Lektin banane (BanLec) pripada podgrupi lektina koji vezuju glukozu ili manoza- u
okviru familije lektina sličnih žakalinu. U plodu banane, koji predstavlja prirodni izvor
BanLec-a, javlja se u više izoformi, a može se proizvesti i rekombinantnom DNK
tehnologijom.
Rekombinantna izoforma BanLec-a (rBanLec) je po svojim strukturnim
karakteristikama i specifičnosti vrlo slična prirodnim izoformama. Kao i većina biljnih
lektina, (r)BanLec ne podleže brzo denaturaciji / razgradnji u uslovima digestivnog
trakta, a pokazano je da se vezuje za epitel tankog creva i postepeno prolazi u subepitelni
prostor. Danas je poznato da BanLec, nezavisno od izoforme, ima imunomodulatorni
potencijal. Dosadašnja istraživanja su se dominantno bavila uticajem (r)BanLec-a na
funkcionalne karakteristike limfocita i pokazano je da stimulacije određene ćelijske
populacije prirodnim i rekombinantnom izoformom kvalitativno imaju isti ishod. Uticaj
(r)BanLec-a na funkcionalne karakteristike drugih ćelija imunskog si...stema nije detaljno
analiziran.
Cilj ove doktorske teze je da se kroz ispitivanja (1) modulatornog dejstva rBanLeca
na funkcionalne karakteristike antigen-prezentujućih ćelija (APĆ) BALB/c miša, (2)
imunomodulatornog dejstva rBanLec-a u debelom crevu BALB/c miša u fiziološkim
uslovima, i (3) efekata profilaktičke i terapijske primene rBanLec-a u modelu TNBS-om
indukovanog kolitisa kod BALB/c miša, dobije uvid u imunomodulatorni efekat rBanLec-a
na imunski sistem u mukozi debelog creva miševa BALB/c soja pod specifičnim uslovima.
Koristeći peritonealne makrofage i dendritske ćelije slezine (DĆ) kao in vitro model
sisteme, pokazano je da rBanLec dozno-zavisno moduliše funkcionalne karakteristike APĆ
BALB/c miševa. Analiza uticaja rBanLec stimulacije na karakteristike rezidentnih (RM) i
tioglikolatom-indukovanh (TGM) peritonealnh makrofaga, pokazala je da ishod
stimulacije određene ćelijske populacije nije jednoznačan već zavisi od njihovog
funkcionalnog stanja. rBanLec na pozitivan dozno-zavisan način pospešuje
proinflamatorni kapacitet RM (povećanje produkcije NO i IL-12 i smanjenje aktivnosti
arginaze i sekrecije IL-4), a na isti način smanjuje proinflamatorni kapacitet TGM
(smanjenje produkcije NO i IL-12 i povećanje aktivnosti arginaze i sekrecije IL-4). Uticaj
rBanLec-a na produkciju TGF-β kod peritonealnih makrofaga takođe zavisi od
funkcionalnog stanja makrofaga (kod RM, rBanLec negativno dozno-zavisan, a kod TGM
pozitivno dozno-zavisno utiče na produkciju TGF-β). I kod RM i kod TGM, rBanLec na
pozitivan dozno-zavisan način podstiče aktivnost mijeloperoksidaze (MPO), produkciju
TNF-α i IL-10. Za stimulaciju produkcije proinflamatornih medijatora, nezavisno od
funkcionalnog stanja makrofaga, značajno je vezivanje rBanLec-a za TLR2 i TLR4 / CD14.
rBanLec utiče na sekreciju efektorskih citokina IFN-γ i IL-4 (negativno dozno-zavisno) i
regulatornog citokina IL-10 (pozitivno dozno-zavisno) od strane DĆ slezine. rBanLec
TLR2-posredovana stimulacija DĆ slezine nije ključna za promene u ekspresiji Ifn-γ i Il-4...
Banana lectin (BanLec) is primarily isolated from the fruit of banana (Musa
paradisiac). It is glucose / mannose-specific lectin that belongs to the jackalin superfamily
of lectins. There are several naturally occurring BanLec isoforms. Nowadays BanLec can
also be produced by recombinant technology (rBanLec). rBanLec possesses structural and
functional characteristics that highly resemble those reported for its natural counterparts.
As most plant lectins, (r)BanLec is resistant to denaturation/proteolytic cleavage in the
gastrointestinal tract. It has been reported that rBanLec attaches specifically to the
mucosal surface of small intestine and passes into subepithelial compartment. (r)BanLec
has been recognized as a potential immunomodulator. It has been shown that (r)BanLec
modulates functional characteristics of lymphocytes but the effects of (r)BanLec
stimulation in other immune cells are not yet elucidated.
The aim of this study was to investigate immunomodulatory / immunostimulat...ory
activity of rBanLec in the colon of BALB/c mice. This aim was accomplished through
evaluation of the impact of rBanLec stimulation on 1) functional characteristics of antigen
presenting cells (APC) isolated from BALB/c mice 2) local immune system in the large
intestine of healthy BALB/c mice, and 3) the course of TNBS-induced experimental colitis
in BALB/c mice.
Peritoneal macrophages and spleen dendritic cells (DC) isolated from BALB/c
mice were used in vitro for the evaluation of rBanLec influence on functional
characteristics of APC. Generally, it is shown that rBanLec in a dose-dependent manner
modulated the functional characteristics of APCs. By using resident (RMs) and
thioglycollate-elicited (TGMs) peritoneal macrophages, it has been shown that effects of
rBanLec stimulation depend on its concentration but also on the functional status of
macrophages. Obtained results have clearly shown that rBanLec, in a positive dosedependent
manner, promotes pro-inflammatory phenotype with BALB/c RMs
(enhancement of NO and IL-12, reduction of arginase activity and IL-4 secretion) while, in
the same manner, it tends to skew BALB/c TGMs towards anti-inflammatory profile
(reduction of NO and IL-12 production, enhancement of arginase activity and IL-4
secretion). The dose-dependent changes in production of TGF-β by peritoneal
macrophages also depended on their functional status: rBanLec stimulated the
production of TGF-β by RMs in a negative dose-dependent manner, while in TGMs
production of TGF-β positively correlated to the rBanLec concentration. The activity of
myeloperoxidase (MPO) and productions of TNF-α and IL-10 were enhanced upon
rBanLec stimulation in a positive dose-dependent manner with both RMs and TGMs.
Further, it was shown that interactions of rBanLec with TLR2 and TLR4 / CD14 are
important for initiations of the production of pro-inflammatory mediators by peritoneal
macrophages irrespective to their functional status. rBanLec also stimulated in a specific
dose-dependent manner the secretion of effector cytokines IFN-γ and IL-4 (negative dose dependent manner) and regulatory cytokine IL-10 (positive dose-dependent manner) by
spleen DCs...
