Objective To investigate the relation between the apotosis of B cells in the peripheral blood (PB) and the expression of interleukin (IL)-17 in patients with rheumatoid arthritis (RA).Methods The proportions of apoptosis of B cells in the PB of 80 patients with RA and 80 healthy controls were measured by flow cytometry.B cells in the PB of 20 RA and 20 healthy individuals were isolated by MACS and Western blotting was used to detect the Bcl-2 and Caspase-3 protein levels.IL-17 levels were detected by enzyme-linked immunosorbent assay (ELISA).T-test and linear regression were used to analyze the data.Results The proportions of apoptosis of B cells in the PB of patients with RA and healthy controls were (14±6)％ and (24±9)％ respectively.The rate of apoptosis of B cells in patients with RA was significantly less than healthy controls (t=2.737,P=0.021).The Bcl-2 protein level of B cells in the PB of patients with RA group was significantly higher than that of control group (26±10,12±6,P＜0.01).Conversely,the Caspase-3 protein level of B cells in the PB of patients with RA group was significantly lower than that of the control group (16±7,31±12,P＜0.01).ELISA detected elevated level of serum IL-17 in the patients with RA as compared with controls [(69±19),(27±10) pg/ml,t=4.631,P=0.014].There was a negative correlation between the level of IL-17 and apoptosis of B cells in patients with RA (r=0.36,P＜0.01).Conclusion The elevated bcl-2 and reduced caspase-3 of B cells in patients with RA further proves there is abnormal apoptosis of B cells in RA patients.There is negative correlation between the expression of IL-17 and apoptosis of B cells in patients with RA and IL-17 can inhibit B cell apoptosis.

Objective To establish a method for detecting urinary vanillylmandelic acid (VMA), homovanillic acid (HVA) and creatinine (Cr) simultaneously by high performance capillary electrophoresis (HPCE). Methods The separations were carried out using a 120 mmol/L phosphate buffer (pH 6.80) in a fused-silica capillary tube of 47 cm×75 μm I.D. by capillary zone electrophoresis (CZE). Injections were made by using the pressure mode for 4 s at 1 p. s. i. after samples were centrifuged and diluted. The detections were monitored by a diode-array detector (DAD) at 200 nm after samples were separated at a voltage of 20 kV. The method developed was validated systematically and applied to urine samples from healthy adults (n = 100) and children (n = 100) for establishing the reference ranges of VMA/Cr and HVA/Cr, respectively. Results Under these conditions, the separations of VMA, HVA and Cr could be completed within 13 min. The linearity ranges of VMA, HVA and Cr were 0-500, 0-500 and 0-4 000 μmol/L, respectively, with the correlation coefficients (r) between 0.997 2 and 0. 999 1 (P < 0.01). The detection limits (S/N= 3) were 1.0 μmol/L for VMA, 1.0 μmol/L for HVA and 50.0 μmol/L for Cr. The mean within-run (n = 10) CVs of migration time for VMA, HVA and Cr in urine were 0.58%, 0.56% and 0.25% respectively, while the mean between-run (n = 10) CVs of migration time were 0.95%, 1.00% and 0.48% respectively. The mean within-run (n = 10) CVs of peak area for VMA, HVA and Cr were 3.78%, 3.97% and 2.76% respectively, while the mean between-rim (n = 10) CVs of peak area were 4.60%, 4.08% and 4.42% respectively. The average recoveries were 98.36% for VMA, 93.56% for HVA and 98.85% for Cr. Other compounds in human urine such as catecholamines, 5-hydroxytryptamine and albumen didn't interfere with the assay. The correlation between CE method and HPLC method was good. And the correlation coefficients (r) of VMA and HVA were 0.954 9(P <0.01) and 0.945 1 (P < 0.01), respectively. Skewness distributions were presented for VMA/Cr and HVA/Cr in random urine from both adults and children, and the 95% reference ranges were established by the percentile method. For adults, the reference ranges of VMA/Cr and HVA/Cr were 0-4. 26 and 0-1.69 (μmol/mmol), respectively. For children, the reference ranges of VMA/Cr and HVA/Cr were 0-10.39 and 0-4.31 (μmol/mmol), respectively. Conclusions The CE method devised here for direct measurement of urinary VMA, HVA and Cr is simple, fast,precise and automatic with good repeatability. It is an ideal method for routine detection and mass screening of pheochromocytoma and neuroblastoms.

Objective To investigate the expression of serum sHLA-G in systemic lupus erythematosus (SLE) patients and the association with the disease activity.Methods The serum concentration of sHLA-G in SLE patients and healthy controls was measured with enzyme-linked immunosorbent assay.Results Significant higher sHLA-G levels were detected in patients of SLE than control group (P＜0.01),The serum concentrations of sHLA-G in active SLE patients were markedly higher than stable SLE patients (P＜0.01).The expression level of sHLA-G showed positive correlations with SLE activity index (SLEDAI)(r=0.30,P=0.01).There was no correlation between sHLA-G levels and serum concentration of Anti-dsDNA,C3,C4 and Anti-ANA in SLE patients (P＞0.05).Conclusion The level of serum sHLA-G is significantly increased in SLE patients.Positive correlations are observed between sHLA-G levels and SLEDAI.These data indicated that sHLA-G may play certain roles in the pathogenesis and progress in SLE.

Objective To investigate the expression of helper T cells (Th)17/regulatory T cells (Treg) balance associated factors in rheumatoid arthritis (RA) patients and their correlation with serum midkine (MK).Methods A total of 60 RA patients were divided into active RA patients (n=32) and inactive RA patients group (n=28).MK level in sera was detected by enzyme linked immunosorbent assay (ELISA) in 60 patients with RA and 30 healthy controls (HCs).The fraction of CD4+CD25+FOXP3+ Treg cells and IL-17+CD4+ Th17 cells in RA patients and healthy controls were determined by flow cytometry (FCM), and the expreasion of Foxp3, RORγt, Signal transducer and activator of transcription (STAT) 3 and STAT5 mRNA were detected with real-time polymerase chain reaction (PCR).Results were evaluated using ANOVA followed by q tests for comparisons of Th17 population between active RA patients, inactive RA patients and HCs, t test was used for comparing of Foxp3, RORγt, STAT3, STAT5 mRNA between RA group and HCs.The correlations between serum MK concentration and peripheral Treg cells, Th17 cells, Foxp3, RORγt, STAT3, STAT5 mRNA were analyzed by Pearson's correlation analysis.Results The percentages of Treg cells from active RA patients, inactive RA patients and HCs were significantly different (F=129.6, P＜0.01), the percentages of Treg cells of active RA patients [(1.41±1.05)％] were lower than that of the inactive RA patients [(3.6±1.6)％;q =7.92, P＜0.05] and healthy group [(7.7±1.7)％;q=22.45, P＜0.05], and there was significant difference between healthy group and inactive RA group (q=14.53, P＜0.05).The percentages of Th17 cells of the three groups were also significantly different (F=36.3, P＜0.01),the percentage of Th17 cells of active RA patients [(1.84±1.01)％] was significantly higher than that of inactive RA patients [(0.71±0.28)％;q=9.59, P＜0.05] and healthy group (0.53±0.16)％ [(P＜0.05;q=1 1.10, P＜0.05], there was no significant difference between the inactive RA group and healthy group (q=1.51, P＞0.05).The expression of RORγt and STAT3 mRNA in RA patients was higher than that of healthy controls (t=5.84, P＜0.01;t=4.52, P＜0.01).The expression of Foxp3 and STAT5 mRNA in RA patients were lower than healthy controls (t=6.01, P＜0.01;t=2.18, P＜0.05).Serum MK values were correlated with STAT5 (r=-0.55, P＜0.01), but not with Foxp3, RORγt, STAT3 mRNA or the percentage of Treg/Th17 cells.Conclusion Serum MK expression and the percentage of Th17 cells increase, while the percentage of Treg cells decrease in RA patients.Serum MK values are negatively correlated with STAT5 mRNA which is associated with Th17/Treg balance.This may be important in the pathogenesis of RA.

Background Most anti-inflammation eyedrops are limited in clinical application owing to multiple adverse effects.A novel peptide GC31 derived from human thrombomodulin has a natural anti-inflammatory activity.Compared with conventional anti-inflammatory eyedrops,GC31 possesses more advantages and potential clinical transforming value.However,relevant study is still lack.Objective The purpose of this study was to evaluate the anti-inflammatory effect of GC31 and the possible mechanisms.Methods Sixty SPF male Wistar rats aged 8-10 weeks were randomized into 6 groups using randomized number table.Non-specific keratitis models were established in 40 rats by intrastromal injection of 10 μl of lipopolysaccharide (LPS) dissolved in PBS.Different doses of GC31 (125 μg or 250 μg) or dexamethason soluble in PBS were sunconjunctically injected in the experimental eyes respectively in the low dose GC31 group,high dose of GC31 group and the dexamethason group,and 10 μl of PBS was used in the same way in the PBS control group.No drug was injected in the model group,and the normal rats were employed as the blank control group.The corneas were examined by slit lamp microscope and were scored based on the criteria of Anand 24 hours after injection.Then the corneas were collected for histopathological examination.Expression of nuclear factor-κB (NF-κB) p65 in the corneas was detected using immunochemistry.Expressions of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) proteins were assayed using ELISA.Real-time PCR was used to detect the expressions of IL-6 mRNA and TNF-α mRNA.The use and care of the experimental animals followed Regulation for the Administration of Affair Concerning Experiment animals by State Science and Techonology Commission.Results A significant difference was seen in the ocular inflammatory scores among the six groups (F =301.238,P =0.000).The inflammatory scores were significantly lower in the high dose of GC31 group than those in the model group (1.85 ± 0.36 versus 2.90± 0.43) (t' =-5.144,P =0.000) ; and the scores in the dexamethason group was lower than those in the high dose of GC31 group(t' =-3.931,P=0.000).Infiltration of inflammatory cells in corneal tissue was milder in the high dose of GC31 and the dexamethason group compared with the model group.The positive response for NF-κB p65 was obviously weaker in the rat corneas in the low and high dose of GC31 groups and the dexamethason group in comparison with the model group.The contents of IL-6 and TNF-α proteins in the corneas were significantly reduced in the low and high dose of GC31 group and the dexamethason group compared with the model group (low dose group:t=-2.626,P=0.009;t'=-2.310,P=0.017.high dose group:t =-3.361,P=0.001 ;t'=-3.151,P=0.002),and the contents of IL-6 and TNF-α proteins in the dexamethason group were lower than those in the high dose of GC31 group (t=-3.361,P=0.001;t'=-3.360,P=0.000).In addition,the expression trend and compared results of IL-6 mRNA and TNF-α mRNA among the groups were similar to those of the IL-6 and TNF-α proteins (all at P＜0.01).Conclusions GC31 suppresses LPS-induced corneal inflammation response by downregulating the expression of inflammatory eytokines.The effect is more dominant in the doses of 250 μg than that in the doses of 125 μg.

Background and purpose:Recently, studies showed that non-steroidal anti-inlfammatory drugs (NSAIDs) could reduce the incidence of cancer. Whether ibuprofen could inhibit the growth of hepatocellular carcinoma cells had not been reported yet. In the current study, we investigated the effects of ibuprofen on hepatoma carcinoma BEL-7402 cells and the relevant mechanisms. Methods: Hepatocellular carcinoma BEL-7402 cells were randomly divided into 7 groups:the control group and the ibuprofen groups (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mmol/L). The effect of ibu-profen on BEL-7402 HCC cells was measured by MTT method, the cell cycles were analyzed by flow cytometry (FCM), cell vitality and apoptosis were determined by cell analyzer. PCNA, Cyclin D1, Bcl-2 and COX-2 protein levels were examined by Western blot, and the expressions of prostaglandin E2 (PGE2) were measured by ELISA. Results:After the exposure to ibuprofen, the suppression ratio of BEL-7402 cells was increased (P<0.05). BEL-7402 cell vitality was decreased by degrees significantly (P<0.05), early apoptosis of BEL-7402 cells was increased (P<0.05), and the G0/Gl phase ratio was increased significantly compared with control group (P<0.05). Ibuprofen effectively decreased PCNA, Cyclin D1, Bcl-2 and COX-2 expressions in BEL-7402 cells (P<0.05), and decreased PGE2 protein expression in cell culture supernatants sig-nificantly (P<0.05). Conclusion:Ibuprofen is effective for inhibiting the proliferations, increasing apoptosis and blocking cell cycles of BEL-7402 HCC cells. The anti-tumor mechanisms of ibuprofen may be related with the inhibition of COX-2 and PGE2 expressions.

Objective To establish a method for detecting the concentration of paraquat (PQ) and creatinine(Cr) in urine simultaneously by capillary electrophoresis.Methods Experimental methodological study.8 acute PQ poisoned patients who were treated in the First People's Hospital of Lianyungang from January 2011 to June 2012 were collected.2 were male,and 6 were female.The separation were carried out using a 25 mmol/L pH1.97 glycine-HCl buffer(containing 40 mmol/L NaCl) in a fused-sillica capillary tube of 47 cm ×75μm I.D.by capillary zone electrophoresis.Urine had been injected by pressure for 4 s after samples were centrifuged and diluted for 10 times with H2O.The detection were monitored by a diode-array detector at 200 nm while samples were separated at a voltage of 20 kV.A systemic methodological evaluation of this method was carried out (The linear range,detection limit,repeatability test,recovery test and interference test).And the method was used to detect the concentration of PQ and Cr in PQ poisoned patients' urine.Results The peaks of PQ and Cr appeared within 5 min.The linear ranges of PQ and Cr were 2-1000,10-5000 μmol/L,respectively,with the correlation coefficients of 0.9997 and 0.9999 (P ＜0.01).The detection limits were 1.0 μmol/L for PQ and 5.0 μmol/L for Cr.The mean within-day(n =10) CVs of peak area for PQ and Cr were 2.84％ and 1.72％,while the mean inter-day(n =10) CVs of peak area were 3.62％ and 3.06％.The average recovery rate of PQ and Cr were 88.6％ and 90.2％ respectively.Diquat(DQ) didn't interfere with the assay.The range of PQ/Cr(μmol/mmol) for 8 cases was 8.9-2215.Conclusions A method was established successfully for the rapid determination of PQ and Cr in urine by capillary electrophoresis.The CE method devised here for direct measurement of urinary PQ and Cr from PQ poisoned patients is simple,fast,automatic and with good repeatability.It is an ideal method for rapid detection of urinary PQ in PQ poisoned patients.

Objective To investigate the clinical significance of apoptosis of B cells in the peripheral blood of patients with rheumatoid arthritis (RA).Methods The proportions of B cells in the peripheral blood (PB) of 51 active and 30 remission patients with RA and 80 healthy controls were detected by flow cytometry.B cells in the PB of 10 active,10 remission and 10 healthy individuals were isolated by MACS.The apoptosis of cultured B cells,which were collected at 24,48,72,96 h respectively,were assessed by flow cytometry.ANOVA,t test and,Spearman correlation analysis were used for statistical analysis.Results The proportions of B cells marked as CD19 and CD22 in the PB of active and remission patients with RA and healthy controls were (26±11)％,(12±8)％,(10±7)％,(26±10)％,(12±8)％,(11±5)％ respectively.The proportions of B cells in the PB of active patients was significantly higher than that of remission patients and healthy controls (P＜0.01).There was positive correlation between B cell proportion in the PB of active patients and DAS 28 and IgG level.The proportion of apoptosis of B cells in the PB with active patients was less than healthy controls.Conclusion The pathway of apoptosis of B cells in the PB of active patients is inhibited,which could increase B cell proportion.Moreover,the high proportion of B cells in the PB of active patients is closely related to disease activity.

Objective To establish a flow cytometric method for the detection of alkaline phosphatase (ALP) expression on the membrane of neutrophils (mNAP).Methods EDTA-K2 anticoagulant venous bloods were collected.Expression of mNAP in peripheral blood was measured by flow cytometry using a phycoerythrin (PE)-labeled anti-ALP monoclonal antibody.BD QuantiBRITE PE was used to generate a calibration curve for PE fluorescence and ratios of PE to anti-ALP antibody and detect the bound ALP antibodies per cell (antibodies bound per cell,AB/c).Preanalytical handling including anticoagulants (EDTA-K2,citrate,and heparin),storage temperature,storage time,and plasma ALP were optimized and measured the precision.The expression levels of mNAP from 481 healthy controls were measured to establish a clinical reference range.The mNAP levels of 84 patients with severe infection and local infection,39 patients with virus infection were determined by this method.Results For preanalytical handling,application of PBS washing can effectively eliminate the interference of plasma ALP.The mNAP levels were not influenced by different anticoagulants and storage conditions (stored for 12 h either at room temperature or 4 ℃).This method had preferable reproducibility (CV in batch were 2.01％-3.33％,average 2.67％ ; CV between batch were 5.80％-6.00％,average 5.90％).The median (quartiles) of mNAP in health controls were 1 758 (1 378-2 310) AB / c for men and 1 897 (1 369-3 249) AB / c for women.There was no significant difference between genders (U =27 140,P =0.243 8).The clinical reference ranges (2.5 percentile to 97.5 percentile) of mALP was 920.5-3 493.0AB / c.The expression levels of mNAP of patients with bacterial infections (13 532,9 756-16 869 AB / c) were significantly higher than those of patients with virus infection(1 143,536-2 012 AB / c) and healthy controls (1879,1399-2497 AB / c) (H=221.5,P＜0.01).Conclusion BD QuantiBRITE PE kit can be used to standardize flow cytometer settings and quantitatively detect molecules per cell.The flow cytometric method for detection of mNAP has important clinical application for differentiating bacterial and virus infection.

Objective To study the potential use of the urinary beta-trace protein ( βTP) for diagnosis of type 2 diabetic renal injury.Methods 174 patients with type 2 diabetic mellitus (T2DM) were classified into 2 groups according to the ratio of urinary albumin to creatinine (Alb/Cr):diabetes without renal injury group (group A) and diabetes with renal injury group (group B).70 healthy subjects served as normal control group ( group C).The level of urinary βTP and αl microglobulin (α1MG) was measured by latex particle enhanced immunoturbidimetry assay.The urinary Alb and Cr were determined by nephelometry and Jaffe method respectively.The level of uriuary βTP among all groups was compared and ROC curve analysis was performed.The relevant analysis on urinary βTP,urinary α1MG and other related indexes was made.Results The median level of urinary βTP/Cr in group B was 9.1mg/g Cr,significantly higher than 3.1mg/g Cr of group A and 2.0mg/g Cr of group C.The difference had statistical significance ( H =45.5,P ＜ 0.01).The other indexes ( Alb/Cr,α1MG/Cr,SCr) were all higher in group B than in the other 2 groups ( H =110.9,38.3,11.4 respectively,P ＜0.01).The relevant analysis showed that urinary βTP/Cr was positively correlated with urinary α1MG/Cr (r =0.894,P ＜ 0.01),SCr (r =0.367,P ＜ 0.05 ),HbA(J) C ( r =0.242,P ＜ 0.05 ),systolic pressure ( r =0.162,P ＜0.05 ),and the course of the disease ( r =0.251,P ＜ 0.05 ).No correlation was found between urinary βTP/Cr and diastolic pressure,fasting blood glucose(FBG) or BMI.ROC curve analysis showed the area under the curve (AUC) was 0.86 (95％CI,0.78-0.93)for urinary βTP/Cr and 0.76 (95％ CI,0.67-0.85) for urinary α1MG/Cr.The best cut-off value of urinary βTP/Cr and α1MG/Cr was 4.1mg/g Cr vs 10.9mg/g Cr,the sensitivity was 68.5％ vs 59.7％,and the specificity was 89.8％ vs 80.3％.The difference had statistical significance (P ＜ 0.05).Conclusions Urinary βTP has better diagnostic value for type 2 diabetic patients with renal injury than urinary α1MG.It can sensitively reflect renal tubular injury and can be used as a novel available biomarker to evaluate the renal tubular injury in clinic.