Objective:To establish a headspace GC method for the determination of 7 kinds of residual solvents in bicalutamide, including dichloromethane, n-hexane, tetrahydrofuran, ethanol, ether, acetone and ethyl acetate. Methods: The residual solvents in the substance were determined by GC equipped with an FID detector and linked with an Agilent DB-624 capillary column (30. 0 m × 0. 25 mm × 1. 4 m). The inlet temperature was 200℃ and the FID detector temperature was 250℃. The column temperature was raised by program:the initial temperature was 35℃, maintained for 13 min, raised to 180℃ with a rate of 100℃/min, and maintained for 5 min. The carrier gas was nitrogen and the flow rate was 1. 2 ml·min-1. The heated temperature of the headspace oven was 90℃, the heated time lasted 30 min, and the injection volume was 1. 0 ml. The solution medium was dimethyl sulphoxide (DMSO). Results:Each solvent could be completely separated, and the calibration curve of each solvent showed good linear relationship with good accuracy. Conclusion:The method can be applied for the determination of residual solvents in bicalutamide.

Objective To generate the spaO-ompA fusion gene of Salmonella paratyphi A and its prokaryotic expression system,and to determine the immunoprotection of the recombinant expression product rSpaO-OmpA.Methods A flexible peptide sequence was used to link spaO and ompA genes and a prokaryotic expression system of spaO-ompA fusion gene was subsequently generated.SDS-PAGE and Bio-Rad Agarose Image Analyzer were applied to examine the expression as well as the yield of the target recombinant protein rSpaO-OmpA.The antigenicity and immunoreactivity of rSpaO-OmpA were determined using immunodiffusion test,Western Blot assay and micro-Widal's test.By a mouse infection model,the immunoprotection of rSpaO-OmpA against the lethal challenge of S.paratyphi A was determined.In the animal protective test,the recombinant expressed SpaO (rSpaO) and OmpA ( rOmpA ) were used as the controls.Results The generated spaO-ompA fusion gene had 100％ nucleotide and amino acid sequence identities compared to the single spaO or ompA gene.The constructed prokaryotic expression system IPTG E.coli BL21DE3pET42a-spaO-ompA expressed the recombinant protein rSpaO-OmpA.rSpaO-OmpA combined with the antiserum against wholecell of S.paratyphi A to present positive hybridization signal and induced specific antibody in the immunized rabbits.Immunization with 100 or 200 μg rSpaO-OmpA contributed 66.7％ (8/12) or 83.3％ (10/12) immunoprotective rates in mice when the animals were attacked with S.paratyphi A.The immunoprotective rates produced by rSpaO-OmpA were significantly higher than that of equal rSpaO or rOmpA( P＜0.05 ).The sera from rSpaO-OmpA immunized mice presented 1∶5-1∶40 agglutination titers to the H antigens of different S.paratyphi species,and 1∶1-1∶16 immunodiffusion titers to rSpaO,rOmpA and rSpaO-OmpA proteins,respectively.Conclusion The artificially fusion antigen,rSpaO-OmpA,has more powerful immunogenicity and immunoprotection that the equal rSpaO or rOmpA.

Objective To explore how neutrophil-lymphocyte ratio (NLR) is related with inflammation and atherosclerosis,and the role of NLR in hospitalization in maintenance hemodialysis (MHD) patients.Methods MHD patients treated in hemodialysis center of Sichuan Provincial People's Hospital from June till November 2013 were enrolled.Patients with severe infection,cardiovascular events and malignant carcinoma were excluded.NLR was determined from complete blood count differential.Clinical parameters such as serum albumin,lipids,intact parathormone,ferritin,C-reactive protein (CRP),25-(OH) vitamin D,interleukin-6 (IL-6) and alkaline phosphatase (ALP) were collected.Pulse wave velocity (PWV) and ankle-brachial index (ABI) were used to evaluate the arterial stiffness.Spearman analysis was used to evaluate the relationship between NLR and these parameters.All patients were divided into low NLR group (NLR≤3.25) and high NLR group (NLR ＞3.25) on the median NLR,and their differences in these indexes were analyzed.During the one-year follow-up,the reasons and rates of hospitalization and survival were analyzed.Results One hundred and thirteen MHD patients including 58 males and 55 females were enrolled with (69±49) dialysis age and (54± 15) average age.(1) The NLR was significantly correlated with whole blood count (WBC,r=0.538,P＜ 0.001),ABIL (r=0.201,P=0.033),ABIR (r=0.235,P=0.012) and total cholesterol (TC,r=-0.414,P＜ 0.001) and low-density lipoprotein cholesterol (LDL-C,r=-0.378,P ＜ 0.001).(2) Low NLR patients had increased TC,LDL-C and IL-6 as compare with high NLR patients,however decreased ABIL and ABIR (all P ＜ 0.05).(3) Forty one patients were hospitalized 63 times during the follow-up period.Annual hospitalization rate was 558/1000 and the mortality rate was 17.7/1000.(4)NLR in patients at least hospitalized once a year was significantly lower than in patients without hospitalization,while ALP was higher (all P ＜ 0.05).Compared with those in other patients,NLR and hemoglobin (Hb) were significantly lower in patients with hospitalization due to infection,while ALP was higher (all P ＜ 0.05).Conclusions NLR is related with WBC,ABI,TC and LDL-C in MHDpatients.Lower NLR may indicate high risk for cardiovascular,atherosclerosis and hospitalization,probably different form non-MHD patients,which needs more studies to verify.

Autoimmune Lymphoproliferative Syndrome ; diagnosis ; genetics ; immunology ; therapy ; Biomarkers ; blood ; Child, Preschool ; Humans ; Male ; Mutation ; Prednisolone ; administration & dosage ; therapeutic use ; Ultrasonography, Doppler, Color ; fas Receptor ; genetics

BACKGROUND:Bone tissue transplantation or osteogenic material fil ing is after used for bone defect repair. To remove autologous bone tissues can lead to additional damage and secondary deformity, therefore, it is extremely urgent to search for a new osteogenic material. OBJECTIVE:To construct the porousβ-tricalcium phosphate (β-TCP)/col agen scaffold modified with human bone morphogenetic protein 2 (hBMP2) gene, and to observe its effects on differentiation of MC3T3-E1 cel lines. METHODS:The porousβ-TCP/col agen scaffold modified with hBMP2 gene was prepared. Then in vitro culture system of MC3T3-E1 cel lines with composite scaffold was established. There were scaffold and plate groups, and each group was divided into two subgroups according to the different concentrations of plasmid. Samples were col ected and observed morphological y by scanning electron microscope and light microscope after complex culture. After 1, 3, 7 and 14 days of induction, calcium nodules were observed through alizarin red staining, the cel cycle was detected by real-time PCR, and expressions ofαI-chain col agen type I gene, Osterix and bone sialoprotein were observed. RESULTS AND CONCLUSION:The number of cel s adhered, differentated and distributed on the composite scaffold was significantly higher than that of the single scaffold (P<0.05). Alizarin red staining and real-time PCR detection showed that the osteogenesis ability of MC3T3-E1 cel lines in the scaffold group was stronger than that in the plate group. To conclude, the porousβ-TCP/col agen scaffold modified with hBMP2 gene is an appropriate candidate for bone defect repair.

Objective To explore the differentiation of allogeneic naive T cells to regulatory T cells (Tregs) and T helper (TH) 1/2/17 cells by coculture with bone marrow-derivedimmature dendritic cells (irnDC).Method Bone marrow-derived imDC were cultivated from Balb/c mice.Lipopolysaccharide-stimulated DC were harvested as mature dendritic cells (mDC) and unstimulated cells were collected as imDC.Then irnDC or mDC were cocultured with allogeniec naive T cells,respectively.TH1 cytokines [interferon-γ (IFN-γ) and interleukin (IL)-2],TH2 cytokines (IL-4 and IL-10),and TH17 cytokine (IL-17) of co-cultured cells were detected by enzyme linked immunospot assay.CD4+ Forkhead box p3 (FoxP3) + Treg proportion in CD4+ cells in the co-cultured system with IL-2 and transforming growth factor-β1 (TGF-β1) was analyzed by flow cytometry.Result As compared with mDC,na(i)ve T cells cocultured with imDC secreted much less IFN-γ (11.67 ± 2.18 vs.182.00±23.71,P＜0.01),IL-2 (26.67±2.96 vs.318.30± 18.62,P＜0.01),IL-4 (17.00±3.78 vs.45.33±3.48,P＜0.01),IL-10(7.00±1.00vs.158.70±10.90,P＜0.001) and IL-17 (0.66 ± 0.33 vs.238.30 ± 24.39,P＜0.001).Furthermore,imDC induced more CD4+ FoxP3+ Tregs than mDC after adding IL-2 and TGF-β1 in the coculture system for 7 days (22.70 ± 1.53 ％ vs.5.42 ± 1.27％,P＜0.01).Conclusion imDC are more effective to induce na ve T cells to Tregs,but not differentiate to TH 1/TH 2/TH 17 cells.These findings provide in vitro experimental evidence for induction of transplant tolerance by adoptive transfer of imDC.

Objective To investigate the relative percentage of normal cognitive function (NCF),age associated memory impairment (AAMI),mild cognitive impairment (MCI) and Alzheimer's disease (AD) in the elderly,and the correlation between cognitive function and ApoE genotypes.Methods A total of 2666 elderly people aged ≥65 years (2132 males and 534 females)were divided into 3 groups according age:65-74-year age group (925 cases),75-84 year age group (1054 cases) and 85-100-year age group (687 cases).ApoE genotypes were determined in the controls and patients with AAMI and MCI.The degrees of fundus arteriosclerosis were detected in all subjects except for patients with AD.Results There were 867 cases with NCF,860 cases with AAMI and 782 cases with MCI.The incidence of AAMI was higher in 65-74-year age group than in the other two groups (42.0％ vs.31.1,20.96).The incidences of MCI and AD were higher in 85-100-year age group than in the other groups (42.5％,13.3％).The major degrees of fundus arteriosclerosis were Ⅰ+,Ⅰ-Ⅱ°,Ⅱ in subjects with AAMI (34.7％,x2=10.02,P＜0.01) and were Ⅱ °/ Ⅱ + / Ⅲ° in subjects with MCI (34.9 ％,x2 =23.39,P＜0.001).The APOEε4 allele frequency was significantly higher in patients with MCI than in the controls (x2=8.31,P＜0.05).However,no significant differences in APOEε4 allele frequency were found between patients with AAMI and the controls.Conclusions The incidence of AAMI is highest in 65-74-year age group,while the incidences of MCI and AD are highest in 85-100-year age group.Compared to patients with AAMI,the more serious fundus arteriosclerosis and higher allele frequency of APOEε4 appear in patients with MCI.

Objective To investigate if the administration of CORM-2 can provide protection against renal ischemia-reperfusion injury (IRI).Method Murine renal ischemia was induced by clamping left renal pedicles for 40 min with vascular micro damps at 32 C,then the contralateral kidney was removed.CORM-2 or vehicle was administered via intravenous infusion 1 h before the onset of ischemia.The blood plasma and renal samples were obtained at 24 h after reperfusion to assess renal function and cellular injury.Plasma Cr and BUN levels,HE and TUNEL were performed to estimate the magnitude of renal damage.Kidneys were retrieved from indicated animals at various time points after renal IRI,and the sections were prepared for histological evaluation.MPO staining procedures were performed to assess the neutrophils infiltration in the renal IRI.Besides,Immunofluorescent stain of TNF-α was performed on the kidneys which were retrieved from indicated animals to determine the production of inflammatory mediators in renal I/R.Results The plasma Cr and BUN were significantly increased at 24 h after reperfusion in IRI control mice,and CORM-2 treatment could markedly diminish the increase of plasma Cr and BUN in mice subjected to I/R.In parallel,histological analysis demonstrated that CORM2 treatment markedly reduced apoptosis of the renal tubular epithelium cells and hemorrhage.IRI caused marked infiltration and accumulation of the MPO-positive neutrophils in renal interstitium.Administration of CORM-2 before ischemia dramatically inhibited neutrophils infiltration as compared with IRI or iCORM-2 group.Furthermore,we confirmed that CORM-2 markedly decreased production of TNF-α.Conclusion Carbon monoxidereleasing molecule CORM-2 could ameliorate inflammation to protect against the renal IRI in mice.

Objective To investigate whether rat adipose-derived stem cells (rASCs) could resist human xenoantibody-dependent complement-mediated lysis and to explore its possible mechanisms.Method SD rat ASCs were isolated,rASCs at passage 2 to 8 were used for the following studies and rat lymphocytes were harvested as control cells.α-Gal expression was detected by flow cytometry.After incubation of rASCs with 20％ normal human serum (NHS) or heat inactivated normal human serum (HINHS),flow cytometry was used to detect cytotoxicity,IgG or IgM binding,and C3c,C4c and C5b-9 deposition.Result We successfully established the method to isolate and culture rASCs.The morphology of rASCs remained unchanged after passages.rASCs were positive for tell surface markers of CD44 and CD90,while negative for CD45 and MHC-Ⅱ.As compared with rLCs,rASCs significantly resisted human natural antibody and complement-mediated lysis when incubated with 20％ NHS in vitro (20.42％ ± 2.80％ vs 51.84％ ± 6.70％,P ＜ 0.01).Mechanistically,rASCs expressed lower level of α-Gal (13.97 ± 0.33 vs.24.47 ± 3.03,P＜0.05),which was correlated with decreased binding of human xenoreactive IgG and IgM (IgM:9.4 ± 2.0 vs.107.2± 4.8,P＜0.01; IgG:5.73 ± 1.0 vs.27.49 ± 3.9,P＜0.01) and reduced deposition of complements C3c,C4c and C5b-9 (C3c:294.6 ± 38.02 vs.1924 ± 509.4,P＜0.05; C4c:35.23 ± 3.1vs.177.3 ± 37.17,P＜0.05; C5b-9:5.63 ± 1.74 vs.37.05 ± 7.4,P＜0.01).Conclusion These data demonstrated that the resistance of rASCs to human xenoantibody and complement-mediated lysis is associated with low expression of xenoantigen a-Gal and inhibition of MAC (membrane attack complex) formation.