Provider: - Institution: - Data provided by Europeana Collections- Probiotics are life microorganisms that, after administration in gastrointestinal (GI) tract, excert positive effects on host’s health. Host reaction to probiotic administration largely depands on ability of bacteria to closely interact with epithelial cells of GI mucosa. On the other hand, considering the unknown antigenic nature of administered bacteria, in immune-compromised host there is a risk of immune reaction of gut tissue which could lead to disturbance of mucosal barrier integrity and chronic inflammation in GI tract. The aim of the study was to examine the effects of the strain Lactobacillus fermentum BGHI14, which is alochtonous to GI tract of rats, after peroral administration in healthy rats and in rats with TNBS (trinitrobenzenesulfonate)-induced colitis. Methodologically, following parameters were examined in experiments: body masses of rats and expression of mRNA of inflammatory cytokines (IL-1β, TNFα, IL-17F), as well as markers of cellular stress (HSP70) and barrier integrity maintenanace (TJP1); next histological and biochemical analyses and microflora profiling were performed. Results of the study indicated immune-stimulating effects of BGHI14 in colon tissue after administration in GI tract of healthy rats in first 16 days of peroral treatment of rats. Although, histologically, with prolongation of BGHI14 treatment of rats for next 12 days, immune reaction in colon tissue had retreated, mRNA levels of proinflammatory IL-1β and TNFα cytokines remained elevated which points to the risk of uncontrolled inflammation in the case of subsequent colon epithelial barrier damage. In research it was shown that after immunostimulation of colon tissue with BGHI14, in the case of colitis induction, but with sequel of treatment with bacteria, transcription of Hsp70 gene was induced, in early (48 h) as well as late (7 days) acute phase of colitis. The increase of Hsp70 gene transcription, according to histological and inflammatory markers, as well as body mass changes during the experiment, was not followed by positive action of the strain. On the other hand, the cessation of treatment with BGHI14 after colitis induction had led to less pronounced body mass decrease compared to control colitic rats, but without concomitant reduction in inflammatory scores in colon tissue, at least in late acute phase of disease (seven days from induction). Based on the above, it could be concluded that mechanisms of adaptive immunity including memory T-cells might had been activated during interaction of BGHI14 with healthy colon tissue and those systems were induced after the injury in the case of continuous presence of same bacterial antigen. Contrary, therapeutic treatment with BGHI14 had brought about the protection in late acute colitis phase according to body mass changes as well as indicators of mucosal barrier disruption. Obtained results implicate that use of immune-stimulating probiotics immediately after colon tissue damage would represent effective approach in treatment of bowel inflammatory diseases, though more detailed research is needed in this area. Considering the importance of interaction of bacteria with gut mucosa in achieving its probiotic potential, in current research mechanisms for improvement of potential probiotic lactobacilli binding to rat’s GI mucosa were tested. In this respect the effects of mucinbinding domain containing protein (MbpL) from strain Lactococcus lactis subsp. lactis BGKP1-20 in ability of bacterial host to bind to ileal and colonic mucosa in vivo and ex vivo, as well as in assays in vitro with isolated mucin and mucin-producing HT29-MTX colonic cells were tested. Aside from using the homologous host, for functional tests we have used potential probiotic strain Lactobacillus salivarius BGHO1. Results have demonstrated that the expression of MbpL protein was not sufficient for binding of BGHO1 to gut mucosa, although it influenced positively the binding of BGKP1-20 to pig gastric mucin and HT29-MTX cells in culture. It is possible that in lactococcal strain BGKP1-20, phenotypic effects of MucBP protein expression are more pronounced compared to BGHO1 strain, because of presence of other potential adhesion factors synthesized in lactobacilli strains. Simultanously, utillizing the same methodology, the role of aggregation factor AggL from BGKP1-20 in binding to GI tract mucosal surfaces was tested. Expression of aggregation factor is commonly correlated to enhanced affinity of bacterial host to GI mucosa. Our results have demonstrated that, although, by increasing surface hydrophobicity, in lactococcal strains it could contribute to binding to colonic surface, the expression of aggregation factor generally aggravates binding of bacteria to surfaces exposed to dynamic peristaltic contractions such as those present in ileum. That way strains expressing aggregation factor could facilitate removal of pathogens but without directly affecting the host’s immune system.- Probiotici predstavljaju žive mikroorganizme koji, nakon unošenja u gastrointestinalni (GI) trakt, ostvaruju pozitivne efekte na zdravlje domaćina. Reakcija domaćina na unos probiotika u velikoj meri zavisi od sposobnosti bliske interakcije bakterija sa epitelnim ćelijama GI mukoze. S druge strane, s obzirom na nepoznatu antigenu prirodu unetih bakterija, kod imunokompromitovanog domaćina je prisutan rizik od imunske reakcije crevnog tkiva što može da dovede do narušavanja integriteta mukozne barijere i hronične inflamacije u GI traktu. Cilj istraživanja bio je ispitivanje efekta soja Lactobacillus fermentum BGHI14, alohtonog za GI trakt pacova, nakon peroralnog unosa u zdrave pacove i u pacove sa kolitisom indukovanim TNBS-om (trinitrobenzensulfonat). Metodološki, u eksperimentima su praćeni: telesna masa pacova i ekspresija iRNK za parametre inflamacije (IL-1β, TNFα, IL-17F), pokazatelje ćelijskog stresa (HSP70) i parametre uključene u održavanje integriteta mukoze (TJP1); zatim su rađene histološke i biohemijske analize i analiza sastava crevne mikroflore. Rezultati istraživanja su pokazali imunostimulišuće delovanje soja BGHI14 na nivou tkiva kolona nakon unosa u GI trakt zdravih pacova u prvih 16 dana peroralnog tretmana pacova. Iako se, histološki, sa produžetkom tretmana pacova sojem BGHI14 tokom narednih 12 dana, imunska reakcija u tkivu kolona povukla, nivoi iRNK za proinflamatorne IL-1β i TNFα citokine ostali su povišeni što ukazuje na rizik od nekontrolisane inflamacije u slučaju naknadnog oštećenja epitelne barijere kolona. U istraživanju je pokazano da je nakon imunostimulacije tkiva kolona sojem BGHI14, u slučaju indukcije kolitisa, ali uz nastavak tretmana sojem BGHI14 došlo do indukcije transkripcije iRNK za HSP70 protein, kako u ranoj (48 h) tako i u kasnoj (7 dana) akutnoj fazi kolitisa. Povećanje transkripcije Hsp70 gena, sudeći po histološkim i inflamatornim pokazateljima, kao i promenama telesnih masa pacova tokom eksperimenta, nije bilo praćeno pozitivnim delovanjem soja. S druge strane, prestanak tretmana sojem BGHI14 nakon indukcije kolitisa doveo je do manje izraženog pada telesnih masa životinja u poređenju sa kontrolnim pacovima sa kolitisom, iako bez pratećeg smanjenja inflamatorne reakcije u tkivu kolona, bar u kasnoj akutnoj fazi bolesti (sedam dana od indukcije). Na osnovu opisanog može se zaključiti da su se u interakciji soja BGHI14 sa zdravim tkivom kolona aktivirali mehanizmi adaptivne imunosti u vidu memorijskih T-limfocita koji su indukovani nakon povrede u slučaju kontinuiranog prisustva istog bakterijskog antigena. Nasuprot, terapijski tretman pacova sojem BGHI14 doveo je do pozitivnog efekta u kasnoj akutnoj fazi kolitisa ocenjujući po promenama telesnih masa životinja kao i na osnovu parametara oštećenja mukozne barijere kolona. Dobijeni rezultati ukazuju da bi primena imunostimulišućih probiotika neposredno po nastanku oštećenja crevnog tkiva predstavljala efikasan pristup u lečenju inflamatornih oboljenja crevnog trakta, iako su na ovu temu potrebna dodatna istraživanja. S obzirom na značaj koji interakcija bakterija sa crevnom mukozom ima u ostvarivanju njihovog probiotičkog potencijala, u istraživanju su testirani mehanizmi kojima se može poboljšati vezivanje laktobacila kao potencijalnih probiotika za GI mukozu pacova. U tom smislu praćeni su efekti proteina sa mucin-vezujućim domenom (MbpL) poreklom iz soja Lactococcus lactis subsp. lactis BGKP1-20 na sposobnost vezivanja bakterijskog domaćina za mukozu ileuma i kolona u uslovima ex vivo i in vivo, kao i u in vitro esejima sa izolovanim želudačnim mucinom i mucin-produkujućim HT29-MTX ćelijama kolona. Osim homolognog domaćina, za funkcionalna ispitivanja korišćen je potencijalni probiotički soj Lactobacillus salivarius BGHO1. Rezultati su pokazali da eksprimirani MbpL protein nije bio dovoljan za vezivanje soja BGHO1 za crevnu mukozu, iako je pozitivno uticao na vezivanje BGKP1-20 soja za svinjski želudačni mucin i HT20-MTX ćelije u kulturi. Moguće je da je fenotipski efekat ekspresije MucBP proteina kod laktokokalnog BGKP1-20 soja veći u odnosu na BGHO1 soj, s obzirom na prisustvo drugih potencijalnih adhezivnih faktora koji se sintetišu u laktobacilima. Koristeći istu metodologiju testirana je uloga koju u vezivanju za mukozne površine GI trakta ostvaruje agregacioni faktor AggL poreklom iz soja BGKP1-20. Agregacioni faktor eksprimiran na površini bakterijskih ćelija često se dovodi u vezu sa povećanim afinitetom bakterijskog domaćina za GI mukozu. Naši rezultati su pokazali da, iako bi, povećanjem površinske hidrofobnosti mogao doprineti vezivanju za površinu kolona kod laktokokalnih sojeva sa niskom ukupnom hidrofobnošću površine, eksprimirani agregacioni faktor generalno otežava vezivanje bakterija za površine izložene dinamičkim peristaltičkim kontrakcijama kakva je u najvećoj meri površina ileuma. Na taj način bi sojevi koji sintetišu faktor agregaci