Insulinoma-glucagonoma clone 20 can express at least 6 isoforms in human cells.These isoforms can affect cell apoptosis and proliferation through apoptosis related signaling pathway,such as TRAIL or TNF signaling pathways.Furthermore,insulinoma-glucagonoma clone 20,as a GTP-GDP exchange factor,participates in the transportation of nerve synaptophysins.

Objective To investigate the prevalence of virulence genes(ply, pspA, nanA, lytA, psaA) among Streptococcus pneumoniae recently isolated from clinical patients. Methods The 133 strains were isolated from patients in three teaching hospitals in Chongqing from 2006 to 2008. Polymerase chain reaction was used to screen for virulence genes (ply, pspA, nanA, lytA, psaA). Results The positive rate of lytA, psaA, ply, nanA and pspA in 133 clinical isolates were 94.7%, 85.0%, 82.7%, 84. 2% and 60.2%, respectively. The positive rates of the lytA, psaA, ply, nanA and pspA genes in 87 common serotypes isolates was 100%, 87.4%, 86.2%, 89.7%, 67.8%, respectively. Conclusion The total positive rates of five virulence genes in the 133 clinical strains were high. The positive rates of five genes in the com-mon serotypes isolates were higher than those in the no-common serotypes. These genes are important virulence genes of Streptococcus pneumoniae. They could be candidates for protein vaccine of Streptococcus pneumoniae.

Objective To investigate long-term biological variability of serum lipids in Chinese. Methods Serum lipids in a Chinese population with relatively stable life styles were monitored with standardized measurements for 1 year (specimens were taken bimonthly) (23 subjects) or 10～15 years (yearly) (100 subjects). Results The total intra-individual variability (analytical and biological variations combined) of total cholesterol, triglycerides, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol averaged 10%, 28%, 16% and 18%, respectively. Conclusion Biological intra-individual variability is a major source of inaccuracy of cardiovascular risk assessments based on lipid measurements. Measures need to be taken to minimize biological variation when medical decisions are to be made in the treatment of lipid disorders.

Decreased serum high-density lipoprotein cholesterol (HDL-C) and increased low-density lipoprotein cholesterol (HDL-C) concentrations are important risk factors for cardiovascular diseases. Achieving accurate and comparable HDL-C and LDL-C results is the primary requirement for the prevention and treatment of cardiovascular diseases, in which reference system including reference method, reference materials and standardization programs are needed. The current reference method for HDL-C and LDL-C measurement is the β-quantification method developed by the United States Center for Disease Control and Prevention (CDC), a multi-step procedure involving ultracentrifugation, chemical precipitation, and cholesterol analysis by CDC reference method. This method uses a large amount of serum and is technically demanding which limited its application in standardization. The reduced volume β-quantification method uses a small volume of serum sample, is simple and high through put and is more applicable. However, both methods are sensitive to serum matrix. Commutability of reference materials should be evaluated when they are used to calibrate routine methods. Fresh (non-frozen) serum samples should be used for a split sample comparison between reference and direct methods. In addition, development of reference measurement procedure based on ultracentrifugation and preparation of commutable reference materials are the important work in lipoprotein standardization.

Objective To establish a method for measuring serum cotinine by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) and provide an assay that can be applied to theevaluation of the level of smoke exposure and to the risk analysis of smoking related diseases.Methods Blood samples were collected from 94 apparently healthy subjects from October to December in 2010 and centrifuged,and the sera were separated.Serum samples were mixed with [ D3 ] -cotinine ( as the internal standard) and treated with acetonitriles to precipitate protein.After centrifugation,the supernatants were transferred and evaporated under a stream of nitrogen until dryness and reconstituted with mobile phase.Then the residuals were analyzed by LC/MS/MS system with multiple reaction monitor model; the concentration of cotinine were quantified by the isotope internal standard method and the stand curve was employed with a series of calibration.To estimate the precision of the method,five frozen serum pools were repeatedly analyzed in five runs,and every pool was analyzed in triplicate.In addition,the recovery rates were analyzed with the serum sample added with different levels of standard.The stability of cotinine in serum preserved at room temperature,4 ℃ and - 80 ℃,respectively.Finally,the levels of cotinine of 94 healthy subjects were measured to evaluate the distribution of cotinine with different smoke statuses.Results Serum cotinine measured by ID-LC/MS/MS was separated well with few interferences.The correlation coefficients between the peak area ratios and cotinine concentrations were higher than 0.9993.The values of within-run coefficients of variation (CV) of five frozen serum pools (0.68,48.42,94.34,250.95 and 287.04 μg/L) were 2.19％,0.78％,0.75％,0.65％ and 0.67％,respectively.The values of total CV were 4.71％,1.40％,1.98％,1.10％ and 1.03％,respectively.The limit of detection (LOD) and limit of quantitation ( LOQ ) were 0.013 and 0.050 μg/L,respectively.The analytical recoveries ranged from 99.22％ to 102.67％.The samples could maintain stability within 2 d at room temperature,7 d at 4 ℃ and 3 months at -80 ℃ resulting the accuracy of measurements from 99.28％ to 100.87％ and the CV＜5％.The levels of cotinine of 94 healthy subjects were measured and shown skewed and leptokurtic distribution.The concentrations of twenty smokers,fourteen former smokers and sixty non-smokers were 116.40 (63.17 -241.12),0.67 (0.15 - 0.95 ) and 0.22 (0.15 - 0.42 ) μg/L,respectively.Furthermore,the level of cotinine of former smokers (Z =-2.12,P ＜0.05) and smokers (Z =-6.67,P ＜0.001) were statistically higher than non-smokers.Conclusions An ID-LC/MS/MS method for serum cotinine detection has been established.It is hoped that the method will be applied to the assessment of smoke exposure and its association with the risks of smoking related diseases since it is simple,specific,precise,sensitive and accurate.

ObjectiveTo investigate the biological variations ( CVI,CVG ) of serum high-density (HDL2-C,HDL3-C ) and low-density (LDLa-C,LDLb-C ) lipoprotein subfractions and high-density lipoprotein cholesterol esterification rates (FERHDL and MERHDL ).MethodsTwenty healthy volunteers,10 males and 10 females,were recruited for this study from September to October,2010.Blood was collected four times from each individual with a 2-week interval between each sampling.Serum lipoprotein subfraction cholesterol levels were measured by ultracentrifugation/HPLC,FERHDL and MERHDL were measured by HPLC.Within-subject ( CVI) and between-subject ( CVG.) biological variations and quality specifications for precision,bias and total error were calculated.Results The average CVI of this group were 5.5％ and 7.2％ for HDL3-C and HDL2-C,11.2％ and 18.7％ for LDLa-C and LDLb-C,11.95％ and 12.3％ for FERHDL and MERHDL,respectively.The CVG for HDL2-C was 45.5％,much higher than that of HDL3-C (8.7％),and FERHDL(49.5％ ) had a higher CVG than MERHDL (30.6％ ).For each analyte,there was a considerable variation of CVIamongindividuals.ConclusionsBiologicalvariationsof lipoprotein subfractions,FERHDI and MERHDL have been estimated.These rsults will play an important role in quality specifications and cardiovascular disease risk assessment.

Objective To develop an HPLC method for the measurement of n-3 fatty acid index of serum cholesteryl esters.Methods Serum triglycerides were hydrolyzed with ethanolic sodium hydroxide and cholesteryl esters (CEs) were extracted with hexane.The extracted CEs were analyzed by reversed phase HPLC with a UV detection at 205 nm.Cholesteryl eicosapentaenoate and docosahexaenoate ( major n-3 fatty acid cholesteryl esters) were identified by liquid chromatography-tandem mass spectrometry and cholesterol in each CE fraction was measured.Peak areas of CEs were corrected for cholesterol and CE n-3 index was calculated using the corrected peak area and expressed as the percentage of n-3 fatty acid CEs in total CEs.Results The HPLC analysis can be finished in 6 minutes.Triglycerides which interfere with the determination of n-3 fatty acid index, were hydrolyzed with ethanolic sodium hydroxide (4 mol/L) in 30 seconds.The within-run and total CVs for CE n-3 index averaged 0.66% and 0.90%, respectively.CE n-3 indexes of 70 volunteers and 36 coronary heart disease patients apparently healthy subjects and patients with coronary heart disease in Beijing Hospital appeared to be positively skewed and leptokurtic distribution ( skewness = 1.25, kurtosis = 1.70 ).The median of n-3 indices were 0.98% ( 0.37% - 2.40% ).The logarithm of n-3 index appeared to be normal distribution and the average is 0.003 7% with standard deviations of 0.15.The distribution of n-3 indices of gender groups was similar with the total.The medians of females and males were 1.08% (0.60% -2.40%) and 0.95% (0.37% -2.11%) respectively, and the former were significantly higher than the latter( t = - 3.021, P = 0.003 ).Conclusion A new method for the measurement of n-3 index of serum cholesteryl esters by HPLC has been established.It is simple and precise and can be used in predicting cardiovascular diseases risks and monitoring dietary intake of n-3 fatty acids.

Objective To study the expression of thymic stromal lymphopoietin(TSLP) and the activation of DCs in OVA-induced murine asthma model, and investigate the effects and underlying mecha-nisms of TSLP on lung inflammation. Methods Thirty BALB/c mice were randomly divided into control group, OVA group and TSLP neutralizing antibody treated group. The asthma model was evaluated by airway responsiveness and histological analysis of lung tissues ; The levels of TSLP mRNA in lungs were determined by quantitative real-time PCR; The expression of TSLP in lungs were determined by immunohistochemistry and Western blot; The expression of CD40, CD80, CD86 in BALF was detected by FACS. Results Both the histological analysis of lung tissues and the airway responsiveness were all consistent with the characteris-tic of murine asthma model. The expression of TSLP and TSLP mRNA in the OVA group was significantly in-creased compared with blank group. The expression of CD40, CD80, CD86 in BALF from OVA group was increased significantly compared with the control group. Furthermore, treating mice with TSLP neutralizing antibody reduced the expression of CD40, CD80, CD86 on dendritic cells, and IL-4, IL-5, IL-13 in the OVA group. Conclusion Our study indicate that TSLP was highly expressed in the bronchial epithelia of murine asthma model, via upregulation of CD40, CD80, CD86, induce DCs to active CD4~+ T cells and pro-duce type 2 responses, so that aggravating the lung inflammation of asthma. Blocking TSLP is capable of in-hibiting the production of Th2 cytokines, thus presents a promising strategy for the treatment of asthma.

Objective To evaluate the analytical performance of 7 homogeneous HDL-cholesterol reagents. Methods An altracentrifugation-HPLC method was used as the comparison method. Fourty fresh patient samples were analyzed by homogeneous methods and the comparison method. The homogeneous methods were all performed on a Hitachi 7170A chemistry analyzer according to the manufacturer's instructions, Precision, accuracy and total errors were analyzed. Results The homogeneous assays typically demonstrated within run coefficient variance(CV) of < 1%, and total CV of < 3%. Methods A, B and D showed average bias, bias at the medical decision points and total errors all within the NCEP performance criteria and method C and F unacceptable biases (-19.74% and 11.46%, respectively) and total errors according to the NCEP criteria. However, all the homogenous methods (A-F) had total errors of < 30%, as required by the US Clinical Laboratory Improvement Amendment (CLIA). Conclusions Homogeneous HDL-C assays have been shown to be reasonably precise, but discrepant results have been observed with some of the assays. Clinical laboratories should pay more attention on selecting and validating homogeneous HDL-C reagents.