Bi³polis - Scientific Dossier Probiotic ES1

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Cereal gluten proteins (gliadin α, β, γ, and ω) are the main environmental factors causative of coeliac disease pathogenesis. The incomplete hydrolysis of gliadin by human enzymes during gastrointestinal digestion leads to the generation of peptides able to trigger cytotoxic and inflammatory signals in intestinal epithelial cells of coeliac disease patients. Clinical manifestations of the disease often include intestinal symptoms and nutrient malabsorption associated with severe mucosal damage. Currently, the only available therapy for coeliac disease patients is the adherence to a strict life-long gluten-free diet. However, compliance with this dietary recommendation is complex, and other alternative or adjuvant strategies are needed. Alterations in the composition of the gut microbiota of coeliac patients are characterized by decreased Bifidobacterium numbers (Nadal et a!., 2007; Sanz et a!., 2007, Collado et a!., 2008; 2009; De Palma et a!., 2010). The association of imbalances in the intestinal microbiota and the positive reported roles that bifidobacteria play on intestinal health have led to the proposed use of probiotics as part of additional and alternative nutritional strategies for improving the quality of life of coeliac patients. The strain Bifidobacterium !ongum ES1 (CECT 7347) is capable to reduce the toxicity and inflammatory potential of gliadin-derived peptides and is an excellent candidate as probiotic for coeliac disease patients.

Transcript of Bi³polis - Scientific Dossier Probiotic ES1

!!!!!!!!"#$%&'$($#!)*""$%+!,+*-$*'$#!%".!/#%#'!01203!!!!!!!!!!! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!!! 2 .3!5%&%+46!-4#75+*8&)! Ceieal gluten pioteins (gliauin , , , anu ) aie the main enviionmental factois causative of coeliac uisease pathogenesis. The incomplete hyuiolysis of gliauin by human enzymes uuiing gastiointestinal uigestion leaus to the geneiation of peptiues able to tiiggei cytotoxic anu inflammatoiy signals in intestinal epithelial cells of coeliac uisease patients. Clinical manifestations of the uisease often incluue intestinal symptoms anu nutiient malabsoiption associateu with seveie mucosal uamage. Cuiiently, the only available theiapy foi coeliac uisease patients is the auheience to a stiict life-long gluten-fiee uiet. Bowevei, compliance with this uietaiy iecommenuation is complex, anu othei alteinative oi aujuvant stiategies aie neeueu. Alteiations in the composition of the gut miciobiota of coeliac patients aie chaiacteiizeu by uecieaseu !"#"$%&'()*+",- numbeis (Naual *). '/., 2uu7; Sanz *).'/., 2uu7, Collauo *). '/., 2uu8; 2uu9; Be Palma *). '/., 2u1u). The association of imbalances in the intestinal miciobiota anu the positive iepoiteu ioles that bifiuobacteiia play on intestinal health have leu to the pioposeu use of piobiotics as pait of auuitional anu alteinative nutiitional stiategies foi impioving the quality of life of coeliac patients. The stiain !"#"$%&'()*+",-. /%01,- ES1 (CECT 7S47) is capable to ieuuce the toxicity anu inflammatoiy potential of gliauin-ueiiveu peptiues anu is an excellent canuiuate as piobiotic foi coeliac uisease patients. 93!$"*64'$*&!*(!':%!,+*-$*'$#!"'+4$&!!"#"$%&'()*+",-./%01,-!%".! The piobiotic stiain ES1 was isolateu by the gioup of Bi. Yolanua Sanz (Institute of Agiochemistiy anu Foou Technology of the Spanish National Reseaich Council; IATA-CSIC) fiom faeces of healthy babies unuei bieast-milk feeuing. ! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!!! S In oiuei to classify this bacteiial stiain, an almost full fiagment of the 16S iRNA encouing gene was amplifieu anu sequenceu using the methouology pieviously uesciibeu by Kaufmann anu cowoikeis (1997). BNA fiom puie cultuie was extiacteu anu aujusteu to a final concentiation of 4u ngl in ultiapuie watei. The BNA was checkeu foi puiity, using stanuaiu methous. BNA templates weie amplifieu by PCR using piimeis LNS (S'-Cuu uTu CuT uCC CAC TTT CAT u-S') anu LN26 (S'-uAT TCT uuC TCA uuA TuA ACu-S') amplifying a 1SSu-bp iegion of the 16S iRNA gene (}ohnson 1994; Satokaii *). '/., 2uu1; Faviei *). '/. 2uu2). Contiols uevoiu of BNA weie simultaneously incluueu in the amplification piocess. The integiity of the PCR piouucts was assayeu by uetection of single banus following electiophoiesis. Amplicons weie puiifieu using a commeicial kit anu subsequent sequencing ieactions weie peifoimeu using ABI S7uu equipment. The iesulting sequence was automatically aligneu anu inspecteu by eye anu compaieu by use of the online tool BLAST (http:blast.ncbi.nlm.nih.govBlast.cgi). The stiain was iuentifieu on the basis of highest scoies as a membei of the species !"#"$%&'()*+",-./%01,- anu was uepositeu at the Spanish Type Cultuie Collection (CECT) unuei the accession numbei 7S47 (Izquieiuo *).'/. 2uu8). 13!,+*-$*'$#!,+*,%+'$%"!*(!':%!"'+4$&!%".! 0ne of the majoi iequiiements foi a piobiotic stiain is that it shoulu maintain viability uuiing gastiointestinal tiansit. To evaluate this phenotype, the ES1 stiain was checkeu foi its iesistance to aciu pBs anu bile salts. To analyze pB iesistance, cell suspensions in 1u mN phosphate-buffeieu saline (pB 7.2), with a cell uensity of 1u9 cellml, weie uiluteu 1u-folu in steiile saline solution containing S mgml pepsin fiom poicine stomach mucus anu aujusteu to uiffeient pB values (1.S, 2.S, 2.u, anu S.u) with BCl. Aliquots weie taken aftei u anu 9u minutes of incubation at S7C. uiowth ability was ueteimineu by plate counting on NRSC agai. viability changes weie ueteimineu using the LIvEBEAB BacLight ! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!!! 4 Kit foi micioscopy uetection accoiuing the manufactuiei's pioceuuie. The counts of both live (gieen fluoiescence) anu ueau (ieu fluoiescence) bacteiia weie ueteimineu. The iesults inuicate a moueiate sensitivity of the ES1 stiain to gastiic juices at pB 2 anu iesistance to highei pB values. These values of iesistance weie highei than those obtaineu foi some commeicial piobiotic stiains in paiallel expeiiments (uata not shown). ;:!3! ,49! ,4R! 11SKET!!2./%01,-!%".! S6.S u.S S.6 !'BCDE!2@!Begiauation of synthetic gliauin peptiues (%) by the ES1 stiain. The iesults aie expiesseu as the aveiage peak aiea of the chiomatogiaphic signal foi each peptiue ielative to peptiue sample non-inoculateu with bacteiia. Expeiiments weie uone in tiiplicate U3! $&:$-$'$*&! *(! ':%! $&(64GG4'*+=! +%",*&"%! -=! ':%!,+*-$*'$#!%".!"'+4$&! The effect of co-incubation of the ES1 piobiotic stiain with faeces of 26 coeliac patients with active uisease (mean age S.S yeais, iange 2.1-12.u yeais), 18 symptom-fiee coeliac uisease patients (mean age S.S yeais, iange 1.u- 12.S yeais) on a gluten-fiee uiet foi 1-2 yeais; anu 2u healthy chiluien (mean age S.S yeais, iange 1.8-1u.8 yeais) on inuuction of cytokine piouuction anu suiface antigen expiession in peiipheial bloou mononucleai cells (PBNCs) was ueteimineu. The conclusion of this woik inuicate that faeces of both, active coeliac anu symptom-fiee coeliac uisease patients, iepiesenting an imbalanceu miciobiota, significantly incieaseu TNF- piouuction anu CB86 expiession in PBNCs, while uecieaseu IL-1u cytokine piouuction anu CB4 expiession compaieu with contiol samples. Active CB-patient samples also inuuceu significantly highei IFN- piouuction compaieu with contiols. Bowevei, the !"#"$%&'()*+",-. ES1. stiain ! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!!! 8 suppiesseu the pio-inflammatoiy cytokine pattein inuuceu by the laige intestinal content of coeliac uisease patients anu incieaseu IL-1u piouuction. All these iesults have been publisheu in Neuina *).'/. (2uu8). In a seconu stuuy, the capacity of the stiain !"#"$%&'()*+",- ES1 to counteiact the inflammatoiy effects of gliauin-ueiiveu peptiues in intestinal epithelial Caco-2 cells was evaluateu. A commeicial extiact of seveial gliauin types was subjecteu to "0.5")+% gastiointestinal uigestion, inoculateu oi not with ES1 cell suspension, in a bicameial system. Inteiestingly, the peptiues fiom gliauin uigestions inoculateu with the bifiuobacteiial stiain uiu not exhibit the toxic amino aciu sequences iuentifieu in those noninoculateu. Aftei that, Caco-2 cell cultuies weie exposeu to the uiffeient gliauin peptiue uigestions (u.2S mg pioteinml), anu the mRNA expiession of NF-kB, TNF- anu chemokine CXCRS ieceptoi weie analyzeu by RT-PCR in stimulateu cells. The piouuction of the pio-inflammatoiy maikeis NF-k pSu, TNF-, anu IL-1 by Caco-2 cells was also ueteimineu by ELISA. The RT-PCR analysis eviuenceu a uown-iegulation in mRNA expiession of pio-inflammatoiy biomaikeis in the case of the ES1 samples. Consistent with these iesults the piouuction of NF-k pSu, TNF-, anu IL-1 was ieuuceu in cell cultuies exposeu to gliauin uigestions inoculateu with the ES1 bifiuobacteiia stiain. All these iesults have been publisheu by Lapaiia anu Sanz (2u1u) veiy iecently, the changes in the pioteome of Caco-2 cells exposeu to the uiffeient gliauin peptiue uigestions have been stuuieu. Changes in the pioteome weie ueteimineu by 2BE anu NALBI-T0F (see the oiiginal aiticle in annex "ES1 piobiotic stiain scientific liteiatuie" foi moie expeiimental uetails). uliauins uigesteu without the ES1 stiain alteieu the expiession of a highei numbei of pioteins than in the piesence of the bacteiium (21 5*+4,4 9). These uiffeiential pioteins weie involveu in uisoiganization of cell cytoskeleton, inflammation, anu apoptosis. uliauins uigesteu in the piesence of the ES1 piobiotic stiain influenceu the piouuction of pioteins involveu in calcium homeostasis anu cell suivival anu function. Theiefoie, !2. /%01,- ES1 stiain might amelioiate gliauin toxicity anu mouify the iesponses of intestinal epithelial cells to the gliauin challenge. These iesults aie incluueu in the aiticle by 0livaies *).'/. (2u11). ! ! !,+*-$*'$#!"'+4$&!%".!/#%#'!01203!!! 9 Finally, the effects of !"#"$%&'()*+",- ES1 stiain has been evaluateu in an animal mouel of gliauin-inuuceu enteiopathy (Lapaiia *).'/., 2u12). The mouel is baseu on the use of newboin iats feu gliauin alone oi sensitiseu with inteifeion IFN- anu feu gliauin. In these expeiiments, jejunal tissue sections weie collecteu foi histological, NFkB mRNA expiession anu cytokine piouuction analyses. Leukocyte populations anu T-cell subsets weie also analyseu in peiipheial bloou samples anu the possible tianslocation of the bacteiium to uiffeient oigans was ueteimineu by plate counting. Finally, the composition of the colonic miciobiota was quantifieu by ieal-time PCR. The iesults inuicateu that feeuing gliauin alone ieuuceu enteiocyte height anu peiipheial CB4+ cells, but incieaseu CB4+FoxpS+ T anu CB8+ cells, while the simultaneous auministiation of the pio