Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648-2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Animals ; Antibodies, Viral ; biosynthesis ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Genetic Vectors ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H9N2 Subtype ; Plasmids ; Rabbits ; Viral Proteins ; immunology

Liver innate immunity plays an important role in the defense against pathogen invasion.The mechanisms of liver innate immunity for defending against pathogen invasion are reviewed,including the special anatomical and physiological characteristics of the liver,the fea-tures of innate immune cells in the liver (liver-settled cells and cells raised from blood circulation)and their function and relationship with each other in pathogen invasion,the process of pathogen identification and effect of liver innate immune cells,and the mechanism of liver immune injury and immune tolerance.Given current research on innate immune cells and innate immune molecules in the liver,further stud-ies should be conducted to understand the connection and difference between liver innate immunity and systemic immunity,thus promoting the diagnosis and treatment of liver diseases and deeper research.

To further analyze immunoglobulin (Ig) properties of panel reactive antibodies (PRA) and their potential harmful effects on allogeneic renal transplants. Methods Since dithiothreitol (DTT) is able to crack disulphuric bond of Ig and to depoly-merize IgM with heavy molecular weight, PRA study was performed with treatment of DTT. Thus, a DTT-ERA method was established, which can identify immunoglobulin class in sera of high PRA patients. Results Among 701 recipients, whose sera were positive by the standard PRA, the positive rate of DTT-PRA was 33.2% . Besides, positive PRA patients could be divided into three groups according to their sera's sensitivity to DTT: Group 1 consisted of patients whose sera contained exclusively IgM antibodies; Group 2 consisted of patients whose sera contained of IgG antibodies only; Group 3 consisted of patients whose sera were found to contain both IgM class antibodies and IgG antibodies. Conclusion IgM class antibodies are not associated with posttrans-plant rejection, while IgG antibodies alone and mixture of IgG and IgM antibodies may mediate acute, or even hyperacute rejection.

Background Angle closure is the pathological basis of primary angle-closure disease (PACD).Understanding different types of angle closure mechanisms is significant for evaluation of risk factors,prevention,diagnosis and treatment of PACD.Ultrasound biomicroscopy (UBM) is commonly used for investigating the types of angle closure mechanisms in clinical works.However,UBM is a contact examination,which limits its application.Anterior segment optical coherence tomography (AS-OCT) is a non-contact examination of anterior segment imaging,through which qualitative and quantitative evaluations of the anterior chamber angle can be made.Objective This study was to investigate the reproducibility of mechanism categories of angle closure based on AS-OCT images and UBM images.Methods Cross-sectional study was performed.PACD outpatients from September to October in 2013 in Beijing Tongren Hospital were enrolled and received both UBM and AS-OCT examinations.UBM images were obtained in superior,inferior,nasal and temporal quadrants of the anterior chamber.AS-OCT images were obtained in the anterior segment quadrant mode at 0-180,45-225,90-270,and 135-315 degree meridians.UBM and ASOCT images were categorized into three dominant angle closure mechanisms:pupil block,anterior located ciliary body and thick peripheral iris roll,separately.And the same mechanism of at least two UBM/AS-OCT images of one eye was defined as the angle closure mechanism of this eye.If both eyes of one PACD patient were eligible for inclusion,the right eye was selected for analysis.The reproducibility of two examinations was evaluated by Kappa coefficients.Results Finally 40 PACD patients (40 eyes,27 right eyes and 13 left eyes) were enrolled for analysis.Based on UBM images,12 eyes (30.0％) were classified as pupil block,23 eyes (57.5％) as anterior located ciliary body and 5 eyes (12.5％) as thick peripheral iris roll.Based on AS-OCT,12 eyes (30.0％) were classified as pupil block,21 eyes (52.5％) as anterior located ciliary body and 7 eyes (17.5％) as thick peripheral iris roll.The Kappa coefficient for the classification of angle closure mechanisms was 0.870.Conclusions Criterion of angle closure mechanism categories based on AS-OCT images was established.Mechanism categories obtained from AS-OCT and UBM images showed a good reproducibility.

ObjectiveTo observe the effect of Spasticity Therapeutic Apparatus on the talipes varus in ptients with stroke. Methods60 patients were divided into treatment group and control group. Patients in the control group accepted routine therapy, while the treatment group were treated with the Spasticity Therapeutic Apparatus in addition. ResultsThe Clinic Spasticity Index (CSI) of ankle significantly improved after treatment (P＜0.05) in both groups, and more in the treatment group than in the control group (P＜0.05). ConclusionThe Spasticity Therapeutic Apparatus can facilitate the recovery of talipes varus of the patients with stroke.

Objective To assess clinicopathological features and prognosis of patients with combined hepatocellular and cholangiocarcinoma (cHCC-CC). Methods Clinicopathological and follow-up data of 115 cHCC-CC patients confirmed pathologically in Liver Cancer Institute of Fudan University from 1995 to 2007 were analyzed. Kaplan-Meier method was used to calculate 1-,3- and 5-year survival rates and tumor-free survival rates. Survival curves were analyzed using the log-rank test. The factors that impacted the prognosis of cHCC-CC were estimated. Results In 115 cases, one was Allen's type A, one was Allen's type B, and the other 113 were Allen's type C. Being with male in predominance, most of the cHCC-CC patients had liver cirrhosis background. They presented with elevated AFP or CA19-9, vascular invasion, resembling hepatocellular carcinoma(HCC)as well as lymph nodes metastasis. One-, 3-, 5-year survival rates of 115 patients were 68. 1%, 38. 1% and 33.6%, respectively, with median survival time of 13.0 months. Whereas the 1-, 3-, 5-year survival rates in radical resected patients were 78.4 % ,44.4 % and 44.4 % ,respectively, with median survival time of 16.0 months. Tumor free survival time at 1-, 3- and 5-year was 57.8 %, 12.6 % and 0.0 %,respectively,with median recurrent time of 10.0 months. One-, 3-, 5-year survival rates of 10 nonsurgical patients were 10/10,10/10 and 0/10,respectively, with median survival time of 5.3 months.TNM stage was independent factor for prognosis of the patients after resection. Whereas the lymph nodes involvement was independent factor for the tumor free survival time of radical resected patients.Conclusions Although clinicopathological characteristics of cHCC-CC are more similar to those of HCC, the prognosis of cHCC-CC is more unfavorable than that of HCC. TNM stage is an independent determinant of long time outcome for patients after resection.

Objective To investigate the relationship between serum homocysteine (Hcy) and large artery atherosclerosis (LAA).Medods The consecutive stroke patients with LAA and small artery occlusion (SAO) were enrolled.The demographics,baseline clinical data,and laboratory test results were compared.Multivariate logistic regression analysis was used to identify the independent risk factors for LAA.Spearman correlation analysis was used to analyze the correlation between serum Hcy concentration and carotid plaque scores.Results A total of 117 patients with LAA and SAO were enrolled,42 were in the LAA group and 75 were in the SAO group.The plasma Hcy levels (median [interquartile range] 15.35 [12.20-20.43] μmol/Lvs.12.40 [10.40-15.00] μmoFL; Z=-2.540,P=0.011),the carotid plaque scores (6.30 ±4.11 mm vs.4.19 ±2.84 mm; t =2.952,P=0.004),and the proportion of plasma Hcy level at tertile groups of the patients (x2 =10.697,P =0.005) of the LAA plasma group were all significantly higher than those of the SAO group.Multivariate logistic regression analysis showed that the plasma Hcy concentration was in the third tertile (＞ 15.3 μmol/L),and it was an independent risk factor for LAA (odds ratio 4.739,95％ confidence interval 1.696-13.241; P =0.003).Moreover,there was a significant positive correlation between the serum Hcy concentration and the carotid plaque scores (r =0.245,P =0.008).Conclusions The plasma Hcy levels were positively correlated with the severity of carotid atherosclerosis.The Hcy ＞ 15.3 μmol/L was an independent risk factor for LAA.

Objective To investigate hospital staffs' appraisals on public hospitals reform.Methods 890 medical staffs were randomly investigated by questionnaire,to learn their comments on the implementation of reform measures,public-benefit nature and reform outcomes of the reform,as well as problems found with the government and improvement suggestions,and difficulties encountered in such a reform.Results The hospital staffs' appraisals on the reform tend to be low,as 36.4％ of them hold that the reform is less effective in its outcomes or a mere formality (19.8％).The staff blamed the lack of system breakthrough (67.9％),lack of financial support (61.1％),and insufficient support from the doctors as stakeholders (49.1 ％),for most of the problems of the reform.Conclusion It is suggested to win over support of the medical staff for the reform; to improve the laws and policies related to the reform; and to establish an effective supervision system for public hospitals.

OBJECTIVE:To determine the plasma mycophenolic acid concentration by HPLC, and study the multidoses pharmacokinetics character of mycophenolic acid in Chinese kidney transplantation patients. METHODS: The samples were precipitated with acetonitrile before injection. Diamonsil C18 column was used. The mobile phase consisted of acetonitrile-10 mmol?L-1 KH2PO4 (5∶6) at a flow rate of 1.1 mL?min-1. The detection wavelength was 254 nm and the column temperature was 40 ℃. This method was used to determine the multidoses pharmacokinetics in 12 kidney transplantation patients. RESULTS: MPA was well-separated from internal standard in chromatography, and endogenous foreign substance in plasma had no interference on the determination. The liner range for MPA was 0.38～59.00 ?g?mL-1,and the lowest detectable concentration of MPA was 0.38 ?g?mL-1. The recovery rate stood at 89.32%～97.63%; Both intra-day and inter-day RSD were less than 8.00%. Significant individual difference was noted among the patients treated with MMF in pharmacokinetic results, which was in line with the literature. CONCLUSION: This method is accurate and simple and applicable for the pharmacokinetics study of mycophenolic acid.

Objective To establish a novel flow cytometric crossmatch assay allowing detection of complement-fixing donor specific anti-HLA IgG alloantibodies (Flow cytometry complement-dependent crossmatch,Flow-CDC). Methods One hundred pretransplant crossmatchings were performed using Flow-CDC and NIH-CDC between 62 patients awaiting renal transplantation and 33 donor cells.These crossmatchings were divided into two groups according to PRA.Group 1 consisted of 25 sera with negative PRA,and group 2 consisted of 75 sera with positive PRA.All of the sera were pretreated with DTT to inactivate IgM. Lymphocytes were isolated from peripheral blood (or,in a few instances,from the spleen) of the cadaveric donors. The correlation between different techniques for detection of donor specific anti-HLA antibodies was evaluated.The effect of both methods on clinical transplantation outcome was observed. Results In group 1,NIH-CDC and Flow-CDC were negative for all 25 sera.In group 2,24 (32.0%) had a positive NIH-CDC,31 (41.3%) had a positive Flow-CDC.There was a significant difference between two methods (?2=5.14, P= 0.016 ).Overall concordance between both tests was 93% with 69 concordant negatives and 24 concordant positives. The correlation coefficient (r) was 0.80.In group 2,5 patients received transplantation. One of them with negative NIH-CDC and positive Flow-CDC suffered from acute rejection after transplantation and lost the graft,and the other patients with negative NIH-CDC and Flow-CDC had good outcome. Conclusions Flow-CDC can detect specifically complement-fixing IgG alloantibodies against donor HLA and is more sensitive than NIH-CDC.Additionally,the computer printouts represent a permanent record of the crossmatch for retrospective review.Flow-CDC may become the standard crossmatch method as a possible alternative to conventional NIH-CDC testing.