Study On The Effects Of Polysaccharides From Trichoderma Pseudokoningii On The Maturation Of Human Umbilical Cord Blood-derived Dendritic Cell And Its Immunocompetence

Objective: This study was designed to investigate the the effects of polysaccharidesfrom Trichoderma pseudokoningii administered in diverse dosage on:(1) thedifferenctiation and maturation of dendritic cells(DC);(2) multiplication-stimulatingactivity of alloreactive T cells; and (3)the activation of DC-derived cytokines ofinterleukin12(IL-12) and tumor necrosis factor-alpha(TNF-α).Methods:(1) Monocytes were initially isolated from five samples of the umbilicalcord blood from full-term healthy neonates, and immature DCs(imDC) were culturedin GM-CSF and IL-4to develop into DCs;(2) Polysaccharide from Trichodermapseudokoningii was used in different dose (50μg/ml,100μg/ml,200μg/ml) to facilitatethe differentiation of imDCs, and flow cytometry was performed to identify themyeloid blood CD11c treated with diverse dosage and changes of related to CD83,CD80and HLA-DR;(3) Peripheral blood mononuclear cells were obtained fromhealthy adults, and T-lymphocytes purified with nylon fiber column were used asresponding cells. DCs induced with different dose of Polysaccharide from Trichodermapseudokoningii were used as irritation cells and mixed and cultured for72h. MTTmethod was used to determine the indexes of the irritation cells, and the activities of Tmymphocyte proliferation induced by DC were compared among groups;(4) DCsupernatant in different groups was obtained at4,8,16,24and48h after treatement,and ELISA was used to determine the level changes of IL-12and TNF-α.Results:(1) The monocytes from the umbilical cord blood were successfullydifferentiated into imDCs under the induction of GM-CSF and IL-4and displayed DC phenotype. Lipopolysaccharide (LPS) was capable of inducing DC maturation and ledto increased expression of the surface maturation factors of HLA-DR, CD80andCD83;(2) By the flow cytometry results of DC phenotype in each group, polysaccharide indose of50μg/ml,100μg/ml and200μg/ml had resulted in significantly increased levelsof surface maturation factors of HLA-DR, CD80and CD83in DCs as compared withpre-treatment(P<0.05), whereas the CD83level in DC induced with LPS remained nosignificant change compared to that treated with50μg/ml of polysaccharide(P>0.05),and HLA-DR and CD80still stayed at lower levels(P<0.05). Although the surfacematuration factors of HLA-DR and CD80in DCs induced with dose of100μg/mlshowed no clear change (P>0.05), yet CD83was markedly up-regulated(P<0.05), andthe HLA-DR and CD80levels in DCs treated with dose of200μg/ml had no significantchange (P>0.05).(3) On the results of the T-lymphocytes in individual group stimulated with mature DCbasis, the reproductive capacity was significantly boosted for the T cells in DCs treatedwith dose of50μg/ml,100μg/ml and200μg/ml, respectively, as compared with theblank control group(P>0.05), whereas the dosage of100μg/ml and200μg/mladministered in DCs produced no obvious effect on the the T-lymphocyte proliferationas compared with LPS treatment(P>0.05), and polysaccharide in dose of50μg/ml ledto the lowest alloreactive T cell levels(P<0.05).(4) By the results of DC IL-12and TNF-αconcentration in the supernatant at48h, thelevels remained no significant change for DC-derived cytokines of IL-12and TNF-αtreated with dose of either100μg/ml or200μg/ml compared with LPStreatment(P<0.05), and the concentration stayed lowest in polysaccharide dose50μg/ml(P<0.05). DC supernatant was respectively obtained at different time point bytreatment with polysaccharide in dose of100μg/ml or LPS for determination of thetime-effect relationship with TNF-αgenerated by DCs, and the results showed thatLPS had caused earlier production of TNF-α, which peaked at around8h, whereas the polysaccharide in dose of100μg/ml had delayed generation of TNF-α, whichtended to increase slowly and peaked at neighboring24h.Conclusions:(1) Polysaccharide from Trichoderma pseudokoningii can facilitatematuration of DCs isolated in human umbilical cord blood, and dosage of100μg/mlmay produce best results;(2) Mature DCs induced by polysaccharide from Trichoderma pseudokoningii arecapable of facilitating the alloreactive T cell proliferation; and(3) Mature DCs induced by polysaccharide from Trichoderma pseudokoningii cangenerate the cytokine IL-12and TNF-αactivity, and TNF-αlevel peaks at around24h, which suggest time-independent effect.