ABSTRACT
Objective To explore the expression of TLR2,4,7 mRNA on peripheral blood mono-nuclear cells (PBMCs) in patients with chronic cystic echinococcosis(CE) infection, and the level of serum IL-10. Methods The expression level of TLR2,4,7 mRNA on peripheral blood mononuclear were tested in 42 chronic CE cases and 28 normal controls (NC) by real-time fluorescent quantitative reverse transcription polymerase chain reaction (FQ-RT-PCR) method. GAPDH was selected as the internal control. The level of serum IL-10 was determined in ELISA. The subjects were determined by t test. The correlations between TLR2, TLR4, TLR7 and IL-10 were determined by differences of expression of TLR2, TLR4, TLR7 on PBMCs and serum IL-10 in two groups of study linear correlation test. Results The expressions of TLR2, TLR4,TLR7 mRNA in chronic CE group were higher than those of in NC group. Compared with the NC group, the expressions of TLR2, TLR4 and TLR7 mRNA increased more than 7.3-, 3.6-, 3.6-fold, respectively. In chronic CE group, TLR2, TLR4 and TLR7 mRNA expressions were 1.0729 ±0.4006, 5.0976 ±1.6682, 0. 6481 ±0. 2574, respectively. TLR2, TLR4 and TLR7 mRNA expressions were 0. 1468 ± 0.0435, 1.4067 ±0. 3279, 0. 1804 ±0. 0568 in NC group, respectively. Compared with NC group, the differences of TLR2 and TLR4 mRNA expression were significant (P = 0.0287, 0. 033), while the expression of TLR7 mRNA was not difference (P =0.0862). Moreover, in chronic CE group, the level of serum IL-10 was higher than that of in NC group. In chronic CE group and NC group, the level of serum IL-10 was (17.6770±1.6298) pg/ml, (9.4898 ±0.7049) pg/ml. Compared with NC group, there was significant difference in chronic group (P<0.01). Significant positive correlation between TLR2 and TLR4 was found in chronic CE group, r = 0. 1135, P =0.036. Others were not correlations. Conclusion In the development of chronic CE, TLR2 and TLR4 participate in this progression. As the receptors of antigen of cystic echinococcus, TLR2 and TLR4 can regulate the immune response through interacting with different antigens from cystic echinococcus. Meanwhile, under the participation of TLR2, TLR4 and increased serum IL-10, they will approach to Th2 immune reaction, which play an important role in chronic CE that can induce immune evasion.
ABSTRACT
Objective To investigate the effects of Th17 cells and Treg cells on immune evasion in patients with hepatic hydatid disease. Methods From August 2008 to September 2009, 54 patients with hepatic hydatid disease who were treated at the First Affiliated Hospital of Xinjiang Medical University and 20 healthy people (control group) were enrolled in this study. Of the 54 patients, 21 had liver cystic enchinococcosis (CE)(CE group), 15 had recurrent cystic echinococcosis (RCE) (RCE group) and 18 had liver alveolar echinococcosis(AE) (AE group). The serum concentrations of interleukin-17 (IL-17), IL-23, transforming growth factor-β1(TGF-β1) and IL-10 were measured by enzyme-linked immunosorbent assay. All data were analysed by one-way analysis of variance, LSD-t test and Pearson correlation analysis. Results Serum IL-17 levels were significantlylower in the AE group [(11±3)ng/L], CE group [(13±4) ng/L] and RCE group [(13 ±5) ng/L]compared with those in the control group [(16±5) ng/L] ( F = 6.53, P ＜ 0.05 ). There was no significant difference in serum IL-17 levels between the CE and RCE groups (t =0.22, P ＞0.05). Serum levels of IL-23were also lower in the AE group [(72±27) ng/L], CE group [( 106±53) ng/L] and RCE group [( 107±48 ) ng/L] compared with those in the control group [( 139±50) ngg/L] ( F = 6.74, P ＜ 0.05 ), while there was no significant difference between the CE and RCE groups (t =0.02, P＞0.05). The serum levels of IL-10 were significantly higher in the AE group [(5.5±2.2) ng/L], CE group [(4.3±2.0) ng/L] and RCE group [(4.2 ± 1.4) ng/L] compared with those in the control group [(3.1 ± 0.8 ) ng/L] ( F = 9.78, P ＜ 0.05 ),with no significant differences between the CE and RCE groups ( t = 0.14, P ＞ 0.05 ). TGF-β1 levels were significantly higher in the AE group [(38±7) μg/L], CE group [(37±7) μg/L] and RCE group [(33±9) μg/L]compared with those in the control group [( 26±7) μg,/L] ( F = 6.73, P＜ 0.05 ), with no significant difference among the AE, CE and RCE groups ( t = 0.56, 1.81, P ＞ 0.05 ). The Th17/Treg (IL-17/IL-10) ratio was significantly decreased in the AE group ( 2.1 ± 0.7 ), CE group ( 3.6 ± 1.5 ) and RCE group ( 3.4 ± 1.9)compared with that in the control group (5.7 ± 2.6) ( F = 13.76, P ＜ 0.05 ), while no significant difference was found between the CE and RCE groups (t = 0.23, P ＞ 0.05). The serum concentrations of IL-17 were negatively correlated with TGF-β1 ( r = - 0.23, P ＜ 0.05 ) and positively correlated with IL-23 ( r = 0.70, P ＜ 0.05 ).Serum concentrations of IL-10 were positively correlated with TGF-β1 ( r = 0.46, P ＜ 0.05 ). Conclusion The overwhelming expression of Treg related cytokines disrupts the Th17/Treg balance in patients with AE or CE,which may have a potential role in immune evasion in the progress of hydatid disease.
ABSTRACT
Objective To establish a economic and stable method to induce and culture dendritic cells (DCs) from peripheral blood of human being, and compare with the magnetic activated cell sorting. Methods Monocytes were isolated from health donors peripheral blood mononuclear cells(PBMC) by density gradient separation,cultured and compared with that of cells isolated by the magnetic activated cell sorting or adherent culture,respectively. PBMC were cultured with recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4(rhIL-4) for 6 days to induce the growth of DCs. Morphological changes was observed under inverted microscope. Meanwhile, cell viability was tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DR were analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. After adding human recombinant cytokines, the phenotypes of acquired cells surface markers, CD14, CD1a, CD86, CD83 and HLA-DR would be detected and compared with flow cytometry. T cells proliferating activity was determined by allogeneic mixed lymphocyte reaction in vitro. Results After adherent culture for 2 h, the acquired DCs showed typical morphology. Cell viability was decreased at days 5th, 6th[(53.333 ±5.774)%,(38.333 ± 7.638)%] than that at day of 3rd[(68.667 ± 3.215)%, all P ＜ 0.05] with the magnetic activated cell sorting, but with adherent culture method, the difference was not statistically significant (F = 0.737,P＞ 0.05) at days of 3rd, 5th, 6th[(92.667 ± 3.055)%,(94.000 ± 1.000)%,(94.667 ± 1.528)%]. Moreover,compared with the magnetic activated cell sorting, there were differences in cell viability of adherent culture method at days of 3rd, 5th, 6th(t = 9.374, 12.021,12.527, all P ＜ 0.05). Before and after using the magnetic activated cell sorting, the expression of CD14 were (32.457 ± 12.351) %, (41.914 ± 14.858)%, respectively. The difference was not statistically significant(t = 1.295, P ＞ 0.05). After culturing for 2 h, the expression of CD14[(35.267 ± 4.658)%]was higher than those of culturing for 1, 5 h[(15.033 ± 6.189)%, (21.233 ± 4.895)%, all P ＜ 0.05]. Compared with the 1st day[(32.328 ± 14.517)%], the CD14 expression level[(2.200 ± 1.356)%] on surface of DCs was significantly reduced(t = 5.467, P ＜ 0.05) at the 6th day of culturing, the CD1a expression level[(43.371 ±16.250)%] was remarkablely increased than that of the 1st day[(12.300 ± 6.223)%, t = 2.545, P ＜ 0.05];while the expressions of CD86, CD83, HLA-DR[(16.857 ± 5.686)%,(9.343 ± 5.230)%,(72.800 ± 17.881)%] were similar(t = 0.652,1.137,0.907, all P ＞ 0.05) compared with that of the 1st day[(12.550 ± 16.758)%, (6.250 ±1.323)%, (64.671 ± 15.588)%]. In mixed lymphocytes reactions, with increasing of lymphocytes, T lymphocytes proliferating activities were reduced. In the magnetic activated cell sorting, when the ratio of DCs and lymphocytes were 1: 50, 1: 100, cells proliferation ability(1.502 ± 0.055,1.507 ± 0.029) were lower than that of ratio of 1: 10(1.859 ± 0.049, all P ＜ 0.05);in adherent culture method, the ratio of DCs and lymphocytes was 1: 100, the cells proliferation ability(1.545 ± 0.066) was decreased than that of ratio 1: 10(2.015 ± 0.301, P ＜ 0.05). When the proportion of DCs and lymphocytes remained the same, the capacity to stimulate T lymphocyte was similar of the two methods(P ＞ 0.05). Conclusions Comparied with the magnetic activated cell sorting, after culture of PBMC for 2 h the induction of DCs can produce better formed and functional cells, and this method is stable, simple,economic, and is a suitable method for basic and clinical research of DCs in vitro.