23. ± ± ± ± d Ascending colon 15.7 ± ± ± ± Trnsverse colon 13.1 ± ± ± ± Descending colon 12.7 ± ± ± ± Sigmoid colon 11.9 ± ± ± ± Rectum 3.3 ± ± ± ± P-vlue between functionl bdominl pin disorders (FAPDs) nd Crohn s disese (CD) except regionl inflmmtory sites ws clculted by independent t test except for d P-vlue by Mnn-Whitney U test. b P-vlue between FAPDs nd ulcertive colitis (UC) except regionl inflmmtory sites ws nlyzed by Mnn-Whitney U test. c P-vlue between FAPDs nd inflmmtory bowel disese (IBD) except regionl inflmmtory sites ws clculted by independent t test except for d P-vlue by Mnn-Whitney U test. Dt indicte the men number of eosinophils/high-power field for ech ntomicl region of the gstrointestinl trct. Dt re shown s men ± SD. between children with FAPDs nd those with CD fter eliminting the specimens of the GI segments with mcroscopic lesions, showed tht the eosinophil counts of the stomch (both gstric ntrum nd body) nd 2 segments of the lrge intestine (trnsverse colon nd rectum) in children with CD were significntly higher thn those observed in children with FAPDs (Tble 3). A comprison between children with FAPDs nd those with UC fter eliminting the specimens of the GI segments with mcroscopic lesions, showed tht the eosinophils in sections of the colon (scending colon, descending colon, sigmoid colon, nd rectum) were significntly higher in children with UC thn those observed in children with FAPDs (Tble 3). A comprison between children with CD nd those with UC showed tht children with UC demonstrted significntly higher numbers of eosinophils in the scending colon, descending colon, sigmoid colon, nd rectum thn those observed in children with CD. However, fter excluding the tissue specimens with mcroscopiclly ctive inflmmtory lesions, there were no significnt differences in tissue eosinophil counts except the scending colon (dt re not shown). Comprison of Tissue Eosinophil Counts Among Norml Controls nd Those With Functionl Abdominl Pin Disorders nd Inflmmtory Bowel Diseses A comprison between children with FAPDs nd norml controls showed tht eosinophil counts of children with FAPDs were significntly higher in the stomch (gstric ntrum), smll bowel (duodenum, terminl ileum), cecum, nd the scending colon thn those observed in norml controls (Tble 4 nd Fig. 2). A comprison between children with IBD nd norml controls showed tht eosinophil counts in ll segments between the stomch nd the rectum were significntly higher in children with IBD thn those observed in norml controls (Tble 4 nd Fig. 2). Becuse the study reported by Sd 13 which we used s the lower GI pthology reference presented rectosigmoid re, nd the region ws not definitively divided into the sigmoid nd rectum, it ws difficult to ccurtely compre the eosinophil counts in the rectum. Eosinophil counts of the rectum were significntly higher in children with IBD thn in norml controls when the rectl eosinophils were compred using the vlues noted by the DeBrosse et l 12 (Tble 4 nd Fig. 2). Comprisons between norml controls nd children with UC s well s those with CD re shown in Tble 4. A comprison between children with UC nd those studied s norml controls showed tht the eosinophil counts throughout the GI trct between the stomch nd rectum were significntly higher in children with UC thn in Vol. 24, No. 4 October, 18 ( ) 619

7 Eun Hye Lee, et l Eosinpohil counts/hpf (men + SE) Eosinpohil counts/hpf (men + SE) 1 1 Gstric ntrum Control FAPDs IBD Control FAPDs IBD Control FAPDs IBD Control FAPDs IBD Cecum Gstric body Duodenum Terminl ileum Control FAPDs IBD Control FAPDs IBD Control FAPDs IBD A T Control FAPDs IBD D Control FAPDs IBD S Control FAPDs IBD Rectum Figure 3. Eosinophil counts in ech prt of the gstrointestinl (GI) trct in norml controls, childrens with functionl bdominl pin disorders (FAPDs), nd children with inflmmtory bowel diseses (IBD) fter eliminting the GI segments with grossly ffected IBD lesion on endoscopy. The order of br grphs is norml controls, FAPDs, nd IBD. In the rectum, eosinophil counts ccording to both upper GI reference nd lower GI reference were presented becuse lower GI trct pthology reference presented the rectosigmoid re, with no cler division between the sigmoid nd the rectum. P-vlue is less thn.5. HPF, high-power field; A, scending colon; T, trnsverse colon; D, descending colon; S, sigmoid colon. those studied s norml controls (Tble 4). A comprison between children with CD nd those studied s norml controls showed tht the eosinophil counts between the stomch nd the sigmoid colon were significntly higher in children with CD thn in those studied s norml controls (Tble 4). Eosinophil counts of the rectum were not significntly different between children with CD nd those studied s norml controls even when the rectl eosinophils were compred using the vlues obtined from the peditric upper GI trct pthology references (Tble 4). After selecting the histopthologicl specimens of the GI trct segments tht endoscopiclly did not show mcroscopic IBD involvement, we nlyzed eosinophil counts between norml controls nd children with IBD, in these segments to minimize the effects of IBD-induced locl inflmmtion. Eosinophil counts in the GI segments without mcroscopic IBD lesions were significntly higher thn those of norml controls in the gstric ntrum, gstric body, terminl ileum, nd throughout the colon except the rectum (Tble 5 nd Fig. 3). Eosinophil counts in the duodenum were lso higher in children with IBD without mcroscopic lesions, but not significntly different when compred to norml controls (Tble 5 nd Fig. 3). Comprisons between norml controls nd children with CD s well s those with UC fter excluding GI segments with mcroscopic IBD lesions on endoscopy re lso shown in Tble 5. A com- 6 Journl of Neurogstroenterology nd Motility

8 GI Eosinophils in Childhood FAPDs Tble 4. Comprison of Tissue Eosinophil Counts of the Gstrointestinl Trct Between Norml Controls nd Children With Functionl Abdominl Pin or Those With Inflmmtory Bowel Diseses Antomicl region Norml control (upper, n = 19/ lower, n = 41) FAPDs (n = 56) CD (n = 52) UC (n = 23) IBD (CD + UC) (n = 75) P-vlue (Control-FAPDs) b P-vlue (Control-CD) c P-vlue (Control-UC) d P-vlue (Control-IBD) Gstric ntrum 1.9 ± ± ± ± ± <.1.1 <.1 Gstric body 2.1 ± ± ± ± ± <.1 Duodenum 9.6 ± ± ± ± ± Terminl ileum 12.4 ± ± ± ± ± <.1.24 <.1 Cecum 14.2 ± ± ± ± ± <.1 <.1 <.1 Ascending colon 12. ± ± ± ± ± <.1 <.1 Trnsverse colon 11.9 ± ± ± ± ± <.1 <.1 <.1 Descending colon 1.7 ± ± ± ± ± <.1 <.1 <.1 Sigmoid colon 12.4 ± 6.1 e 11.9 ± ± ± ± <.1 <.1 Rectum 12.4 ± 6.1 e 3.3 ± ± ± ± 18.1 < ± 5.9 f P-vlue between norml control nd functionl bdominl pin disorders (FAPDs) ws clculted by independent t test. b P-vlue between norml control nd Crohn s disese (CD) ws clculted by independent t test. c P-vlue between norml control nd ulcertive colitis (UC) ws clculted by independent t test. d P vlue between norml control nd IBD (CD + UC) ws clculted by independent t test. e Tissue eosinophil counts in the rectosigmoid ccording to norml peditric lower gstrointestinl (GI) trct pthology references which we dopted s substitution for the dt of norml controls. This reference presented rectosigmoid re, not divided into sigmoid nd rectum. Tissue eosinophil counts in the rectum ccording to norml peditric GI trct pthology references which we dopted s substitution for upper GI trct dt of norml controls. upper, upper GI region including gstric ntrum, gstric body, duodenum, nd terminl ileum; lower, lower GI region including cecum, scending colon, trnsverse colon, descending colon, nd rectosigmoid colon. Dt indicte the men number of eosinophils/high-power field for ech ntomicl region of the GI trct. Dt re shown s men ± SD. f Vol. 24, No. 4 October, 18 ( ) 621

9 Eun Hye Lee, et l Tble 5. Comprison of Tissue Eosinophil Counts Among Norml Controls, Children With Functionl Abdominl Pin, nd Those With Inflmmtory Bowel Diseses Except Regionl Inflmmtion Site Antomicl region Norml control (upper, n = 19/ lower, n = 41) FAPDs (n = 56) CD except regionl inflmmtion (n = 52) UC except regionl inflmmtion (n = 23) IBD except regionl inflmmtion (n = 75) P-vlue (Control- FAPDs) b P-vlue (Control-CD) c P-vlue (Control-UC) d P-vlue (Control-IBD) Gstric ntrum 1.9 ± ± ± ± ± <.1.1 <.1 Gstric body 2.1 ± ± ± ± ± Duodenum 9.6 ± ± ± ± ± Terminl ileum 12.4 ± ± ± ± ± <.1.56 <.1 Cecum 14.2 ± ± ± ± ± <.1 <.1 <.1 Ascending colon 12. ± ± ± ± ± Trnsverse colon 11.9 ± ± ± ± ± Descending colon 1.7 ± ± ± ± ± Sigmoid colon 12.4 ± 6.1 e 11.9 ± ± ± ± Rectum 12.4 ± 6.1 e 3.3 ± ± ± ± 16.3 <.1 < ± 5.9 f P-vlue between norml control nd functionl bdominl pin disorders (FAPDs) ws clculted by independent t test. b P-vlue between norml control nd Crohn s disese (CD) except regionl inflmmtion ws clculted by independent t test. c P-vlue between norml control nd ulcertive colitis (UC) except regionl inflmmtion ws clculted by independent t test. d P-vlue between norml control nd inflmmtory bowel diseses (IBD) (CD + UC) except regionl inflmmtion ws clculted by independent t test. e Tissue eosinophil counts in the rectosigmoid ccording to norml peditric lower gstrointestinl (GI) trct pthology references which we dopted s substitution for the dt of norml controls. This reference presented rectosigmoid re, not divided into sigmoid nd rectum. Tissue eosinophil counts in the rectum ccording to norml peditric GI trct pthology references which we dopted s substitution for upper GI trct dt of norml controls. upper, upper GI region including gstric ntrum, gstric body, duodenum, nd terminl ileum; lower, lower GI region including cecum, scending colon, trnsverse colon, descending colon, nd rectosigmoid colon. Dt indicte the men number of eosinophils/high-power field for ech ntomicl region of the GI trct. Dt re shown s men ± SD. f 622 Journl of Neurogstroenterology nd Motility

10 GI Eosinophils in Childhood FAPDs prison between CD pthology without mcroscopic lesions nd those studied s norml control showed tht the eosinophil counts of the stomch (gstric ntrum nd body), terminl ileum, cecum, scending colon, trnsverse colon, nd descending colon were significntly higher in those with CD without mcroscopic lesions thn in those studied s norml controls (Tble 5). A comprison between children with UC nd norml controls fter excluding the tissue specimens with mcroscopiclly observed ctive inflmmtory lesions showed tht the eosinophil counts of the stomch (gstric ntrum nd body), duodenum, cecum, scending colon, descending colon, nd sigmoid colon were significntly higher in those with UC thn those observed in norml controls (Tble 5). Eosinophil counts of rectum in children with UC without mcroscopic lesions were much higher thn in those studied s norml controls, but this difference ws not sttisticlly significnt becuse the number of eliminted slides ws too lrge to show sttisticl power (Tble 5). A Eosinophil counts/hpf (men + SE) C Eosinophil counts/hpf (men + SE) 1 1 Gstric ntrum Gstric body Gstric ntrum Gstric body Duodenum Duodenum Terminl ileum Terminl ileum Cecum Cecum A A Rectum Rectum B Eosinophil counts/hpf (men + SE) D Eosinophil counts/hpf (men + SE) 1 1 Gstric ntrum Gstric body Gstric ntrum Gstric body Duodenum Duodenum Terminl ileum Terminl ileum Cecum Cecum A A Rectum Rectum Figure 4. Eosinophils distribution of whole gstrointestinl (GI) trct in norml controls (A), functionl bdominl pin disorders (FAPDs) (B), inflmmtory bowel diseses (IBD) (C), nd IBD fter selecting the GI trct segments endoscopiclly did not show mcroscopic IBD involvement (D). The grph shows the tendency tht eosinophils incresed from the stomch to the cecum nd decresed from the cecum to the rectum long the colon. The degree of this tendency ws more prominent in the FAPDs nd IBD thn tht observed in norml controls. Eosinophil counts were significntly high in the order of IBD, FAPDs, nd norml controls, regrdless of endoscopiclly detected mcroscopic IBD lesions. In the rectum, eosinophil counts ccording to both upper GI reference nd lower GI reference were presented becuse lower GI references presented the rectosigmoid re, with no cler division between the sigmoid nd the rectum. HPF, high-power field; A, scending colon; T, trnsverse colon; D, descending colon; S, sigmoid colon. The sterisk () mens norml reference of rectum on the study by Debrosse et l. 12 Vol. 24, No. 4 October, 18 ( ) 623

11 Eun Hye Lee, et l Eosinophilic distributions of the entire GI trct in norml controls, children with FAPDs, those with IBD before nd fter eliminting GI segments with mcroscopic IBD lesions on endoscopy re shown in Figure 4. Eosinophils re not uniformly distributed through the length of the intestine (Fig. 4). Eosinophils incresed from the stomch to the cecum nd gin decresed from the cecum to the rectum long the colon nd this tendency ws more prominent in the FAPDs nd IBD thn tht observed in norml controls (Fig. 4). Eosinophil counts were compred in ech specific region of the GI trct nd significntly high eosinophil counts of the GI trct were noted in the order of IBD, FAPDs, nd norml controls, regrdless of endoscopiclly detected mcroscopic IBD lesions (Fig. 4). Comprison of Tissue Eosinophil Counts According to the Subtypes of Functionl Abdominl Pin Disorders nd Sex To compre tissue eosinophil counts in ech specific region ccording to the subtypes of FAPDs, 56 children with FAPDs were clssified into the 3 groups; IBS group (n = 37), overlp group (IBS + FD, IBS + FAP-NOS, IBS + AM; n = 9), nd non- IBS group (FAP-NOS, FD, nd AM; n = 1) (Tble 6). Eosinophil counts of ech region of the GI trct were not significntly different mong the 3 groups (Tble 6). To identify the difference in tissue eosinophil counts of GI trct between mle nd femle ptients, tissue eosinophil counts in ech region were compred ccording to sex in 56 children with FAPDs. Eosinophil counts of ech region of the GI trct were not significntly different between boys nd girls (Tble 7). Discussion Eosinophils re known to be involved with immune response nd ffect tissue dmge nd repir processes. 17 Becuse eosinophils contribute to multiple phses of the immune response, they cn significntly influence disese processes. 17 The ccumultion nd degrdtion of eosinophils cuses neurl stimultion nd smooth muscle contrction, which consequently produces GI symptoms Tble 7. Comprison of Tissue Eosinophil Counts According to Sex of Children With Functionl Abdominl Pin Disorders Antomicl region Boys (n = 39) Girls (n = 17) P-vlue Gstric ntrum 4. ± ± Gstric body 2.5 ± ± Duodenum 13.5 ± ± Terminl ileum 26.1 ± ± Cecum 25. ± ± Ascending colon 16.4 ± ± Trnsverse colon 13.6 ± ± Descending colon 11.4 ± ± Sigmoid colon 9.9 ± ± Rectum 3.1 ± ± P-vlue between norml control nd functionl bdominl pin disorders ws clculted by Mnn-Whitney U test. Tble 6. Comprison of Tissue Eosinophil Counts Among the Subtypes of Functionl Abdominl Pin Disorders Antomicl region IBS (n = 37) Overlp (n = 9) Non-IBS (n = 1) P-vlue Gstric ntrum 3.6 ± ± ± Gstric body 2.1 ± ± ± Duodenum 12.8 ± ± ± Terminl ileum 24.1 ± ± ± Cecum 22.5 ± ± ± Ascending colon 17.2 ± ± ± Trnsverse colon 14. ± ± ± Descending colon 13.3 ± ± ± Sigmoid colon 12.8 ± ± ± Rectum 3.3 ± ± ± P-vlue between norml control nd with functionl bdominl pin disorders (FAPDs) ws clculted by Kruskl-Wllis test. IBS, irritble bowel syndrome; FD, functionl dyspepsi; FAP-NOS, functionl bdominl pin not otherwise specified; AM, bdominl migrine. Totl of 56 children with FAPDs were clssified s IBS (n = 37), IBS + FD (n = 5), IBS + FAP-NOS (n = 3), IBS + AM (n = 1), FAP-NOS (n = 6), FD (n = 3), nd AM (n = 1). Overlp group included IBS + FD (n = 5), IBS + FAP-NOS (n = 3), nd IBS + AM (n = 1). Non-IBS group included FAP-NOS (n = 6), FD (n = 3), nd AM (n = 1). 624 Journl of Neurogstroenterology nd Motility

12 GI Eosinophils in Childhood FAPDs including bdominl pin in ptients with FGIDs. 1,18- To dte, studies involving ptients with IBS hve not demonstrted significnt difference in the tissue eosinophil counts. 6,21-23 Furthermore, histopthologicl studies in children with FAPDs re reltively rre, nd peditric studies hve primrily been limited to studies investigting FD, focusing on the stomch nd duodenum. 8,9,24-27 Therefore, the histopthologicl fetures of the GI trct beyond the duodenum in children with FAPDs remin uncler. In the present study, we showed tht eosinophil counts of the stomch (gstric ntrum), smll bowel (duodenum, terminl ileum), nd proximl colon (cecum, scending colon) were significntly higher in children with FAPDs including IBS thn in norml controls. Our results suggest tht GI eosinophils my be ssocited with FAPDs in children nd my contribute to its pthogenesis. Severl studies hve shown tht mucosl eosinophili ws prominent in IBD ptients compred to helthy controls or those with IBS. 28 Furthermore, eosinophil counts or eosinophil ctionic protein levels positively correlted with disese ctivity nd negtively correlted with tretment response in IBD ptients. 17,29, Therefore, eosinophils re known to ply role in the pthogenesis of IBD, even though their exct reltionship with IBD remins uncler. 11,28,31,32 We hypothesized tht micro-inflmmtion my be the primry pthogenesis tht cuses GI symptoms in FAPDs. 6,7,18,21,33-39 Bsed on this hypothesis, we presumed the density of inflmmtory cells in the GI trct of the FAPDs group to be higher thn tht observed in norml controls without ny inflmmtion nd lower thn tht observed in the IBD group with definite inflmmtion of the GI trct. Study results showed significntly high eosinophil counts of the stomch nd colon in the order of IBD, FAPDs, nd norml controls, regrdless of endoscopiclly detected mcroscopic IBD lesions. Our study results re in greement with those of previous studies reported by Bss et l, Pensbene et l, 41 nd Flores et l 42 in tht eosinophil counts of the colonic re were significntly higher in children with IBD thn in those with FAPDs or IBS or norml controls, lthough study by Bss et l described tht tissue eosinophil counts of the stomch were not significntly different between children with IBD nd those with FAPDs, unlike our results. Furthermore, study reported by Crvlho et l 43 hs shown the results consistent with those of our study in tht high eosinophil counts of the colon showed definite order in terms of the disese entity UC, followed by CD, non-inflmed CD, nd IBS. Their results suggested tht the mcroscopic uninvolved mucos my not be entirely norml in ptients with IBD nd tht eosinophils could ply some roles in the pthogenesis of the erly lesions in IBD. 43 To our knowledge, the present study is the first comprtive study completed wherein the entire GI trct ws evluted to determine differences in tissue eosinophil infiltrtion in ptients with FGIDs. A grdient of eosinophil density is known to exist from the proximl to the distl colon 12,13,31,41,44 ; therefore, it is importnt tht GI trct eosinophils should be nlyzed through comprisons performed in ech specific region, even in the colonic re. In severl studies performed on dults with IBS, the biopsy specimens studied were primrily from the descending colon or rectosigmoid re. 37 In severl comprtive studies involving ptients with IBS nd/ or IBD, some studies hve not clerly described which prt of the colon ws nlyzed, nd others were performed with tissue obtined only from the rectosigmoid/rectl re. 29,-43 An inpproprite comprison of eosinophils within different sections of the colon cn led to inccurte results. In this regrd, our study involved ssessment of the entire GI trct nd compred eosinophils in ech segment of the gut, which is strength of our study. The present study hs some limittions. First, this study could not recruit helthy individuls s norml controls. Although the comprison with ge- nd sex-mtched helthy children s norml controls would be idel, this ws not performed becuse of ethicl considertions regrding subjecting helthy children without ny GI symptoms to endoscopic exmintion. Insted, we dopted norml reference vlues bsed on previously published study, which evluted endoscopic mucosl biopsies from ech segment of the GI trct for children with no identified orgnic findings, nd performed the finl clinicl ssessment. Furthermore, we used two references 12,13 ; one study s the upper GI trct reference nd the other one s the lower GI trct reference. Since these reference groups did not comprise of Koren children, they my not ccurtely represent norml controls. The study reported by DeBrosse et l 12 ws lredy used s norml reference in our previous study 1 published in 16 so it cn be good lterntive s upper GI reference. However, we should find nother norml reference to compre eosinophils of the colonic re becuse the study reported by DeBrosse et l 12 does not provide informtion regrding the cecum nd the descending nd sigmoid colon. A study published in 16 by Chernetsov et l 45 showed eosinophil counts of the whole GI trct in helthy Cndin children, however the study used different stin methods, hemtoxylin-phloxine-sffron nd Giems stin to enhnce cells detection, so their results present much higher cell counts thn the study by DeBrosse et l 12 nd the study by Sd. 13 The study by Chernestov et l 45 compred their results with the forementioned two studies nd dmitted differences. Thus, we used the study reported by DeBrosse et l 12 s n upper GI refer- Vol. 24, No. 4 October, 18 ( ) 625

13 Eun Hye Lee, et l ence to compre eosinophil infiltrtion between the stomch nd ileum, nd nother study reported by Sd 13 s lower GI reference to compre colonic eosinophil counts. Becuse of the study reported by Sd 13 which we used s lower GI pthology reference presented rectosigmoid re, nd the region ws not definitively divided into the sigmoid nd rectum, it ws difficult to ccurtely compre the eosinophil counts in the rectum. So, when the rectl eosinophils were compred using the reference vlues, rectl eosinophil counts reported in the study by DeBrosse et l 12 were lso presented s norml reference vlues in Tble 4 nd Tble 5. Second, this ws retrospective study with reltively smll smple size, nd evluted only eosinophils without other inflmmtory cells. Third, the mjority (46 [82.1%]) of 56 children with FAPDs were ptients with IBS. This is due to the fct tht children with IBS mostly underwent colonoscopic exmintion. Further prospective studies including lrger number of children, blnced recruitment of other subtypes of FAPDs, nd the nlysis of dditionl cell types such s mst cells or intrepithelil lymphocytes re needed to confirm low-grde inflmmtion in children with FAPDs. The present study evluted tissue eosinophil infiltrtion throughout the entire GI trct in children with FAPDs compred to those with IBD, nd to norml reference vlues in ech specific region of the GI trct. Our results suggest GI trct eosinophili my contribute to the development of childhood FAPDs s one of key mechnisms of the pthogenesis. Acknowledgements: The uthors would like to thnk Dr Je Bong Lee t Seoul Ntionl University Bundng Hospitl Medicl Reserch Collborting Center for her ssistnce in sttisticl nlyses. Finncil support: This study ws supported by grnt from Seoul Ntionl University Bundng Hospitl Reserch Fund (Grnt No ). Conflicts of interest: None. Author contributions: Eun Hye Lee: conducting the study, collecting nd interpreting dt, drfting the mnuscript; Hye Rn Yng: designing the study, interpreting dt, drfting the mnuscript; nd Hye Seung Lee: conducting the study, collecting nd interpreting dt. References 1. Korterink JJ, Diederen K, Benning MA, Tbbers MM. Epidemiology of peditric functionl bdominl pin disorders: met-nlysis. PLoS One 15;1:e Mdni S, Prikh S, Mdni RS, Krselp A. Long-term study of children with rome III functionl gstrointestinl disorders mnged symptomticlly in biopsychosocil model. Gstroenterology Res 17;1: Horst S, Shelby G, Anderson J, et l. Predicting persistence of functionl bdominl pin from childhood into young dulthood. Clin Gstroenterol Heptol 14;12: Hyms JS, Di Lorenzo C, Sps M, Shulmn RJ, Stino A, vn Tilburg M. Functionl disorders: children nd dolescents. Gstroenterology 16;15: , e2. 5. Schmulson MJ, Drossmn DA. Wht is new in rome IV. J Neurogstroenterol Motil 17;23: Ford AC, Tlley NJ. Mucosl inflmmtion s potentil etiologicl fctor in irritble bowel syndrome: systemtic review. J Gstroenterol 11;46: Vnheel H, Vicrio M, Vnuytsel T, et l. Impired duodenl mucosl integrity nd low-grde inflmmtion in functionl dyspepsi. Gut 14;63: Wuters L, Nightingle S, Tlley NJ, Sulimn B, Wlker MM. Functionl dyspepsi is ssocited with duodenl eosinophili in n Austrlin peditric cohort. Aliment Phrmcol Ther 17;45: Schurmn JV, Singh M, Singh V, Neiln N, Friesen CA. Symptoms nd subtypes in peditric functionl dyspepsi: reltion to mucosl inflmmtion nd psychologicl functioning. J Peditr Gstroenterol Nutr 1;51: Lee EH, Yng HR, Lee HS. Anlysis of gstric nd duodenl eosinophils in children with bdominl pin relted functionl gstrointestinl disorders ccording to rome III criteri. J Neurogstroenterol Motil 16;22: Yntiss RK. Eosinophils in the GI trct: how mny is too mny nd wht do they men? Mod Pthol 15;28(suppl 1):S7-S DeBrosse CW, Cse JW, Putnm PE, Collins MH, Rothenberg ME. Quntity nd distribution of eosinophils in the gstrointestinl trct of children. Peditr Dev Pthol 6;9: Sd AG. Norml quntity nd distribution of mst cells nd eosinophils in the peditric colon. Peditr Dev Pthol 11;14: Dperno M, D Hens G, Vn Assche G, et l. Development nd vlidtion of new, simplified endoscopic ctivity score for Crohn s disese: the SES-CD. Gstrointest Endosc 4;6: Trvis SP, Schnell D, Krzeski P, et l. Developing n instrument to ssess the endoscopic severity of ulcertive colitis: the Ulcertive Colitis Endoscopic Index of Severity (UCEIS). Gut 12;61: Trvis SP, Schnell D, Krzeski P, et l. Relibility nd initil vlidtion of the ulcertive colitis endoscopic index of severity. Gstroenterology 13;145: Trvers J, Rothenberg ME. Eosinophils in mucosl immune responses. Mucosl Immunol 15;8: Wng X, Li X, Ge W, et l. Quntittive evlution of duodenl eo- 626 Journl of Neurogstroenterology nd Motility

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