Akemi Imaoka, Tatsuichiro Shima, Kimitoshi Kato, Shigeaki Mizuno, Toshiki Uehara, Satoshi Matsumoto, Hiromi Setoyama, Taeko Hara, Yoshinori Umesaki

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1 Online Sumissions: wjg.wjgnet.com World J Gstroenterol 28 April 28; 14(16): World Journl of Gstroenterology ISSN wjg@wjgnet.com 28 WJG. All rights reserved. CLINICAL RESEARCH Anti-inflmmtory ctivity of proiotic Bifidocterium : Enhncement of IL-1 production in peripherl lood mononucler cells from ulcertive colitis ptients nd inhiition of IL-8 secretion in HT-29 cells Akemi Imok, Ttsuichiro Shim, Kimitoshi Kto, Shigeki Mizuno, Toshiki Uehr, Stoshi Mtsumoto, Hiromi Setoym, Teko Hr, Yoshinori Umeski Akemi Imok, Ttsuichiro Shim, Stoshi Mtsumoto, Hiromi Setoym, Teko Hr, Yoshinori Umeski, Ykult Centrl Institute for Microiologicl Reserch, Yho, Kunitchi, Tokyo , Jpn Kimitoshi Kto, Shigeki Mizuno, Toshiki Uehr, Deprtment of Gstroenterology nd Heptology, Nihon University School of Medicine, Tokyo , Jpn Author contriutions: Imok A, Kto K nd Umeski Y designed the reserch; Imok A, Shim T, Kto K, Mizuno S, Uehr T, Mtsumoto S, Setoym H nd Hr T performed the reserch; Imok A, Kto K nd Umeski Y wrote the pper. Correspondence to: Akemi Imok, Ykult Centrl Institute for Microiologicl Reserch, 1796 Yho, Kunitchi, Tokyo , Jpn. kemi-imok@ykult.co.jp Telephone: Fx: Received: Septemer 2, 27 Revised: Mrch 11, 28 Astrct AIM: To determine the nti-inflmmtory ctivity of proiotic Bifidocteri in Bifidocteri-fermented milk (BFM) which is effective ginst ctive ulcertive colitis (UC) nd excertions of UC, nd to explore the immunoregultory mechnisms. METHODS: Peripherl lood mononucler cells (PBMNC) from UC ptients or HT-29 cells were co-cultured with het-killed proiotic cteri or culture superntnt of Bifidocterium reve strin Ykult (BrY) or Bifidocterium ifidum strin Ykult (BiY) to estimte the mount of IL-1 or IL-8 secreted. RESULTS: Both strins of proiotic Bifidocteri contined in the BFM induced IL-1 production in PBMNC from UC ptients, though BrY ws more effective thn BiY. Conditioned medium (CM) nd DNA of oth strins inhiited IL-8 secretion in HT-29 cells stimulted with TNF-α, wheres no such effect ws oserved with hetkilled cteri. The inhiitory effect of CM derived from BiY ws greter thn tht of CM derived from BrY. DNAs of the two strins hd comprle inhiitory ctivity ginst the secretion of IL-8. CM of BiY induced repression of IL-8 gene expression with higher expression of IκB-ζ mrna 4 h fter culture of HT-29 cells compred to tht in the sence of CM. CONCLUSION: Proiotic Bifidocterium strins in BFM enhnce IL-1 production in PBMNC nd inhiit IL-8 secretion in intestinl epithelil cells, suggesting tht BFM hs nti-inflmmtory effects ginst ulcertive colitis. 28 WJG. All rights reserved. Key words: Bifidocterium ; Ulcertive colitis; Antiinflmmtory; Interleukin-1; Interleukin-8 Peer reviewers: Jy Prvd, MD, Inflmmtory Disese Reserch Center, Ginesville, Florid, , United Sttes; Frncesco Cost, Dr, Diprtimento di Medicin Intern-U.O. di Gstroenterologi Università di Pis-Vi Rom, Pis, Itly Imok A, Shim T, Kto K, Mizuno S, Uehr T, Mtsumoto S, Setoym H, Hr T, Umeski Y. Anti-inflmmtory ctivity of proiotic Bifidocterium: Enhncement of IL-1 production in peripherl lood mononucler cells from ulcertive colitis ptients nd inhiition of IL-8 secretion in HT-29 cells. World J Gstroenterol 28; 14(16): Aville from: URL: DOI: org/1.3748/wjg INTRODUCTION Ulcertive colitis (UC) is ssumed to e result of n impired intestinl immune response to intestinl environmentl ntigens such s uiquitous microflor [1,2]. There is evidence tht proiotic tretment is effective ginst UC [3-6]. We hve previously shown tht tretment with ifidocterifermented milk (BFM) contining live ifidocteri is more effective thn the usul tretment in inducing [7] nd mintining remission [8] of ulcertive colitis. An improvement in the composition of intestinl flor y BFM my prevent the overgrowth of potentilly pthogenic cteri such s Bcteroides vulgtus, ut we hypothesized tht proiotic strins of ifidocteri in BFM my directly interfere with the host signling events tht drive the intestinl inflmmtory response. In inflmmtory owel disese, IL-1 is cytokine of prticulr therpeutic interest since it hs een shown in niml models tht interleukin (IL)-1(-/-) mice spontne

2 2512 ISSN CN /R World J Gstroenterol April 28, 28 Volume 14 Numer 16 ously develop intestinl inflmmtion. IL-1 plys key role in the control of inflmmtory responses to enteric orgnisms [9-12]. More recently, it hs een shown in niml models tht proiotic strins displying n in vitro potentil to induce higher levels of the nti-inflmmtory cytokine IL-1 nd lower levels of the inflmmtory cytokine IL-12, offer the est protection ginst in vivo colitis in the model [13]. A geneticlly engineered Lctococcus lctis thy12 producing IL-1 meliorted colitis in two models of experimentl colitis, providing proof of principl tht topiclly delivers IL-1, cn e therpeuticlly efficcious [14] nd recent proof-or-principle experiment using this trnsgenic cterium expressing IL-1 in 1 ptients with Crohn s disese showed efficcy [15]. In ddition, there is n incresing mount of evidence to suggest tht the potent neutrophil chemottrctnt, IL-8, hs n importnt role in the pthogenesis of inflmmtory owel disese (IBD) [16-19]. Recently, higher concentrtion of IL-8 ws found in more histologiclly inflmed tissue segments from peditric IBD ptients, suggesting tht IL-8-specific therpies my universlly modify the inflmmtory ctivity in IBD ptients [2]. In this study, we focused on the effect of proiotic strins on the secretion of IL-1 y peripherl lood mononucler cells nd lso the production of IL-8 y intestinl epithelil cells. MATERIALS AND METHODS Bcteri nd relted preprtions Bifidocterium ifidum strin Ykult (BiY) nd Bifidocterium reve strin Ykult (BrY) were grown in MRS roth (Becton, Dickinson nd Compny, Sprks, MD). Hetkilled BiY or BrY ws prepred y heting cteri resuspended in distilled wter t 1 for 3 min, nd then lyophilized [21]. For the preprtion of conditioned medium (CM), cteri grown in MRS roth were collected y centrifugtion nd cultured over 16 h in RPMI-164 medium (Sigm-Aldrich, St Louis, MO) contining 1% fetl clf serum (FCS) nd 2% lctose, then centrifuged [22]. The superntnt ws filtrted on.22 μm memrne nd neutrlized with sodium hydroxide. To chrcterize the ctive component in CM, it ws seprted into frctions of more thn nd less thn 3 kd through Centricon YM-3 (Millipore, Bedford, MA), djusted to the initil volume, nd then to het tretment t 1 for 15 min [23]. DNA ws isolted using the method of Yuki [24] with slight modifiction. Briefly, cteril cells were suspended in Tris- EDTA uffer (ph 8.) contining.5 mol/l sucrose nd treted with N-cetylmurmidse SG (Seikgku Corp., Tokyo, Jpn) nd lysozyme (Sigm-Aldrich) t 37 for 1 h. The cells were lysed y ddition of sodium dodecyl sulfte nd proteinse K (Sigm-Aldrich) followed y 6-min incution t 65. Deproteiniztion ws done y extrction with Tris-sturted phenol nd phenol/chloroform/ isomyl lchol (25:24:1). Finlly, DNA ws precipitted y ethnol. Peripherl lood mononucler cells Peripherl lood mononucler cells (PBMNC) were isolted from peripherl lood of UC ptients y Ficoll- Conry (Lymphosepr I; Immuno-Biologicl Lortories, Tkski, Jpn) density grdient centrifugtion. Tle 1 summrizes the ptient chrcteristics. All 9 ptients (outptient) hd ctive UC which ws moderte or mild (1 mild, 8 moderte) ccording to the criteri of Truelove & Witts [25]. All ptients received stndrd therpeutic regimen consisting of orl 5-ASA (meslzine) nd five of the 9 ptients with ctive UC took low dose of orl predonine. Cells (2 1 5 ) were cultured with het-killed cteri (1 µg/ml) in 2 µl of AIM-V medium (Invitrogen Corp., Crlsd, CA) in flt-ottomed 96-well culture plte (Nunc, Roskilde, Denmrk) for 48 h. Superntnt ws collected nd frozen until cytokine levels were quntified. In ech ssy, positive control with lipopolyscchride (LPS, 1 µg/ml) dded to PBMNC nd negtive control with no stimuli were included. Quntifiction of cytokine levels in culture superntnt IL-1 nd IL-8 levels in the culture superntnt were determined y sndwich enzyme-linked immunosorent ssy (ELISA). IL-1 ws detected using nti-humn IL-1 ntiody ( E, BD Biosciences PhrMingen, Frnklin Lkes, NJ) nd iotinylted ntiody ( E, BD Biosciences PhrMingen). IL-8 ws detected using ntihumn IL-8 ntiody (AHC932, Invitrogen BioSource) nd iotinylted nti-humn IL-8 ntiody (AHC789, Invitrogen BioSource). HT-29 cell culture HT-29 cells were cultured t 37 in n tmosphere contining 5 ml/l CO2 in RPMI-164 medium contining 1% FCS, 1 mmol/l sodium pyruvte (Invitrogen) nd 1 mmol/l 4-(2-hydroxyethyl)-1-piperzineethnesulfonic cid (HEPES). Confluent monolyers were incuted in 96-well or 24-well tissue culture pltes with humn TNF-α (1 ng/ml, PeproTech, London, UK). Quntittive RT-PCR Totl RNA ws isolted from HT-29 cells using the TRIzol regent (Invitrogen) ccording to the mnufcturer s instructions. The RNA ws reverse trnscried into cdna using SuperScript First-Strnd Synthesis System kit (Invitrogen). Rel-time quntittive PCR ws performed in n ABI prism 75 Rel Time PCR System (Applied Biosystems, Foster City, CA) with the SYBR Premix Ex Tq (Tkr Bio, Shig, Jpn). The following primers were used to mplify cdna frgments: IL-8: forwrd: 5'-ACACTGCGCCAACACAGAAATTA-3', reverse: 5'-TTTGCTTGAAGTTTCACTGGCATC-3'; GAPDH: forwrd: 5'-GCACCGTCAAGGCTGAGAAC-3', reverse: 5'-ATGGTGGTGAAGACGCCAGT-3'; IκB-ζ: forwrd: 5'-ACGCGCAAACATGAGTCCAG-3', reverse: 5'-CT- CAGCAGCAGCAACAGCATC-3'. All results were finlly determined fter correction with GAPDH expression. Sttisticl nlysis Results were expressed s men ± SD. Differences in men vlues etween groups were nlyzed with Student s t-test. RESULTS Effect of proiotic cteri on IL-1 secretion in PBMNC The secretion of IL-1 incresed in ll the cultures of

3 Imok A et l. Anti-inflmmtory ctivity of proiotics 2513 Tle 1 Chrcteristics of the ptients with ulcertive colitis Ptient No. Age Sex Disese extent Clinicl pttern Disese durtion (yr) Disese severity Tretment 5-ASA (meslzine) (SASP) Steroid (predonine) 1 23 Mle Totl Relpsing 6 Moderte Femle Totl Relpsing 2 Moderte Mle Totl Relpsing 2 Moderte Femle Proctitis Relpsing 15 Mild Mle Totl First 3 Moderte Mle Totl Relpsing 2 Moderte Mle Totl First.5 Moderte Mle Left-sided Chronic 7 Moderte Mle Left-sided Relpsing 14 Moderte + + SASP: Slzosulphpyridine. IL-1 (pg/ml) IL-8 (pg/ml) B. ifidum B. reve LPS Bsl d,d Het-killed cteri B. ifidum B. reve Figure 2 Effects of proiotic ifidocteri on IL-8 production in HT-29 cells. HT-29 cells were stimulted with TNF-α (1 ng/ml) in the sence or presence of vrious concentrtions of proiotic cteri. Six hours fter incution, the IL-8 concentrtion ws determined y ELISA (men ± SD, n = 4). PBMNC isolted from the nine UC ptients with historicl record of the tretment s shown in Tle 1 in the presence of het-killed BrY or BiY, compred to the sl secretion in the sence of proiotic cteri (Figure 1). BrY induced significntly higher levels of IL-1 thn BiY in 8 out of the 9 PBMNC preprtions. Incution with BrY lso elicited significntly higher levels of IL-1 thn tht with LPS (1 µg/ml) in 6 out of the 9 PBMNC preprtions.,d No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 Figure 1 Effects of proiotic ifidocteri on IL-1 production in PBMNC. PBMNC were isolted from 9 ulcertive-colitis ptients nd incuted with het-killed proiotic BiY or BrY (1 µg/ml), or LPS. At forty-eight hours fter incution, the IL-1 concentrtion ws determined y ELISA (men ± SD, n = 3). P <.5, P <.1 vs BiY; c P <.5, d P <.1 LPS.,d,d,c,c B. ifidum B. reve Control 1 1/2 1/5 1/1 Figure 3 Effects of proiotic ifidocteri-derived CMs on IL-8 production in HT-29 cells. HT-29 cells were stimulted with TNF-α in the sence or presence of grded dilutions of CMs. CMs were prepred s descried in the Methods. Other experimentl conditions re shown in Figure 2. P <.5, P <.1 vs control. Effect of proiotic Bifidocterium on IL-8 secretion in TNF--stimulted HT-29 cells HT-29 cells were incuted with TNF-α for six hours in the presence or sence of het-killed BiY nd BrY. Neither of the het-killed proiotic cteri hd n effect on the secretion of IL-8 t the concentrtion rnging from 1 µg/ml- 1 µg/ml (Figure 2). Next, we exmined the effect of conditioned medium (CM) on IL-8 secretion y HT-29 cells. Both CMs of BiY nd BrY inhiited TNF-α-induced secretion of IL-8 in dose-dependent mnner (Figure 3). Concentrtions of BiY nd BrY cultured over 16 h in RPMI-164 medium were CFU/mL nd CFU/mL, respectively. Nture of the inhiitory effect on IL-8 secretion in proiotic-derived CM To investigte the nture of this solule fctor, BiY-derived CM ws sujected to moleculr sieve nd het tretment. The frctions of less thn nd more thn 3 kd oth retined the inhiitory ctivity towrd the secretion of IL-8 in HT-29 cells, though the former frction ws greter thn the ltter one (Figure 4). The inhiitory effect of the ltter frction ut not the former one ws diminished y hettretment (dt not shown). We checked the effect of cetic nd lctic cid with their mjor constituents in less thn 3 kd frction. Acetic cid ut not lctic cid inhiited the IL-8 secretion in HT-29 cells, when dded t the sme

4 2514 ISSN CN /R World J Gstroenterol April 28, 28 Volume 14 Numer IL-8/GAPDH IL-8 IkB-z/GAPDH IkB-z TNF TNF + CM Medium BiY CM > 3 kd BiY CM < 3 kd BiY CM Control Figure 4 Assessment of the moleculr weight of ctive component in proioticderived CMs. CM ws seprted into frctions of more thn nd less thn 3 kd, djusted to the initil volume. The inhiitory effect on the secretion of IL-8 ws determined s descried in Figure 2. P <.1 vs control t /h t /h Figure 6 IL-8 nd IκB-ζ mrna expression during the culture of HT-29 cells in the presence of proitic-derived CMs. The mrna expression ws quntified s descried in Mterils nd Methods. Results re representtive of three seprte experiments A DNA (mg/ml) B. ifidum B. reve L. cidophilus Acetic cid + Lctic cid Acetic cid Lctic cid BiY CM Control B 15 P <.1 P <.1 DNA DNA + DNse Figure 5 Effects of cetic cid nd/or lctic cid on IL-8 secretion in HT-29 cells. HT-29 cells were incuted with pproximtely the sme concentrtion of cetic cid (11 mmol/l) nd/or lctic cid (4 mmol/l) to tht of CMs. The inhiitory effect on the secretion of IL-8 ws determined s descried in Figure 2. P <.5, P <.1 vs control. 1 5 concentrtion s in the CM, 11 mmol/l nd 4 mmol/l, respectively (Figure 5). IL-8 nd IκB-ζ expression during incution with proiotic ctei in HT-29 cells MRNA levels of IL-8 nd IκB-ζ were determined in TNFα-stimulted HT-29 cells in the presence or sence of BiY-derived CM (Figure 6). A repression of IL-8 expression occurred with n up-regultion of IκB-z expression 4 h fter incution of HT-29 cells with proiotic-cm. Proiotic DNA inhiited epithelil secretion of IL-8 HT-29 cells were stimulted with TNF-α in the presence or sence of cteril DNA isolted from BiY or BrY. DNA from oth BiY nd BrY significntly inhiited the IL-8 secretion in HT-29 cells, t the concentrtion rnging from 1 µg/ml-1 µg/ml, while DNA from L. cidophilus (YIT 168) hd no effect (Figure 7A). Tretment of proiotic DNAs with DNse olished their inhiitory effects on the secretion of IL-8 (Figure 7B). DISCUSSION The efficcy of ifidocteri-fermented milk (BFM) in the tretment of ulcertive colitis hs een reported B. ifidum B. reve Control Figure 7 Effects of proiotic DNAs on IL-8 production in HT-29 cells. HT-29 cells were stimulted with TNF-α in the sence or presence of vrious concentrtions of proiotic DNA without (A) or with DNse tretment (B). The inhiitory effect on IL-8 secretion ws determined s descried in Figure 2. P <.5, P <.1 vs control. elsewhere [7,8]. To determine the nti-inflmmtory ctivity of proiotic Bifidocterium strins in the BFM, we firstly investigted the effects of these strins on the production of nti-inflmmtory cytokine IL-1 y PBMNC isolted from UC ptients in vitro. IL-1 down regultes the TNF-α secretion y mcrophges [26]. IL-1 knockout mice develop IBD under conventionl conditions, indicting the importnce of IL-1 in the control of intestinl inflmmtion [27]. It ws reported tht intrgstric dministrtion of IL-1- secreting Lctococcus lctis cuses 5% reduction in colitis in mice treted with dextrn sulfte sodium [14] nd BFM contining BiY nd BrY meliortes IBD in SAMP1/Yit mice with n up-regultion of IL-1 synthesis nd downregultion of IFN-γ production in mesenteric lymph node cells [28]. In this study, the two het-killed proiotic cteril strins in BFM induced the secretion of IL-1 in PBMNC isolted from UC ptients.

5 Imok A et l. Anti-inflmmtory ctivity of proiotics 2515 It hs een found tht the degree of locl inflmmtion nd tissue dmge in ptients with IBD is dependent on the locl expression of specific chemokines within ffected tissues [16]. IL-8 protein [17] nd mrna [18] expression re ssocited with inflmmtion in UC. The fct tht conditioned medi of BiY nd BrY reduced the TNFα-induced secretion of IL-8 y HT-29 cells suggests tht oth proiotic strins in the BFM could produce solule nti-inflmmtory fctors tht inhiit IL-8 secretion in intestinl epithelil cells. No single fctor produced y the proiotic strin seems to e responsile, ecuse the frctions of less thn nd more thn 3 kd, differing in hetsensitivity, were found to hve inhiitory effects on IL-8 secretion in this study. Acetic cid ut not lctic cid, mjor component of CM, ws likely to lrgely contriute to the inhiitory ctivity of the less thn 3 kd frction. Components responsile for the inhiition of IL-8 in the more thn 3 kd frction remin to e investigted. IκB-ζ is n inducile nucler fctor-κb (NF-κB) regultor, which is ssocited with the p65 nd p5 suunits of NF-κB nd inhiits trnscription s well s the DNAinding of the trnscription fctor [29,3]. Incresed expression of IκB-ζ mrna in the presence of proiotic-derived CM ws oserved with repression of IL-8 gene expression in HT-29 cells compred to tht in the sence of CM, indicting tht NF-κB-medited IL-8 gene expression in HT-29 cells is inhiited through the induction of IκB-ζ expression y the CM of the Bifidocterium strin. The present study hs shown tht the Bifidocterium strins proved to e eneficil in inducing nd mintining remission of ulcertive colitis exhiit regultory ctivities tht contriute to the control of intestinl inflmmtion, thus unrveling the iologicl sis of the eneficil effects of proiotics in humn disese. ACKNOWLEDGMENTS The uthors re grteful to Dr. Pen AS (VU University Medicl Center Amsterdm) for criticl reviewing of the mnuscript nd to Dr. H Ishikw H (Kyoto Prefecturl University of Medicine) for helpful discussion nd dvice regrding the mnuscript. COMMENTS Bckground We hve previously shown tht tretment with ifidocteri-fermented milk (BFM) contining live ifidocteri is more effective thn usul tretment in inducing nd mintining remission of ulcertive colitis (UC). We hypothesized tht proiotic strins of Bifidocteri in BFM my interfere with the intestinl inflmmtory response s well s prevention of the overgrowth of potentilly pthogenic cteri such s Bcteroides vulgtus. In this study, we focused on the effect of proiotic strins on the secretion of IL-1 y peripherl lood mononucler cells (PBMNC) nd lso the production of IL-8 y intestinl epithelil cells. Reserch frontiers There is evidence tht proiotic tretment is effective in ptients with UC. The precise mechnisms y which proiotic microorgnisms used in clinicl trils exert their eneficil effect hve not een well defined yet. Innovtions nd rekthroughs This study hs shown tht Bifidocterium strins tht proved to e eneficil in inducing nd mintining remission of ulcertive colitis exhiit regultory ctivities tht contriute to the control of intestinl inflmmtion, thus unrveling the iologicl sis of the eneficil effects of proiotics in humn disese. Applictions Anti-inflmmtory ctivity of proiotic ifidocteri shown in this study supports the eneficil effects of BFM on in clinicl trils. Therpeutic pplictions of BFM in the tretment of UC re promising. Peer review Proiotic Bifidocterium strins in BFM tht proved to e eneficil in inducing nd mintining remission of UC enhnced IL-1 production in PBMNC nd inhiited IL-8 secretion in intestinl epithelil cells, suggesting tht BFM hs nti-inflmmtory effects ginst UC. The mechnism of ction regrding the immunosuppressive effect of proiotics hs wider therpeutic implictions for the tretment of inflmmtory owel disese in generl. REFERENCES 1 Srtor RB. Therpeutic mnipultion of the enteric microflor in inflmmtory owel diseses: ntiiotics, proiotics, nd preiotics. Gstroenterology 24; 126: Cmpieri M, Gionchetti P. Proiotics in inflmmtory owel disese: new insight to pthogenesis or possile therpeutic lterntive? Gstroenterology 1999; 116: Kruis W, Fric P, Pokrotnieks J, Luks M, Fix B, Ksck M, Kmm MA, Weismueller J, Beglinger C, Stolte M, Wolff C, Schulze J. Mintining remission of ulcertive colitis with the proiotic Escherichi coli Nissle 1917 is s effective s with stndrd meslzine. Gut 24; 53: Biiloni R, Fedork RN, Tnnock GW, Mdsen KL, Gionchetti P, Cmpieri M, De Simone C, Srtor RB. VSL#3 proioticmixture induces remission in ptients with ctive ulcertive colitis. 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6 2516 ISSN CN /R World J Gstroenterol April 28, 28 Volume 14 Numer 16 etween in vitro nd in vivo immunomodultory properties of lctic cid cteri. World J Gstroenterol 27; 13: Steidler L, Hns W, Schotte L, Neirynck S, Oermeier F, Flk W, Fiers W, Remut E. Tretment of murine colitis y Lctococcus lctis secreting interleukin-1. Science 2; 289: Brt H, Rottiers P, Hommes DW, Huygheert N, Remut E, Remon JP, vn Deventer SJ, Neirynck S, Peppelenosch MP, Steidler L. A phse I tril with trnsgenic cteri expressing interleukin-1 in Crohn's disese. Clin Gstroenterol Heptol 26; 4: Bnks C, Btemn A, Pyne R, Johnson P, Sheron N. Chemokine expression in IBD. Mucosl chemokine expression is unselectively incresed in oth ulcertive colitis nd Crohn's disese. J Pthol 23; 199: Izzo RS, Witkon K, Chen AI, Hdjiyne C, Weinstein MI, Pellecchi C. Interleukin-8 nd neutrophil mrkers in colonic mucos from ptients with ulcertive colitis. Am J Gstroenterol 1992; 87: Ktsut T, Lim C, Shimod K, Shiut K, Mitr P, Bnner BF, Mori M, Brnrd GF. Interleukin-8 nd SDF1-lph mrna expression in colonic iopsies from ptients with inflmmtory owel disese. Am J Gstroenterol 2; 95: Umehr Y, Kudo M, Nkok R, Kwski T, Shiomi M. Serum proinflmmtory cytokines nd dhesion molecules in ulcertive colitis. Heptogstroenterology 26; 53: Reddy KP, Mrkowitz JE, Ruchelli ED, Bldssno RN, Brown KA. Lmin propri nd circulting interleukin-8 in newly nd previously dignosed peditric inflmmtory owel disese ptients. Dig Dis Sci 27; 52: Ysui H, Ngok N, Hykw K. Augmenttion of ntiinfluenz virus hemgglutinin ntiody production y Peyer's ptch cells with Bifidocterium reve YIT464. Clin Dign L Immunol 1994; 1: Menrd S, Cndlh C, Bmou JC, Terpend K, Cerf-Bensussn N, Heymn M. Lctic cid cteri secrete metolites retining nti-inflmmtory properties fter intestinl trnsport. Gut 24; 53: Mdsen K, Cornish A, Soper P, McKigney C, Jijon H, Ychimec C, Doyle J, Jewell L, De Simone C. Proiotic cteri enhnce murine nd humn intestinl epithelil rrier function. Gstroenterology 21; 121: Yuki N, Shimzki T, Kushiro A, Wtne K, Uchid K, Yuym T, Morotomi M. Coloniztion of the strtified squmous epithelium of the nonsecreting re of horse stomch y lctocilli. Appl Environ Microiol 2; 66: Truelove SC, Witts LJ. Cortisone in ulcertive colitis; finl report on therpeutic tril. Br Med J 1955; 2: Schreier S, Heinig T, Thiele HG, Redler A. Immunoregultory role of interleukin 1 in ptients with inflmmtory owel disese. Gstroenterology 1995; 18: Kuhn R, Lohler J, Rennick D, Rjewsky K, Muller W. Interleukin-1-deficient mice develop chronic enterocolitis. Cell 1993; 75: Mtsumoto S, Wtne N, Imok A, Oke Y. Preventive effects of Bifidocterium- nd Lctocillus-fermented milk on the development of inflmmtory owel disese in senescence-ccelerted mouse P1/Yit strin mice. Digestion 21; 64: Ymzki S, Mut T, Tkeshige K. A novel IkppB protein, IkppB-zet, induced y proinflmmtory stimuli, negtively regultes nucler fctor-kppb in the nuclei. J Biol Chem 21; 276: Totzke G, Essmnn F, Pohlmnn S, Lindenltt C, Jnicke RU, Schulze-Osthoff K. A novel memer of the IkppB fmily, humn IkppB-zet, inhiits trnsctivtion of p65 nd its DNA inding. J Biol Chem 26; 281: S- Editor Zhong XY L- Editor Wng XL E- Editor Liu Y