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Objective:To investigate the role of peptidyl arginine deiminase 4 (PAD4) in anti-β 2GP1/β 2GP1 complex-induced formation of neutrophil extracellular trapping networks (NETs). Methods:Peripheral blood neutrophils were isolated from healthy humans by density gradient centrifugation. PAD4 expression was detected by Western blot after the neutrophils were incubated with anti-β 2GP1/β 2GP1 complex (100 μg/ml) for a certain period of time. PAD4 inhibitor Cl-amidine (10 μmol/L) was used to pretreat neutrophils. Changes in the expression of citrullinated histone 3 (CitH3) at protein level and the relative content of myeloperoxidase (MPO)-DNA were detected by Western blot and ELISA, respectively. A mouse thrombus model of antiphospholipid syndrome (APS) was established by inferior vena cava stenosis. Intervention experiments were performed by intraperitoneal injection of Cl-amidine (50 mg/kg). The expression of CitH3 at protein level in plasma was detected by Western blot. The concentration of circulating free DNA (cf-DNA) in plasma was measured with fluorescent staining. Thrombus in inferior vena cava was collected and weighted to evaluate whether inhibiting the activity of PAD4 would suppress the APS-IgG-induced formation of NETs and thrombosis. Differences among groups were analyzed by t test or one-way analysis of variance (ANOVA). Results:The expression of PAD4 induced by anti-β 2GP1/β 2GP1 complex was significantly down-regulated in the cytoplasm, but increased in the nucleus [(3.67±0.32) vs (1.47±0.19), t=10.22, P<0.05; (0.57±0.19) vs (2.97±0.31), t=11.49, P<0.05]. Cl-amidine significantly inhibited the anti-β 2GP1/β 2GP1 complex-induced expression of CitH3 protein by neutrophils [(2.46±0.47) vs (0.46±0.13), t=12.24, P<0.01], and reduced the MPO-DNA content in the culture supernatants [(4.09±0.94) vs (2.80±0.57), t=4.23, P<0.05]. In vivo, Cl-amidine significantly inhibited the expression of CitH3 protein [(3.97±0.56) vs (1.09±0.45), t=11.83, P<0.01] and decreased the content of cf-DNA [(2 685.0±735.8) vs (1 784.0±577.0), t=3.93, P<0.05] in plasma of APS mice. Compared with the experimental APS mice in the control group, the weight of thrombus in the APS mice pretreated with Cl-amidine was significantly reduced [(8.22±3.06) vs (4.89±1.90), t=2.27, P<0.05]. Conclusions:PAD4 was involved in the formation of NETs induced by anti-β 2GP1/β 2GP1 complex, which might play an important role in APS thrombosis.
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Objective To detect the levels of neutrophil extracellular traps (NETs) in patients with antiphospholipid syndrome (APS) and to preliminarily explore its formation mechanism.Methods Plasma samples from 27 APS patients and 30 healthy controls were collected.The circulating free DNA (cf-DNA) in plasma was detected by the PicoGreen nucleic acid quantitative assay kit,and the concentration of citrulline histone 3 (CitH3) was analyzed by enzyme-linked immuno sorbent assay (ELISA).The association of cf-DNA/NETs with thrombotic events in APS patients was further analyzed.The neutrophils in healthy controls were separated by density gradient centrifugation and stimulated with anti-β2GPl/β2GPI complex (100 μg/mL) for 4 h,and the cf-DNA/NETs in the culture supernatant was determined.TLR-4 inhibitor-TAK242 (5 μmol/L) was further used to observe whether the stimulation of the anti-β2GPI/β2GPI complex on cells could be intervened.The differences between groups were analyzed by analysis of variance (ANOVA) or rank sum test,Sidak or Dunnett's test were used to compare the mean of multiple samples and the correlation between variables was analyzed by Spearman's correlation test.Results The concentration of cf-DNA/NETs and CitH3 were significantly increased in plasma of APS patients compared with that in healthy controls [175.7(70.6,205.7) ng/ml vs 29.8(7.6,115.7) ng/ml,Z=-3.654,P＜0.05;19.5(7.8,26.4) ng/ml vs 3.3(0.84,10.3) ng/ml,Z=-3.932,P＜0.05],and there was a significant positive correlation between the cf-DNA/NETs and CitH3 (r=0.447,P=0.019).In the APS group,there was no significant difference in cf-DNA/NETs between patients with arterial thrombosis and those with venous thrombosis [177.1(67.8,297.2) ng/ml vs 184.7(82.4,233.9) ng/ml,Z=-0.301,P=0.786],whereas cf-DNA/NETs in the patients who experienced a new thrombotic event in 1 month was significantly higher than those with a history of thrombosis [192.1(83.6,328.8) ng/ml vs 90.0(42.8,184.7) ng/ml,Z=-2.006,P=0.046].In vitro,anti-β2GPI/β2GPI complex (100 μg/ml) stimulated the release of cf-DNA/NETs from neutrophils,which was significantly increased compared with the control group (t=10.39,P＜0.05),while TAK242 significantly inhibited the stimulating effects of anti-β2GPI/β2GPI complex on cells (t=4.22,P＜0.05).Conclusion The level of cf-DNA/NETs in peripheral blood of APS patients is significantly increased,which may play an important role in APS thrombosis.Anti-β2GPI/β2GPI complex induces the formation of cf-DNA/NETs through TLR4 and participates in the pathological process of APS.
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Objective To establish and assess the new method of APTT assay based on the combinations of Mg2+and Ca2+for lupus anticoagulants(LA)measurements.Methods This prospective study included 309 trisodium citrate anticoagulated plasma samples from 244 random patients and 65 patients with different autoimmune diseases(AID)to establish and assess the method of LA measurement, respectively.Final concentrations of 0,2.0, 4.0, 8.0,16.0 mmol/L Mg2+were added into 25 mmol/L Ca2+solution, and Actin reagent was used to measured plasma APTT of 94 patients.The applied concentration of Mg2+-Ca2+solution was confirmed through the special and significant alteration of APTT from LA-positive and -negative plasma observed in the presence of Mg 2+(test solution).Based on Actin reagent use,the test solution and 25 mmol/L Ca2+solution were applied to measure APTT of patients and normal individuals, respectively, and the ratio of Mg2+-Ca2+-APTT to Ca2+-APTT(Mg2+-Ca2+-APTT indices)and normalized Mg2+-Ca2+-APTT indices(NAR)were calculated, respectively.Mixed plasma NAR was measured,and CV%was calculated to evaluate the repeatability and stability of Mg 2+-Ca2+-APTT method.APTT of 150 patients was measured with the test solution and Actin reagent to calculate Mg 2+-Ca2+-APTT indices, and normalized LA ratio was determined with dRVVT method.The applicability of Mg2+-Ca2+-APTT assay was assessed through comparisons of the results from the two methods.Finally, NAR and NLR of 65 patients with AID(including 26 SLE patients)were measured with Mg2+-Ca2+-APTT assay and dRVVT method, respectively, and ROC curve was also used to assess the efficacy of the two methods for LA measurements.Results In all LA-negative plasma,APTT increased from 28.1 ±4.5 s to 61.2 ±7.9 s in normal APTT group,47.2 ±8.9 s to 97.5 ±10.3 s in increased APTT group,and 27.6 ± 5.1 s to 61.2 ±7.9 s in ACA-positive group when Mg2+increased from 0 to 8 mmol/L in Mg2+-Ca2+solution(F=34.12, 38.9 and 28.35,P<0.01).Following increased Mg2+concentration, APTT shortened from 0 to 4.0 mmol/L, but simultaneously prolonged from 4.0 to 16.0 mmol/L in LA-positive plasma with prolonged or normal APTT(F=31.55 and 39.51, P<0.01), and APTT was significantly higher in 8.0 mmol/L than that in 4.0 mmol/L(P<0.001).The test concentration of Mg 2+/Ca2+solution was 4.0 mmol/L.The within, inter-day CV% of NAR was 1.39%,2.30%, and 3.44%, respectively. According to the judging criteria of <0.966 and >1.034 of Mg2+/Ca2+indices, there was 141 patients with increased indices and NLR <1.20, and 9 patients with decreased ones and NLR≥1.20 in all 150 patients.The area under ROC curve of NAR and NLR for LA detection was 0.913(95%CI:0.848-0.978) and 0.892(95%CI:0.817-0.966), respectively, and the cut-off value was 0.87 and 1.13, respectively. The sensitivity and specificity of NAR(85% and 77%)was higher than that of NLR(81% and 74%), respectively.The accordant rate of positive,negative,and total results between NAR and NLR was 94.4%, 98.5%,and 98%,respectively.Conclusion The method of APTT assay based on Mg2+combining Ca2+for LA measurements is feasible,and can be used to detect plasma LA of patients.
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Objective To investigate the correlation between serum total bile acid level and coronary atherosclerosis. Methods The clinical data of 1408 patients who had underwent coronary angiography were retrospectively analyzed. The patients were divided into coronary atherosclerosis group (stenosis ≥ 50%, 681 cases) and coronary normal group (stenosis 0.05). The rate of smoking, rate of hypertension, rate of diabetes, age, body mass index (BMI), creatinine, fasting plasma glucose, total bile acid and low density lipoprotein cholesterol in coronary atherosclerosis group were significantly higher than those in coronary normal group:18.6%(127/681) vs. 14.2%(103/727), 64.6%(440/681) vs. 45.8%(333/727), 48.5%(330/681) vs. 22.7%(165/727), (58.9 ± 12.2) years vs. (56.7 ± 13.1) years, (25.6 ± 4.3) kg/m2 vs. (24.9 ± 4.5) kg/m2, (70.28 ± 15.94)μmol/L vs. (52.79 ± 12.75)μmol/L, (6.82 ± 2.73) mmol/L vs. (5.57 ± 2.35) mmol/L, (7.86 ± 4.38)μmol/L vs. (5.63 ± 3.71)μmol/L and (3.32 ± 0.69) mmol/L vs. (2.28 ± 0.57) mmol/L, and there were statistical differences (P24 kg/m2) and total bile acid levels were risk factors of coronary atherosclerosis (P<0.05 or<0.01). Conclusions The serum total bile acid level is positively correlated with the severity of coronary atherosclerosis, which may be one of the independent risk factors for coronary atherosclerosis.
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Objective To study the value of deferred stent implantation in patients with high thrombus burden of acute ST-segment elevation myocardial infarction (STEMI). Methods Select 106 cases with a high thrombus burden within 12 hours of onset in patients with STEMI ,the infarct-related artery thrombus aspiration after antegrade flow of TIMI 2-3 and≤2 points of the thrombus aspiration(TS)patients were randomly divided into immediate stenting group(n = 43)and deferred stenting group(n = 40). Two groups of patients were compared with the myocardial blush grade(MBG),the incidence of slow-/no-reflow ,the incidence of compound endpoints in 6 months and the cardiac function after PCI for 6 months. Results After stenting,the MBG of deferred group was significantly higher than that of immediate group ,the incidence of slow-/no-reflow and the compound endpoints events within 6 months in deferred group was significantly lower than that in the immediate group. After PCI for 6 months,the improvement of LVEF in the deferred group was significantly higher than that in the immediate group, the left ventricular end diastolic dimension(LVEDD)in deferred group was significantly lower than that in immedi-ate group,and the differences were statistically significant(P < 0.05). Conclusions The high thrombus burden in patients with acute STEMI after thrombus aspiration ,deferred stent implantation can significantly reduce the rate of slow-/no-reflow ,improve myocardial perfusion ,reduce the incidence of compound endpoints events ,im-prove cardiac function in patients.
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Objective To investigate the levels of immunoglobulins and complement in children with cerebral palsy. Methods 59 chil-dren with cerebral palsy were assessed with Gross Motor Function Classification System (GMFCS), and the serum levels of immunoglobu-lin (Ig)G, IgA, lgM, complements C3 and C4 were measured in the children with cerebral palsy and other 61 children without cerebral palsy (controls). Results The serum levels of IgG, IgA, lgM, complement C3 and C4 decreased significantly in the children with cerebral palsy compared with the controls (P0.05). Conclusion There is humoral im-mune dysfunction in some children with cerebral palsy, which may associated with the severity of the disease.
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@#Objective To investigate the levels of immunoglobulins and complement in children with cerebral palsy. Methods 59 children with cerebral palsy were assessed with Gross Motor Function Classification System (GMFCS), and the serum levels of immunoglobulin (Ig)G, IgA, lgM, complements C3 and C4 were measured in the children with cerebral palsy and other 61 children without cerebral palsy (controls). Results The serum levels of IgG, IgA, lgM, complement C3 and C4 decreased significantly in the children with cerebral palsy compared with the controls (P<0.001). There was significant difference in the levels of IgG, IgM, and complement C4 among cerebral palsy children of different grades of GMFCS (P<0.05), but not in the levels of IgA and complement C3 (P>0.05). Conclusion There is humoral immune dysfunction in some children with cerebral palsy, which may associated with the severity of the disease.
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<p><b>OBJECTIVE</b>To explore the role of toll-like receptor 4 (TLR4) on oxidized low-density/β₂-glycoprotein I/β₂-glycoprotein I (ox-LDL/β₂GPI/anti-β₂GPI) antibodies complex induced macrophage foam cell formation.</p><p><b>METHODS</b>The peritoneal macrophages were separated from TLR4 intact C3H/HeN mice and TLR4 defective C3H/HeJ mice. The cells were treated with ox-LDL, ox-LDL/β₂GPI, ox-LDL/anti-β₂GPI, anti-β₂GPI/β₂GPI, ox-LDL/β₂GPI/anti-β₂GPI, lipopolysaccharide (LPS) for 48 h and the foam cells were identified by Oil red O staining for intracellular lipids. The total cellular RNA and the protein lysates were collected. The levels of tissue factor (TF) mRNA in two groups were detected by real-time PCR (RT-PCR), and the expression of phosphorylated nuclear factor-κB (NF-κB) p65 was detected by Western blotting. Monocyte chemotactic protein-1 (MCP-1) secretion from peritoneal macrophages was determined by enzyme linked immunosorbent assay (ELISA) kits.</p><p><b>RESULTS</b>Compared with C3H/HeJ mice, lipid droplets in the cytoplasm of peritoneal macrophages from C3H/HeN mice were significantly increased and phosphorylation-NF-κB expression was significantly upregulated after stimulating by ox-LDL/β₂GPI/anti-β₂GPI complex (P < 0.01). TF mRNA and MCP-1 expression were also upregulated post ox-LDL/β₂GPI/anti-β₂GPI complex stimulation [TF mRNA: 0.041 ± 0.023 vs. 0.005 ± 0.003; MCP-1: (6 200.2 ± 6.4) pg/ml vs. (803.3 ± 5.5) pg/ml, P < 0.01].</p><p><b>CONCLUSION</b>TLR4 can enhance ox-LDL/β₂GPI/anti-β₂GPI complex induced peritoneal macrophage foam cell formation via upregulating phosphorylation-NF-κB, TF and MCP-1 expression.</p>
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Animals , Male , Mice , Antigen-Antibody Complex , Allergy and Immunology , Atherosclerosis , Allergy and Immunology , Metabolism , Cells, Cultured , Foam Cells , Allergy and Immunology , Metabolism , Lipoproteins, LDL , Allergy and Immunology , Macrophages, Peritoneal , Allergy and Immunology , Metabolism , Mice, Inbred C3H , NF-kappa B , Metabolism , Thromboplastin , Metabolism , Toll-Like Receptor 4 , Allergy and Immunology , Metabolism , beta 2-Glycoprotein I , Allergy and ImmunologyABSTRACT
@#Objective To observe the effect of conductive education on gross motor function for children with cerebral palsy. Methods 98 children with cerebral palsy were divided into control group (n=48) and observation group (n=50) according to the wish of their parents.All the children received conventional rehabilitation, while the observation group accepted conductive education in addition. They were assessed with the total score of Gross Motor Function Measure (GMFM-88) before and 6 months after treatment. Results The total score of GMFM-88 was not significantly different between the groups before treatment (P>0.05), but it was different after treatment (P<0.01). Conclusion Conductive education may promote the recovery of gross motor function for children with cerebral palsy.
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This study is to explore the interventional effects of fluvastatin on anti-beta2GPI/beta2GPI-induced activation in THP-1 mononuclear cells. In vitro, human mononuclear cells THP-1 were treated with fluvastatin, LPS and anti-beta2GPI/beta2GPI, then the TF expression on THP-1 cells was detected by real-time quantitative PCR (RT-qPCR) or TF activity was detected by kit. TNF-alpha mRNA and its protein expression were investigated by RT-PCR and ELISA kit. The expression of phospho-NF-kappaB p65 and inhibitory protein of NF-kappaB (IkappaB-alpha) were measured by Western blotting. The results suggested that the expression of TF and TNF-alpha on THP-1 cells was significantly up-regulated with treatment of anti-beta2GPI/beta2GPI complex (100 mg x L(-1)), compared with that of untreated cells (P < 0.05). Fluvastatin (50 mg x L(-1)) could decrease TF (mRNA and activity) expression and the level of TNF-alpha (mRNA and protein) in THP-1 cells with anti-beta2GPI/beta2GPI complex. The expression of TF and TNF-alpha was shown in a concentration-dependent manner. Moreover, anti-beta2GPI/beta2GPI complex could downregulate IkappaB-alpha levels and increase the levels of phospho-NF-kappaB p65. And these effects of anti-beta2GPI/beta2GPI complex could be blocked by fluvastatin. In conclusion, fluvastatin may interfere the expression and regulation of NF-kappaB signal transduction pathway, thereby inhibit the effects of anti-beta2GPI/beta2GPI on activation of THP-1 cells, by decreasing the expression of TF and TNF-alpha.
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Objective To discover regularities behind critical values in an effort to provide scientific evidences for a better critical value report system.Methods Collection, analysis and statistical assessments for critical value items and ranges appropriate for the hospital Results The occurrence rate of critical values is found to be 0.14%.By means of clinical evaluation, statistic analysis and reference to literature and clinical specialists, the clinical labs revised the ranges of PO2, GLU, PT and APTT, and added ALT, MYO, CTnl and K+ , GLU, BILl for newborns, and canceled AMY.All of these changes were put in practice upon approval of the Medical Department of the hospital Conclusion Regular evaluation and continuous improvement of the critical value report system may help with saving lives, improving quality of care of the labs and doctors" diagnosis as well
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Objective To understand the current status of blood pressure and to analyze its influence factors in rural residents in Tianjin.Methods Total 2 390 754 rural residents aged over 15 yrs were detected on their blood pressure by the trained professional staffs with standard method,then calculated the mean of blood pressure and hypertension prevalent rate,compared with results of Village I of National Nutrition and Health Situation Investigation in 2002.After population standardized,a multivariate analysis was done on hypertension in subjects.Results The average SBP was(125.91?18.99)mm Hg,average DBP was(80.44?10.77)mm Hg;hypertension prevalent rate was 31.57%(33.08% for male and 30.18% for female).The general hypertension prevalence was higher than that of the national level;after standardized with China population in 2000,the prevalent rate of hypertension in the group aged over 18 yrs was nearly 50% higher than that in national level.The multivariate analysis showed that the affected factors for hypertension of rural residents included age,gender,region,education level,smoking,alcohol drinking and obesity.Conclusions Hypertension had become a popular and serious public health problem.To prevent and control hypertension should be one of the key issues of chronic diseases prevention in rural area.