OBJECTIVES: To investigate the effect of icariin (ICA) on early β-defensin-2 and T cell subsets in rats after tracheotomy. METHODS: A total of 54 SPF male Sprague-Dawley rats were randomly divided into a normal control group (group A), a model group (group B), and a model+ICA treatment group (group C), with 18 rats in each group. A tracheotomy intubation model of the B and C group was prepared. After 6 h of surgery, ICA intervention was given to group C. Groups A and B were given the same amount of normal saline. Lung tissue, alveolar lavage fluid and peripheral blood were taken at 24 h, 72 h and 168 h, respectively. The expression of rat β-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of β-defensin-2 in alveolar lavage fluid and peripheral blood serum was detected by ELISA. The content of peripheral blood T cell subsets (CD3, CD4, CD8) was detected by flow cytometry, and the ratio of CD4/CD8 was calculated. RESULTS: After tracheotomy, the levels of β-defensin-2 mRNA and β-defensin-2 in lung tissue from the group B were increased significantly at 24 h, then they were decreased gradually, and decreased most significantly at 168 h (<0.05). The content of β-defensin-2 in peripheral blood of group B decreased gradually, and the content of β-defensin-2 in 168 h was significantly lower than that in 24 h (<0.05), but there was no significant difference between group B and group A (>0.05). The level of CD3 T cells in peripheral blood was significantly lower than that in the group A (<0.05), but their was no significant difference in CD4 and CD8 T cells compared with group A (>0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum β-defensin-2 content, and peripheral blood CD3 and CD4 T cell levels were gradually increased, significantly higher than those in the group B (<0.05). CD8 T cell level was significantly lower than that in the group A at 24 h (<0.05), the CD4/CD8 ratio was significantly higher at 168 h than those in the group A or B (both <0.01). CONCLUSIONS ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of β-defensin-2 in lung tissue and alveolar lavage fluid, concomitant with increases in CD3 and CD4 T cells and CD4/CD8 ratio in peripheral blood while reduction in CD8 cells.
Animals ; Flavonoids ; Male ; Rats ; Rats, Sprague-Dawley ; T-Lymphocyte Subsets ; Tracheotomy ; beta-Defensins

Objective To evaluate the changes of adenosine metabolism pathway related molecules and their contribution to inflammatory injury in primary biliary cholangitis (PBC).Methods The consecutive samples of 49 subjects with PBC from The First People's Hospital of Taicang and The Second People's Hospital of Changshu were recruited from October 2016 to October 2017,and 36 healthy controls were involved in this study.The expression of CD39 and CD73 on CD4+T cells and Foxp3 + regulatory T cells were assayed by flow cytometry and the concentration of adenosine triphosphate (ATP) in serum was analyzed by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS).The correlations between Tregs,ATP and liver function were analyzed,i.e.,alanine aminotransferase (ALT),aspartate aminotransferase (AST),gamma-glutamyltransferase (GGT),alkaline phosphatase (ALP) and Mayo scores.Results In the patients with PBC,low proportions of CD4+CD39+T cells were noted compared with healthy controls [(5.28 ± 1.92) ％ vs (11.0l ± 3.19) ％,t =10.25,P ＜ 0.01].The patients with PBC also had significantly low proportion of CD4+CD25 + Foxp3+ CD39+ T cells compared with healthy controls [(23.75 ± 9.48) ％ vs (54.68 ± 5.18) ％,t =13.79,P ＜0.01].No significant difference of the proportion of CD4+CD73+T or CD4+CD25+ Foxp3+CD39+T cells was found between PBC and control groups (t values were 2.235 and 1.083,P ＞ 0.05).The level of serum ATP was higher in the patients with PBC than that of healthy controls [(200.28 ± 79.41) μg/L vs (89.20 ± 33.76) μg/L,t =8.367,P ＜ 0.01].A significant correlation was demonstrated between the proportion of CD39 + Treg in total Treg cells and the levels of ATP (r =-0.413,P =0.003),GGT (r=-0.378,P=0.007) and Mayo score (r=-0.382,P=0.007).Conclusion The low proportion of CD39+ Treg cells may contribute to the down-regulation of ATP hydrolysis in the patients with PBC.No significant change of CD73 + Treg cells was found in PBC patients.

Objective Analyzing the lncRNA expression profile in peripheral blood mononuclear cells(PBMC)of primary biliary cirrhosis(PBC)patients to provide new ideas for the pathogenesis,clinical diagnosis and treatment of PBC.Methods Collected peripheral blood from 30 PBC patients and 30 healthy volunteers, then separated PBMC.Four cases from each group were selected for long-noncoding RNA (lncRNA)expression microarray detection.Reverse transcription-PCR technology in a larger sample size was used to verify the microarray results.Bioinformatic analysis such as Cis-/Trans-target genes Gene ontology(GO)and pathway analysis, co-expression networks were conducted in order to provide a theoretical basis in the pathogenesis of PBC.Transfecting small interfering RNAs(siRNAs)for linc-pbc to see changes in the expression of nuclear receptor 4A group 3(NR4A3)and forkhead boxP3(FOXP3)and cell apoptosis in transfected PBMC.Results Compared to the healthy group, 749 lncRNA and 230 coding messenger RNA(mRNA)genes were abnormally expressed.Interestingly,NR4A3 gene was down-regulated by 78%.While linc-pbc, which was about 20 000 bp downstream of NR4A3 gene, increased 2.56-fold. Then design siRNA for linc-pbc.After transfection, mRNA and protein levels of NR4A3 and FOXP3 were up-regulated.Conclusions By recruiting PRC2 complex, linc-pbc may increased the methylation level of NR4A3 gene promoter region,thus decreasing the expression of NR4A3 in PBC patients and reduced NR4A3 futher downregulated the expression of FOXP 3 and reduced the amounts of immunosuppressive Treg cells in peripheral blood and liver tissue, breaking the equilibrium state of immune tolerance, and promoted the occurrence and development of the disease.

Objective To explore the establishment of methods to evaluate the quality of two different separate gel tubes .Meth-ods An evaluation comparing two separate gel tubes with silicate glass tubes ,which are the standard tubes ,was performed using data from 50 subjects .The serum samples were assayed for routine chemistry or immunology after centrifugation using the quantita-tive or qualitative detection methods such as ECL ,enzyme-linked immunosorbent assay (ELISA) ,reflection luminescence or immu-nofluorescence .The data were collect for further statistical analysis .Results For quantitative results of the items ,the two separate gel tubes ,compared with the standard glass tube ,there was no significant difference between the test results (P>0 .05);for quali-tative projects ,two separate plastic tube ,compared with the standard glass tube ,really the positive rate and the true negative rate was no significant difference (P>0 .05 ,K>0 .75) .Conclusion By using different detection methods and test items ,it is demon-strated that using two separate gel tube for items assay does not affect the accuracy and consistency of the test results .As a result , the method for external supplies quality assessment is established in the laboratory for ISO15189 quality control .

Primary biliary cirrhosis (PBC) is a chronic cholestic autoimmune liver disease and ursodeoxycholic acid (UDCA) is the first-line drug for the treatment.Currently there are several standards applied for the prognostic and therapeutic predictions of PBC,as well as for the guidance of personalized treatment.This paper elucidated the prognostic predicting factors in PBC and their applications in therapy monitoring with UDCA.Current challenges and future research interests are also put forward.

In recent years, the discovery of thousands of long non-coding RNAs ( lncRNAs) has certainly changed human′s view of the complexity of mammalian genomes and transcriptome, as they play an important role in the regulation of transcription, post-transcription as well as epigenetic level, widely involved in various physiological process and diseases.Here this review summarized the lncRNAs associated with cancers, discussed the established methods used to detect and quantify lncRNAs, and evaluated the clinical application of lncRNAs as biomarker.

Autoantibodies are useful laboratory parameters for diagnosis of autoimmune diseases (AID).Methods for the detection of autoantibodies have achieved quantitative or semi-quantitative to some extent.Furthermore,with the development of detection technology,high-throughout and quantitative technology has been the trend in autoantibody measurement.Compared with qualitative results,the quantitative ones may provide more values for diagnosis,prediction,prognosis and therapeutic monitoring for AID.Therefore,although quantitative technology of autoantibodies is still faced a number of challenges,the detection for autoantibodies has come into the era of quantitation.

Objective To investigate the population and role of IL-10+ CD19+ regulatory B cell (Breg) in patients with chronic hepatitis B.Methods Patients with acute hepatitis B (AHB) (n =28),chronic hepatitis B (CHB) (n =31) and normal subjects (n =25) were collected from Changshu No.2 People's Hospital between 2011 June and 2012 October.Peripheral blood mononuclear cells (PBMC) were isolated and stimulated with CpG ODN 2006 and PMA.Flow cytometry was used to analyze the population of IL-10-CD19 + Breg,CD4 + CD25high Treg,and ELISA was used to analyze the concentration of IL-10 in culture supernatant.Results The population of Breg in Peripheral blood of the CHB group [1.28％ (1.05％-2.20％)] was higher than that in the AHB group [0.87％(0.55％-1.22％)] and the HCs group [0.89％ (0.51％-1.37％)] (P =0.001,0.006),and the difference between the AHB group and the HCs group was not statistically significant (P=0.669).Breg in the CHB group [14.30％ (10.70％-16.70％)] was higher than that in the AHB group [10.30％ (7.05％-13.30％)] and the HCs group [10.40％(6.85％-12.60％)] (P =0.003,0.001),treg in the CHB group [5.80％ (4.20％-9.10％)] was also higher than that in the AHB group [4.05％ (2.53％-5.40％)] and the HCs group [4.50％ (2.55％-5.50％)] (P ＜0.001,P =0.005),and there was no significantly difference between the AHB group and the HCs group (Breg:P =0.796 ; Treg:P =0.227).Spearman correlation analysis showed that Breg was positively correlated with Treg in the CHB group (r =0.50,P =0.004),however there was no significantly correlation in the AHB group and the HCs group (r =-0.15,P =0.462; r =0.09,P =0.669).The concentration of IL-10 in the CHB group was higher than that in the AHB group and the HCs group (P ＜ 0.001),and the difference between the AHB group and the HCs group was not statistically significant (P=0.341).Spearman correlation analysis showed that IL-10 were positively correlated with the population of Breg in the CHB group (r =0.409,P =0.022).Conclusion The poluations of regulatory B cell and regulatory T cell increased in patients with chronic hepatitis B,and Breg cell might play the immune regulation role through secreting IL-10 in chronic HBV infection.

Objective To investigate the expression of sialic acid-binding immunoglobulin-like lectin-1 (Siglec-1) in the peripheral blood mononuclear cells (PBMCs) in patients with rheumatoid arthritis (RA),osteoarthritis (OA) and healthy controls and to explore the relationship between Siglec-1 expression and disease activity in RA.Methods Siglec-1 protein and mRNA levels were measured by flow cytometry and real-time quantitative reversetranscription-polymerase chain reaction (qRT-PCR) in 42 RA patients,28 OA patients and 26 healthy controls,respectively.The correlation studies between Siglec-1 and disease activity score 28 (DAS28) or C-reactive protein were performed.T-test was used for comparisons between groups and Pearson's correlation test was used for correlation analysis.Results The percentage of Siglec-1 positive cells of PBMCs in RA group [(15.2±7.6)％] was significantly higher than that in the OA group [(2.3 ±2.6)％] or healthy controls [(2.1±1.6)％,t=8.615,8.661; all P＜0.01].And the major cell type in PBMCs that expressed Siglec-1 was monocytes.The relative Siglec-1 mRNA expression in PBMCs in the RA group (3.4±1.5) was also significantly higher than that in the OA group (1.2±0.4) or healthy controls [(1.0± 0.4),t=3.446,3.966; all P＜0.05].But no significant differences of Siglec-1 protein and mRNA between the OA group and healthy controls were found.Furthermore,positive correlations between Siglec-1 protein and DAS28 or hs-CRP were found in RA patients (r=0.89,P＜0.01; r=0.48,P＜0.01).Conclusion PBMCs are activated which are characterized by elevated expression of Siglec-1 in RA patients.Circulating Siglec-1 may be considered as a potential noninvasive biomarker for monitoring disease activity in RA.

Objective To investigate the role of atherosclerotic monocytes Siglec-1 in stimulating CD4 + and CD8 + T lymphocytes proliferation and activation.Methods Experimental research.Cluster of differentiation antigen 14 (CD14) positive monocytes of 18 acute coronary syndrome (ACS),41 stable angina (SA) and 32 healthy volunteers were separated by magnetic-activated cell sorting.Different concentration of interferon-α (IFN-α,0,2,5,10 ng/ml) were used to up-regulate Siglec-1 and small interfering RNA (siRNA) or blocking antibody were used to down-regulate Siglec-1.Then monocytes were cocultured with CD4 + T/CD8 + T cells from a third healthy volunteer for 5 days.The experiment was designed for 11 groups (n=10 for each group),that was (1) normal CD14,(2) normal CD14 + IFN-α 5 ng/ml,(3) normal CD14 + IFN-α 5 ng/ml + anti-Siglec-1 2 μg/ml,(4) ACS CD14,(5) ACS CD14 + control siRNA group (Mock),(6) ACS CD14 + siRNA 679 40 nmol/L,(7) ACS CD14 + anti-Siglec-1 2 μg/ml,(8) SA CD14,(9) SA CD14 + Mock,(10) SA CD14 + siRNA 679 40 nmol/L and (11) SA CD14 + antiSiglec-1 2 μg/ml.Cell Counting Kit-8 (CCK-8) was used to determine T cells proliferation and ELISA was used to detect Interleukin-2 (IL-2),IL-10,IL-12 and IFN-γ of culture supematant.Data of cytokines detection were expressed as medium (quartiles) and analyzed by nonparametric rank sum test.Results By the blockage of Siglec-1 (group 6),the proliferation of CD4 and CD8 were decreased.Secretion of IL-2,IL-12 and IFN-γ by CD4 cells [67.00(62.50-87.30),0.86(0-1.63),and 47.82(37.60-56.67) pg/ml,respectively] were decreased and IL-10 [56.00(46.25-67.40) pg/ml] was increased compared with those in control group [group 4,213.70 (187.50-275.30),6.87 (4.90-8.93),114.90 (89.50-167.40),and 21.08 (15.70-33.20) pg/ml,respectively,with U =8.50,17.00,8.50,and 87.50,respectively.all P ＜ 0.05].When monocytes Siglec-1 from control group was up-regulated by IFN-α (group 2),secretion of IL-2,IL-12 and IFN-γ [220.44(174.30-312.30),7.90(6.540-10.40) and 143.75(78.20-210.00) pg/ml,respectively] were increased and IL-10 [21.95 (16.30-25.00) pg/ml] was decreased (compared with group 1,with U =89.50,98.00,100.00,and 0,respectively.all P ＜ 0.05).Regulation of Siglec-1 had no role in cytokines production in cocultured CD8 + T cells (all P ＞ 0.05).Conclusions IFN-α can upregulate monocytes Siglec-1.Siglec-1 may participate in the pathogenesis of AS via promoting proliferation of CD4 +/CD8 + T cells and Thl cytokines secretion of CD4 + T cells.