Surface epithelium related activation of complement

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1 92 Gut, 1992,33,92-98 Surfce epithelium relted ctivtion of complement differs in Crohn's disese nd ulcertive colitis T S Hlstensen, T E Mollnes, P Grred, Fus, P Brndtzeg Lbortory for Immunohistochemistry nd Immunopthology (LIIPAT), Institute of.pthology T S Hlstensen P Brndtzeg Institute of Immunology nd Rheumtology T E Mollnes P Grred Section of Gstroenterology Fus Medicl Deprtment A, University of Oslo, The Ntionl Hospitl, Rikshospitlet, Oslo, Norwy Correspondence to: Dr Trond S Hlstensen, LIIPAT, Rikshospitlet, N-27 Oslo 1, Norwy. Accepted for publiction 7 October 1991 Abstrct IgGI nd ctivted complement re coloclised on the colonic epithelil brush border in ctive ulcertive colitis. To investigte whether such deposition is specific for ulcertive colitis, we exmined ethnol fixed mucosl specimens from 18 ptients with Crohn's colitis nd 14 with terminl ileitis by indirect two colour immunofluorescence stining. Monoclonl ntibodies to the IgG subclsses nd to neoepitopes of ctivted complement C3b nd the terminl complement complex were used in combintion with rbbit ntiserum to Clq, C4c or cytokertin. Grnulr deposition of C3b nd terminl complement complex were observed t the luminl fce of the surfce epithelium in 1 of 18 ptients with Crohn's colitis. Specimens from eight of 14 ptients with ilel involvement were intensely stined for ctivted complement (primrily C3b) within the surfce mucus lyer. No epithelil IgG, Clq or C4c deposition ws observed. The results suggest tht erly nd lte phse complement ctivtion tkes plce t the luminl fce of the epithelium in Crohn's disese. The bsence of coloclised IgG nd complement components involved in the clssicl ctivtion pthwy (Clq nd C4c), however, suggest tht other immunopthologicl mechnisms (the lterntive pthwy?) re primrily involved in Crohn's disese in contrst with ulcertive colitis. Crohn's disese is chronic inflmmtory bowel disese of unknown etiology nd pthogenesis.' Immunohistochemicl studies hve reveled strikingly incresed locl immunoglobulin G (IgG) production,2 predominntly of the complement binding IgGI subclss lthough Crohn's disese mucos contins higher percentge of IgG2 contining immunocytes thn ulcertive colitis.3 The sme result hs been obtined by cultivtion of mononucler cells from the lesion.' Ptients with Crohn's disese re reported to hve impired C3b inctivtion' nd incresed complement C3 ctbolism`6 with rised serum concentrtions of complement C3 ctivtion split products.9 "' These findings suggest tht complement ctivtion tkes plce in the ffected intestinl mucos. To ddress this issue we exmined directly ethnol fixed s well s extensively pre-wshed mucosl specimens from ptients with Crohn's disese in the ileum nd/or colon. Monoclonl ntibody (mab) to ctivtion neoepitopes in the erly (C3b) nd lte (terminl complement complex complement ctivtion pthwy were used. The results showed deposition of both C3b nd terminl complement complex in these deposits, however, in contrst with our previous observtions in ulcertive colitis." Methods TISSUE SPECIMENS Mucosl tissue smples were immeditely excised from surgiclly resected colon (n=56) or terminl ileum (n=26) from 21 ptients with Crohn's disese (medin ge 29 yr, rnge 14-64). Three ptients hd only ilel involvement, one only colonic, wheres 17 hd both segments ffected (ilel biopsy vilble only in 11). The ptients hd been observed cliniclly for two to 13 yers (medin five yers). Ulcertive colitis specimens (five) with epithelil complement nd IgGI deposits were selected from 23 previously reported ptients. " Control mteril ws obtined from histologiclly norml colonic mucos of 26 ptients s described elsewhere" nd norml ilel mucos (six). All tissue specimens were plced in ice cold isotonic sline nd brought to the lbortory within one hour for preprtion in two lterntive wys. Trimmed tissue blocks (bout 2 x4x 5 mm) were either fixed directly in cold 96% ethnol for 18 hours to preserve soluble immune complexes (17 ilel nd 33 colonic smples), or wshed for 48 hours t 4 C in.1 M phosphte buffer (ph 7T5) contining 15 M NCl (phosphte buffered sline to extrct diffusible proteins before cold ethnol fixtion (nine ilel nd 23 colonic smples). All tissue smples were finlly dehydrted in cold bsolute ethnol, clered in xylene, embedded in prffin for three to four hours t 56 C, nd stored t 4 C until sectioning. " STAINING PROCEDURES AND IMMUNOLOGICAL REAGENTS Dewxed sections cut t 6 um from directly fixed tissue were incubted for 2 hours t room temperture with monoclonl ntibody to C9 neoepitope of terminl complement complex (Ell; 2.5 mg/l)'" in combintion with rbbit ntiserum to S-protein/vitronectin (1:5; kindly provided by B Dhlbck, Deprtment of Clinicl Chemistry, Mlmo Generl Hospitl, Sweden). 14 An lterntive combintion ws monoclonl ntibody to C3b neoepitope in the C3c prt of C3b/iC3b (bh6, 7.5 mg/l)' nd rbbit ntiserum to cytokertin (1:1). 6 Dewxed sections of prewshed tissue were exmined both for IgG deposits nd complement ctivtion products. Monoclonl ntibodies to

2 Surfce epithelium relted ctivtion ofcomplement differs in Crohn's disese nd ulcertive colitis 93 C3b, TCC, IgGl (clone 267, 1:8; HP 67),'7 IgG2 (clone GOM2, 1:8; HP69),'7 OgG3 (clone CBI-AH7, 1:8; HP648)'7 nd IgG4 (clone RJ4, 1:8; HP611)`7 were ll used in combintion with rbbit ntiserum to C3c (1:5; Behring, Mrburg, Germny) s previously described." Selected colonic (three) nd ilel (two) specimens from five ptients with Crohn's disese nd five with ulcertive colitis found to hve picl complement deposition (see below) were, in ddition, exmined with rbbit ntiserum to Clq (1:5; Dkoptts, Glostrup, Denmrk); C3c (1:5; Behring) nd C4c (1:5, Dkoptts) in combintion with monoclonl ntibody to C3b or terminl complement complex. Secondry regents were biotinylted horse ntimouse IgG ( 25 g/l; Vector Lbortories, Burlingme, CA, USA), followed either by swine ntirbbit IgG ( 14 g IgG/l; Dkoptts) conjugted with rhodmine"8 in combintion with fluorescein Isothiocynte (FITC) conjugted streptvidin (-2 g/l; Boehringer Mnheim, Germny), or by fluorescein isothiocynte conjugted swine ntirbbit IgG (1:1, Dkoptts) combined with Streptvidin-Texs Red (.25 g/l; BRL, Githersburg, MD, USA). The regents were pplied in three step two colour immunofluorescence stining method principlly s described elsewhere. 8 The selected prewshed specimens were lso subjected to three colour stining in which monoclonl ntibody to humn IgGI (murine IgGI) ws combined with monoclonl ntibody bh6 to C3b (IgG2) or monoclonl ntibody E 11 to terminl complement complex (IgG2), nd mixed with rbbit ntiserum to C3c or C4c. Secondry regents were biotinylted nd fluorescein Isothiocynte conjugted subclss specific got ntimouse IgG2 nd IgGI (Southern Biotechnology, Birminghm, AL, USA) followed by 7-mino-4-methylcoumrin-3- cetic cid (conjugted got ntirbbit IgG (1:2, Vector Lbortories) in combintion with Streptvidin-Texs Red (.25 g/l; BRL). Selected colonic specimens (six) with C3bpositive globulr elements in the lmin propri (see lter) were lso exmined for terminl complement complex nd C3b deposition by immunoenzyme stining; the lkline phosphj-u Figure 1: Intense C3b positivity () ws noted in the mucus lyer nd piclly on the epithelium, wheres terminl complement complex (TCC) (b) ws selectively deposited on the epithelil surfce. Broken line indictes the bsement membrne zone. Pired immunofluorescence stining in section ofdirectly ethnolfixed ilel mucos from ptient with Crohn's disese ofthe ileum. tse ntilkline phosphtse'9 nd the vidinbiotin complex peroxidse methods were both pplied, the ltter ccording to the instructions given by the mnufcturer (Dkoptts). IMMUNOHISTOCHEMICAL EVALUATION, CONTROLS, AND STATISTICAL ANALYSIS Immunofluorescence ws exmined in Leitz Orthopln microscope equipped with Ploem type verticl illumintor for selective observtion of red (rhodmine), green fluorescein Isothiocynte or blue 7-mino-4-methylcoumrin-3- cetic cid emmission. For every immunofluorescence mrker, ech specimen ws given semiquntittive score rnging from no (-) to intense (3+) stining. Scoring of the unselected Crohn sections ws done blind by the sme investigtor. Mucus ssocited nd the strictly epithelium relted complement deposits were scored seprtely. One section from every series ws subjected to blind histopthologicl evlution fter hemtoxylin nd eosin stining; ech specimen thereby received n rbitrry inflmmtion score from negtive (-) to intense (3+) ccording to cellulr infiltrtion nd mucosl destruction. Primry incubtion with murine control scites or norml rbbit serum, t dilutions similr to those used for monoclonl ntibodies nd rbbit ntiser, did not produce immunofluorescence. The reltion between mucus ssocited nd/or epithelium relted terminl complement complex/c3b deposits nd inflmmtion ws bsed on Kendll's correltion nlysis. Results ILEAL COMPLEMENT DEPOSITS Stining for C3b-neo (monoclonl ntibody bh6) ws observed within the ilel surfce mucus lyer in directly fixed specimens from eight of 14 ptients. Additionl but weker nd scttered stining for terminl complement complex ws observed in five of these ptients. A distinct epithelium relted picl stining for C3b ws observed in seven ptients, nd four of them showed terminl complement complex t the sme loction (Tble) C3b often dominted throughout the mucus lyer wheres terminl complement complex ppered to be locted closer to the epithelil surfce nd ws then scored s epithelium relted (Fig 1). Only the stining intensity of epithelium relted nd mucus ssocited terminl complement complex ws significntly correlted with the topicl degree of inflmmtion (Fig 2), lthough wide sctter ws observed. Fine grnulr stining for terminl complement complex nd S-protein ws present in the bsement membrne zone of five ptients, wheres C3b ws seen in only one. No epithelil deposition of Clq, C4c or ny of the four IgG subclsses ws detected in the prewshed specimens. An pproximtely 1,um brod zone of the mucosl wll surrounding ilel ulcers stined

3 94 Hlstensen, Mollnes, Grred, Fus, Brndtzeg Clinicopthologicl informtion bout the ptients with Crohn's disese of the colon nd scoring of inflmmtion nd epithelium relted immune deposits Disese Medictiont Mucus Epithelium Ptient Sexlge* durtion Clinicl Inflm- BMZ no (yr) (yr) S P ctivity Loction: mtion C3b TCC C3b TCC TCC 1 WM47 4 No No Mild Ileum M/26 3 No No Mild Ileum M/47 4 No Yes Moderte Ileum M/4 4 No No Mild Ileum Coecum M/15 3 No No Mild Ileum Colon S F/25 4 No Yes Moderte Ileum ColonT M/18 13 Yes No Moderte Ileum Colon A F/35 5 No No Moderte Ileum Colon S F/17 2 No Yes Mild Ileum Colon T M/29 1 No No Moderte Ileum ColonD F/23 2 No Yes Mild Ileum Coecum M/3 7 Yes No Moderte Ileum Colon A M/64 1 Yes Yes Moderte Ileum Colon A 14 F/14 6 No Yes Mild Ileum Colon A F/21 5 Yes Yes Severe Colon D F/24 8 Yes Yes Severe ColonA M/38 9 No No Mild Colon A 18 M/19 4 Yes Yesl Severe Rectum F/3 4 Yes Yes Moderte Rectum M/36 9 No Yes Mild Colon S F/64 13 No No Mild Colon S * M=mle, F=femle, ts=slzopyrin, P=prednisone, tcolon S=sigmoid colon, colon A- SBMZ=bsement membrne zone, 1ptient received Imurel (zthioprin). =scending colon, colon T= trnsverse colon, diffusely for terminl complement complex, nd numerous terminl complement complex positive globulr elements were observed in the luminl content (not shown). COLONIC COMPLEMENTS DEPOSITS Crohn's disese Often grnulr nd prllel positivity for C3b (Fig 3) nd terminl complement complex ws observed t the luminl fce of the epithelium in 1 of 18 ptients with Crohn's disese of the colon (Tble). The stining intensity for strictly epithelium-relted (Fig 2b) C3b nd terminl complement complex ws better correlted with the topicl degree of inflmmtion (Fig 2) thn the mucus ssocited. Grnulr terminl complement complex positivity in the bsement membrne zone ws often observed beneth intct epithelium in nine of the ptients. The content in colonic fissures ws, like tht in crypt bscesses, often intensely positive for terminl complement complex but mostly negtive for C3b. Globulr elements with vrious combintion of peripherl positivity for IgG subclsses (IgG2>IgG3>IgGl) nd/or C3b were observed within the mucus ssocited content, wheres c E 3- A * A [. c 3- G) E B n v 2- O o f. COl) CO E 1- A1 A * * A *A E C v7 2- x, = 4-'O oo.. ov A'*UVT A * XUD 1- A *A St-\ O JA. - LA AL LA AA 1*AAU I OL] *A*mV A\ SOAL *@AA **AA._ ] m [- I 2- D v / 3 3 Li - -E~ L\ AA AA L] *-AA Degree of inflmmtion Figure 2: Sctter digrm showing intensity of() epithelium relted nd (b) mucus ssocited complement relted to the degree of inflmmtion in Crohn ptients (controls not shown). Epithelium relted nd mucus ssocited terminl complement complex (but not C3b) correlted with the topicl degree of ilel inflmmtion (T=O33 nd T=41, respectively; p< 1), wheres both the epithelium-relted C3b (T=O45) nd terminl complement complex(t=o38) correlted with the degree of inflmmtion in the lrge bowel (p<1). The mucus ssocited terminl complement complex (but not C3b) showed wek correltion with the topicl degree of colonic inflmmtion (T=O27; p<o5). Filled symbols represent C3b, open symbols TCC. Ileum, * O; cecum nd scending colon, A A; trnsverse colon, * <; descending nd sigmoid colon, M [; rectum, V V. A eoa *@A *-m~ * A*

4 Surfce epithelium relted ctivtion ofcomplement differs in Crohn's disese nd ulcertive colitis 95 Figure 3: Two colour immunofluorescence stining for C3b (A) nd kertin (B) on directly ethnolfixed colonic mucos from ptient with Crohn's disese ofthe colon. C3b positive globulr elements (rrow) were ttched to nd prtly locted within the epithelium. terminl complement complex positivity ws more diffuse (not shown). Similr C3b positive elements were observed ttched to nd/or locted within the epithelium in nine ptients. Aggregtes of C3b positive globulr elements were observed in the lmin propri in directly ethnol fixed specimens from six ptients (Fig 4). These ggregtes were pprently locted within lymphtic vessels s visulised lso with vidin biotin complex peroxidse (Fig 4C) nd lkline phosphtse ntilkline phosphtse stining technique in three ptients exmined. No epithelil deposition of IgG, Clq, or C4c ws observed in prewshed tissue smples from Crohn's disese (Fig 5). Ulcertive colitis All selected ulcertive colitis smples showed deposition of IgGl, C3b nd terminl complement complex on the luminl fce of individul enterocytes, only interrupted by goblet cells s previously reported. " Crypt bscesses nd luminl contents often stined for terminl complement complex but only wek nd scttered C3b positivity ws observed, contrsting the intense mucus-ssocited stining for C3b in Crohn's disese. Epithelium relted stining for C4c ws observed in three of the five ptients but it ws less prominent thn seen for C3c. Suprepithelil Clq ws wek nd observed only occsionlly. Three colour stining for IgGl, C3b nd C4c or Clq reveled tht the IgGI nd C3b positive epithelil deposits often costined for C4c (though weker thn for C3b/C3c). Such deposits were observed in ll three smples (Fig 6). C3b/ terminl complement complex positive deposits in the bsence of both IgGI nd C4c/C lq were lso observed. The other two smples contined either epithelil C3b nd terminl complement complex deposits in the bsence of IgGi nd Clq/ C4c, or only IgG 1 in the bsence of complement components. C3b positive globulr elements were often observed in the lumen nd were ttched to the surfce epithelium in one of the ulcertive colitis smples. VASCULAR COMPLEMENT Submucosl blood vessels in both ileum nd s..s F* Figure 4: Two colour immunofluorescence stining for C3b (A) nd kertin (B) on directly ethnol fixed colonic mucos from ptient with Crohn's disese ofthe colon. Aggregte of C3b positive globulr elements (rrow) ws present in the lmin propri nd it ws lso visulised with immunoperoxidse stining (rrow) (C). Figure 5: Two colour immunofluorescence stiningfor (A) IgGI nd (B) C3b in prewshed colonic mucos from ptient with Crohn's colitis. No IgGI (or ny other IgG subclss) ws observed within the C3b-positive epithelil deposits (lrge rrow). Epithelil cell (smll rrows) with diffuse stiningfor IgGI in the cytoplsm hd tken up this serum protein in vivo, perhps becuse ofcomplement induced lekge.

5 96 Hlstensen, Mollnes, Grred, Fus, Brndtzeg Figure 6: Immunofluorescence stining for (A) IgGI, (B) C4c nd (C) C3b on two seril sections of colonic mucos from ptient with ulcertive colitis ( nd b, pired stining). In this disorder, the epithelil C3b deposits (c) often costinedfor both IgGI () nd the clssicl ctivtion pthwy component C4c (b). Epithelil detchment hd presumbly been induced in vivo s the epithelium stined diffusely for serum proteins such s IgGl (rched rrow). Note segmentl costining piclly on the epithelil surfce (rrows). colon were intensely positive for terminl complement complex, nd to lesser extent for C3b, s previously reported.22' Some of the C3b positive submucosl blood vessels in both Crohn's disese nd ulcertive colitis showed segmentl costining for Clq nd C4c. Two colonic smples from Crohn's disese contined submucosl blood vessel tht stined for ll complement components (Clq, C3b, C4c nd terminl complement complex) in ddition to some wek IgGI positivity (not shown). Numerous cells with cytoplsmtic stining for C3c, C4c, Clq nd occsionlly lso IgGl were observed deep in the lmin propri nd between the smooth musculr cells in inflmed sections (not shown). CONTROLS Epithelil complement deposition ws not observed in six non-inflmed ilel smples from six controls nd not in 42 of 44 colonic smples from 26 control ptients." Discussion Our previous observtion of epithelium relted IgGI nd ctivted complement (C3b nd terminl complement complex) in ctive ulcertive colitis reflected potentilly destructive immune rection cused by utontibodies to epithelil brush border ssocited protein(s). " Here we report tht luminl complement ctivtion lso occurs in Crohn's disese of the ileum nd colon. Lck of IgG nd clssiclly complement ctivtion components (Clq nd C4c) within the epithelil C3b/termninl complement complex deposits, however, suggests tht inititors of the lterntive pthwy re more importnt in Crohn's disese thn in ulcertive colitis. In the ltter disorder epithelil immune deposits often costin for IgG 1 nd C4c nd lso occsionlly for Clq, strongly suggesting tht IgGI induces clssicl pthwy ctivtion. Epithelil immune deposits negtive for IgGI, Clq nd C4c re lso observed, however, probbly reflecting tht lterntive pthwy ctivtion lso occurs in ulcertive colitis. The two complement ctivtion pthwys re not strictly seprte becuse ntibodies nd immune complexes my lso ctivte the lterntive pthwy,22 nd gents such s crdiolipin,2' C-rective protein2425 nd bcteril (Escherichi coli) surfce ntigens26 my initite the clssicl pthwy. In ddition, C3b produced by clssicl ctivtion my complex with fctor B to generte the lterntive pthwy C3 convertse (C3bBb), resulting in incresed C3 ctivtion (lterntive pthwy mplifiction). It is therefore difficult to determine the min ctivtion pthwy on the bsis of immunohistochemicl stining for complement components in immune deposits. Nevertheless, codeposition of IgGI in ulcertive colitis but not in Crohn's disese, strongly suggests tht utontibodies to the colonic epithelium re involved in the epithelil complement deposition only in the former disese. Also, ptients with Crohn's disese hve been found to hve higher serum levels of the lterntive pthwy ctivtion product Bb thn ptients with ulcertive colitis.8 The subepithelil deposition of C3d, terminl complement complex nd S-protein (presumbly representing the soluble form of terminl complement complex, SC5b-9) observed in ffected ileum nd colon might hve represented fluid fce complement ctivtion secondry to epithelil destruction. Alterntively, soluble immune complexes generted in the bsement membrne zone could hve induced subepithelil compliment ctivtion nd induced epithelil dmge. These deposits, however, were minly seen beneth intct epithelium in Crohn's disese, contrsting with ulcertive colitis where such deposits primrily were observed beneth or close to dmged epithelium. Ahrenstedt et 127 recently reported tht ptients with Crohn's disese of the ileum hve incresed concentrtions of C3 in jejunl lvge fluid. It ws suggested tht this finding reflected ctivted C3 becuse most ws of moleculr size similr to the complement split product C3c. This does not seem to be generl smll intestinl phenomenon, however, becuse C3b/C3c positivity (mab bh6) ws observed only in mucus of ffected ileum. The epithelil complement deposition in Crohn's colitis tended to be more grnulr thn in ulcertive colitis. The globulr elements positive for IgGI, IgG2, IgG3, nd C3b observed in the mucus, nd the C3b positive elements on the epithelium nd in the lmin propri, resembled surfce stining of microorgnisms. This sug-

6 Surfce epithelium relted ctivtion ofcomplement differs in Crohn's disese nd ulcertive colitis 97 gested n immune ttck but the puttive microorgnisms might hve bound C3b by complement receptor like structures s recently shown for Cndid lbicns.28 Such C3b coting does not fford opsonistion nd the microorgnisms my escpe phgocytosis. In n electron microscopic study of Crohn's disese, Thyberg et lf9 observed prtly degrded bcteri in lmin propri mcrophges nd epithelil cells regrdless of the severity of the lesion; nd Aluwihre3 found intrmurl bcteri in six of 11 colonic specimens with intct epithelium nd miniml inflmmtory chnges. Ptients with Crohn's disese hve, in ddition, higher serum concentrtions of gglutinting ntibodies to nerobic coccoid roods (Eubcterium contortum, Coprococcus comes, Peptostreptococcus productus) thn ptients with ulcertive colitis.3' Furthermore, only Crohn ffected mucos contins 16- kd, 12-kD, nd 1 1-kD proteins tht re exclusively precipitted by ser from ptients,32 suggesting n immune response to foreign ntigens selectively locted in the Crohn lesion. Tken together, these results support the suggestion tht Crohn's disese my be cused by immune responses to reltively non-pthogenic replicting microorgnism(s). The ctul lesion my be produced by n ctivted immune system rther thn directly by the microorgnism. Mny microbil cndidtes hve been suggested, including Mycobcteri, Spheroblsts, nd wll deficient Slmonell species, but their pthogenetic significnce remins obscure."" This is lso true for the C3b coted elements seen on the epithelium in our study, the sme observtion ws mde in some ulcertive colitis specimens. Conversely, the ggregtes of C3b coted globulr elements in the lmin propri hve not been observed in ulcertive colitis despite severe epithelil dmge. Their nture therefore needs further elucidtion. We hve previously reported tht ptients with Crohn's disese nd ulcertive colitis hve incresed deposition of terminl complement complex2 nd C3b" in submucosl blood vessels, suggesting tht vsculr complement ctivtion tkes prt in the pthogenesis. The vsculr immune deposits do not generlly contin detectble Ig components.4' In this limited study of prewshed tissue specimens, however, we observed tht the vsculr C3b deposits occsionlly costined for Clq nd C4c, suggesting clssicl complement ctivtion. Furthermore, by three colour immunofluorescence stining, mking it possible to focus on the reltively few terminl complement complex positive vessels contining both C3b nd C4c, we observed some wek segmentl IgGI positively in submucosl vessels of two colonic specimens from Crohn's disese. The nture of this observtion needs further exmintion, but vsculr complement ctivtion my be involved in the multiple intestinl infrction tht seems to be feture of Crohn' disese.4' Although ctivted T cells hve been suggested to be prticulrly importnt in the pthogenesis of Crohn's disese,4' locl complement ctivtion my contribute to the immunopthology. The bsence of IgG, Clq, nd C4c within the epithelium relted immune deposits suggests tht other immunopthologicl mechnisms operte in Crohn's disese thn in ulcertive colitis. TSH is reserch fellow of the Norwegin Cncer Society. 1 Elson CO. The immunology of inflmmtory bowel disese. In: Kirsner JB, Shorter RG, eds. Inflmmtory bowel disese. 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