Važnost mikrobiote u očuvanju zdravlja
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1 Važnost mikrobiote u očuvanju zdravlja Donatella Verbanac Mihaela Perić, Hana Čipčić Paljetak, Mario Matijašić, Marina Panek, Ivana Kraljević, Dunja Rogić, Dina Ljubas Kelečić, Darija Vranešić Bender i Željko Krznarić
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3 Personalizirana medicina Mnogo je važnije dobro upoznati onoga tko boluje od pojedine bolesti, nego potpuno dokučiti bolest od koje netko boluje. Hipokrat
4 Schwartz MW, Morton GJ. Nature Aug 8;418(6898): HRANA + O 2 CO 2 + H 2 O + E
5 Ljudska mikroflora Mikroorganizmi koji nastanjuju dijelove ljudskog tijela nazivaju se skupno mikrobiota Koža, dišni trakt, usta, crijevo, urogenitalni trakt... Većinu mikrobiote čine bakterije (sve stanice ljudskog organizma ) Crijevna mikrobiota ekosistem mikroorganizama koji koloniziraju GI trakt i žive u simbiozi sa svojim domaćinom Zavisno od metode detekcije bakterijskih vrsta 99% tih organizama su anaerobi Metagenomika analiza ukupnog genetskog materijala nekog kompleksnog ekosistema u svom prirodnom sastavu Ljudski genom mikrobiom Ljudskih gena Mikrobnih gena >
6 Mikrobiota i mikrobiom Human microbiome project (NIH, SAD) Ljudski organizam: 10% humanih stanica 90% bakterijskih stanica Promjena unutar mikrobioma u vezi je sa zdravljem i bolesti The international MetaHIT (Metagenomics of the Human Intestinal Tract) project has published a gene catalogue of the human gut microbiome derived from 124 healthy, overweight and obese human adults, as well as inflammatory disease patients, from Denmark and Spain. The data provide the first insights into this gene set - over 150 times larger than the human gene complement - and permit the definition of both a minimal gut metagenome and a minimal gut bacterial genome. Project Coordinator Prof. S.D. Ehrlich Mikrobiom u crijevima = 150x brojniji od humanog genoma! Mikrobiota = humani organ! Nat Biotechnol Aug;32(8):
7 Metode istraživanja mikrobiote Filogenetska analiza Sekvenciranje gena za 16S rrna Sastav, relativna zastupljenost Metagenomika Sekvenciranje genomske DNA Analiza setova gena funkcionalna metagenomika Filogenetski odnosi Metatranskriptomika Analiza cjelokupne mrna, uvid u diferencijalnu ekspresiju gena Metaproteomika Analiza ukupnih proteina različita ekspresija ovisno o uvjetima Metabolomika Profiliranje metabolizma domaćina i mikrobiote Lepage, P., Gut, 2013
8 Uloga mikrobiote Zaštitna Otklanja patogene bakterije Kompeticija za hranjiva Kompeticija za receptore Lučenje antimikrobnih čimbenika Strukturna Prirodna granica Tight junctons među stanicama epitela Razvoj imunog sustava Sinteza IgA Metabolička Sinteza kratkolančanih MK Metabolizam UH iz hrane Sinetza vitamina K, folata i biotina Apsorpcija Fe 2++ Mowat and Agace, Nat.rev. Immunol. 2014
9 Usvajanje mikrobiote kod novorođenčadi Reid Nature Rev Microbiol, 2011
10 Razvoj mikrobiote u najranijoj dobi majčino mlijeko groznica rižine pahuljice dohrana i kruta hrana amoksicili n cefdinir, kravlje mlijeko i prehrana odraslih Prestanak dojenja i dohrane Čimbenici koji moduliraju crijevnu mikrobiotu kod novorođenčadi POZITIVNI vaginalni porod terminski porod dojenje izlaganje mikroorganizmima NEGATIVNI porod na carski rez terapija antibioticima dohrana prijevremeni porod Koenig PNAS 108, (2011)
11 Razvoj mikrobiote do kraja puberteta Novorođenče 3 tj 1 godina 1-7 godina 2-18 godina Odrasla osoba Bifidobacteria Bacterioide Staphilococcus Lactobacillus Bifidobacteria Clostridium Počinje se formirati mikroflora odrasle osobe Stabilni odnos Bifidobacteria i Lactobacillus Stabilna flora tijekom dužeg razdoblja
12 Bolesti povezane s promjenom mikrobiote Odnos crijevo-mozak 1. Autizam 2. Raspoloženje Astma Atopičke bolesti Hipertenzija Ishemija srca HIV Nekrotizirajući enterokolitis Karcinom debelog crijeva Celijakija Žučne bolesti Reumatoidni artritis Promijenjen metabolizam ksenobiotika/lijekova Bolesti perfienih žila Pretilost Metabolički sindrom Dijabetes IBD Iritabilini kolon Gastroenteritis Sanders ME, J Clin Gastroenterol 45 (2011)
13 Utjecaj na mikrofloru Čimbenici utjecaja Vanjski Unutarnji Terapija antibioticima Pretjerana higijena Emocionalni stres Starenje Putovanje Poremećaji peristaltike Kirurške operacije Bolesti jetre i bubrega Radijacijska terapija Kemoterapija Anemija Poremećaji imunog sustava Dostupnost nutrijenata Tip prehrane ph u crijevima Redoks potencijal Proljev Bakterijski antagonizam Bakterijska kooperacija Mucin Lizozim Defensini
14 Intervencije na mikrobiotu Prehrambene Probiotici Lactobacillus, Bifidobacterium, Bacillus Prebiotici Polioli - ksilitol, sorbitol, manitol Disaharidi (laktuloza) oligosaharidi (rafinoza, oligofruktoza) polisaharidi (inulin) Oligosaharidi majčinog mlijeka Simbiotici Novija istraživanja Bolja selekcija pojedinih vrsta GMO koji stvaraju protuupalne spojeve, helminti Transplantacija fekalne mikrobiote Translacijski i holistički pristup Weinstick Eliott,Int. J. Parasitol 2013; Scholmerich, Dig Dis 2013.; Ianiro et al, Medicine, 2014.
15 Iznimni probiotički pripravci Probiotički pripravci s najviše sojeva dobrih bakterija Probiotički pripravci s posebnim obilježjima formulacijama i sinergističkim principima Probiotički pripravci s posebnim obilježjima naseljavanja sluznica Probiotički pripravci s najvećim brojem bakterija Postoje dokazane kliničke studije
16 Mikrobiota, prehrana i debljina Istraživanja odnosa crijevne mikrobiote, prehrane i debljine Kod eksperimentalnih životinja Genetički pretili miševi Humanizirani miševi Kod vitkih i pretilih osoba Razlike u sastavu, složenosti i broju gena Utjecaj prehrane na crijevnu mikrobiotu Intervencije u sastav crijevne mikrobiote Mehanizmi putem kojih crijevna mikrobiota utječe na dobitak tjelesne težine Clarke, S.F., Gut Microbes, 2013
17 Promjena sastava mikrobiote - pretilost Istraživanja su pokazala da se udio bakterija u crijevu mijenja promjenom tjelesne mase: Povećanje Bacteroidetes i smanjenje Firmicutes mršavljenjem (Nadal, 2008) Kod pretile djece više Firmicutes (Kalliomaki 2008) Smanjenje Firmicutes nakon barijatričke kirurgije i mršavljenja (Zhang, 2009), te povećanje Bacteroides/Prevotella (Furet, 2010) Mikrobiom kod pretilih ima nižu raznolikost (prilagodba na jednoličan okoliš tj. hranu) (Greenblum, 2012) Bacteroidetes Debljanje Bacteroidetes Druge bakterije Firmicutes Mršavljenje Druge bakterije Firmicutes Tsukumo, 2009
18 Prehrana i metabolizam Međudjelovanje prehrane i mikrobiote utječe na metabolizam domaćina Kovatcheva-Datchary, 2013
19 Projektni tim - MINUTE for IBD Assessment of Microbiota, Inflammatory Markers, Nutritional and Endocrinological Status in IBD Patients - HRZZ Medicinski fakultet SuZ- Mihaela Perić, Mario Matijašić, Hana Čipčić Paljetak, Marina Panek, Vera Kufner, Donatella Verbanac Imperial College London, UK - Anja Barešić KBC Zagreb - Željko Krznarić, Silvija Čuković Čavka, Darija Vranešić Bender, Marko Brinar, Mirjana Kalauz, Dora Grgić, Nikša Turk, Ivana Kraljević, Dunja Rogić, Dina Ljubas Kelečić, Ana Kunović KB Dubrava - Marija Crnčević Urek, Marko Banić Poliklinika Dr. Zora Profozić Tomislav Meštrović Fidelta d.o.o. - Karmen Brajša Xellia Pharmaceuticals - Gabrijela Ergović
20 Dosadašnji rezultati Do sada je regrutirano 63 ispitanika (16 s IBD dijagnozom) Analiza rezultata sekvenciranja uzoraka fecesa na Illumina platformi je provedena Pilot analiza sekvenciranja uzoraka tkiva ukazuje na razlike u relativnoj zastupljenosti pojedinih bakterijskih razreda kod oboljelih u odnosu na kontrolne ispitanike Preliminarne analize upalnih biomarkera iz seruma upućuju na postojanje značajnih razlika među grupama
21 Zbirna analiza podaci sumirani prema dijagnozi Crohn, UC i zdravi ispitanici (H) n=5, IBS n=27
22 Crohn i IBS n=1 Crohn IBS 1. terminal ileum i caecum 2. colon ascendens 3. colon transversum 4. colon descendens 5. colon sigmoideum 6. rectum
23 Proseek Multiplex Inflammation I panel (92 proteinska markera) Crohn (CD) n=4, Ulcerozni kolitis (UC) n=6, IBS n=32 i zdravi (H) n=6 H v UC H vs CD H vs IBS UC vs CD UC vs IBS CD vs IBS IL-7 * AXIN * * ** TRAIL * * CST-5 * TNFSF14 **** *** **** CXCL5 ** ** MMP-10 * CCL23 * * CXCL10 * EN-RAGE * ** ** CASP-8 * * TNFRSF9 ** TWEAK * ST1A1 ** ** **** ADA * * * <0.05, ** <0.005, *** <0.0005, **** <0.0001
24 Dolazi se i do novih spoznaja MIKROBIOTA I REGULACIJA KOŠTANE MASE
25 Journal of Bone and Mineral Research, Vol. 27, No. 6, June 2012, pp Trends Endocrinol Metab Feb;26(2):69-74.
26 Novija istraživanja Journal of Bone and Mineral Research, Journal of the American College of Nutrition 2017.
27 Mogući mehanizam djelovanja Trends Endocrinol Metab Feb;26(2): Claes Ohlsson i Klara Sjögren (2015) Mikrobiota crijeva (MC) jedan je od predloženih regulatora koštane mase Nedavna istraživanja na GF miševima to potvrđuju Međuodnos je posredovan imunološkim sustavom odgovornim za regulaciju osteoklastogeneze Prehrana i okolišni čimbenici utječu na sastav MC. Intervencijska istraživanja autora pokazala su da antibiotici, probiotici i prebiotic koji mijenjaju MC, reguliraju i metabolizam kosti Vlakna (prebiotici) djeluju pozitivno na MC, potiču sintezu kratko lančanih masnih kiselina, povećavaju apsorpciju kalcija, povećavaju gustoću kostiju i utječu na zdravlje kostiju. Primjena bakterija (probiotika, Lactobacillusi) u pred kliničkim studijama utječe na veću mineralizaciju i snagu kostiju. We propose a new cross-disciplinary GM bone research field called osteo-microbiology, bridging the gaps between bone physiology, gastroenterology, immunology, and microbiology. Future studies are clearly warranted in this research field to determine if the GM composition might be used as a biomarker for fracture risk prediction and to validate the GM as a possible novel therapeutic target for osteoporosis.
28 Pregled studija koje povezuju mikrobiotu crijeva i zdravlje kostiju. Glavni zaključci Autori MC regulira koštanu masu kod miševa preko imunološkog sustava, djelujući na osteoklaste koji igraju glavnu ulogu u razgradnji kostiju. Promicanjem koštanog rasta, Lactobacillus reuteri održava zdravlje kostiju kod muškaraca,ali ne i kod žena. Probiotici utječu na zdravlje kostiju redukcijom upalnih citokina i povećanjem aktivnosti osteoprotegerina (OPG). Lactobacillus reuteri smanjuje gubitak koštane mase sprječavanjem aktivnosti osteoklasta i manipulacijom mikrobiote crijeva. (Sjogren et al., 2012) (McCabe et al.,2013) (Ohlsson et al., 2014) (Britton et al., 2014) Promjena sastava mikrobiote crijeva tijekom rasta može dovesti do velikih promjena u masi kostiju. (Guss et al., 2017) Villa et al., Food Science and Nutrition 2017
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30 Mikrobiota i imunološki/metabolički sustav Jesu li naša crijeva zapravo igralište na kojem se odvija stalna utakmica između imunološkog sustava i mikrobiote? Razvoj intestinalnog epitela Reguliraju funkciju Utječu na propusnost Razvoj imunološkog sustava Lokalno Sistemski Metaboličke bolesti Prerada ugljikohidrata u kratko lančane masne kiseline, metabolizam dušičnih spojeva, aktivacija vitamina B skupine i sinteza i sazrijevanje vitamina K Resta SC. J Physiol Hooper, Nat Rev Microbiol 2009
31 Kliničke studije koje ukazuju na korist od uzimanja probiotika Probiotik Način korištenja Učinak L. rhamnosus, B. animalis Oralni probiotik/1x, 256 trudnica, 1 tromj. Smanjenje gastacijskog dijabetesa L. rhamnosus Oralni dodatak probiotika, 6-mjesečne bebe B. longum, B. animalis, L. acidophillus Oralni probiotik, trudnice/dojilje/dojenčad1-2 mj. prije i 6 mj. nakon poroda Povećanje otpornosti na perturbacije mikrobiote i prerastanje patogena, sprječavanje alergijskih bolesti Smanjena učestalost ekcema kod novorođenčadi L. rhamnosus, L. reuteri 5 dana/1x/vaginalete Veća uspješnost izlječenja bakterijske vaginoze nego s metronidazolom Reid Nature Rev Microbiol 2011
32 Očuvanje bakterijske homeostaze - mogući mehanizmi djelovanja probiotika Ko-agregacija Stvaranje biosurfaktanata Stvaranje bakteriocina i H 2 O 2 Bakterijski signali Bakteriocini Kompetitivno isključivanje Imunomodulacija Modulacija tight junctions Alkalna fosfataza Defenzini i lizozim LPS laktobacili nepatogeni koki bifidobakterije G+ patogeni G- patogeni NF-kB-ovisni antimikrobni geni makrofag Reid Nature Rev Microbiol (2011)
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