OBJECTIVE:To establish a method for the determination of dendrobine and its metabolites M-250 and M-280 in mice plasma for the first time. METHODS:Mice were given dendrobine 60 mg/kg by intragastric administration,1 h later plasma were collected and treated. Using pseudoephedrine hydrochloride as internal standard and dendrobine reference substance as control, the plasma concentrations of dendrobine and its metabolites M-250 and M-280 were determined by UPLC-MS combined with quantitative analysis of multi-components by single marker. The separation was performed on Hypersil Gold C18 column with 0.1％formic acid-acetonitrile(gradient elution)at the flow rate of 0.3 mL/min. The column temperature was set at 40℃,and sample size was 5 μL. Heatable electrospray ionization (HESI) source, scan/ESI + were applied and operated in positive ion mode with atomization temperature of 300℃,ion transmission tube temperature of 350℃,the sheath gas velocity of 35 arb,the auxiliary air velocity of 15 arb,the spray voltage of 3.5 kV,the collision voltage of 30,40,50 eV. The mass-to-charge ratio of detection range were 100-1500. RESULTS:The endogenous substances of mice plasma had no interference with the content determination of dendrobine and its metabolites M-250 and M-280. The linear range of dendrobine were 9.13-912.94 ng/mL(r=0.9996). The limit of quantitation was 3.04 ng/mL. RSDs of intra-day and inter-day were all less than 7.5％(n=5 or n=3). The accuracy were 96.8％-107.5％(n=5). Matrix effects were 97.1％-106.0％(RSD=1.8％-4.7％,n=5). RSDs of the content of sample at 15℃ for 24 h,at -70 ℃ after three times freeze-thaw,at -70 ℃ for 15 d were lower than 12.8％ (n=3). The content of dendrobine in plasma sample of mice was (41.3 ± 5.7) ng/mL (n=12). The contents of its metabolites M-250 and M-280 were (493.0 ± 73.1) and (41.4 ± 3.0) ng/mL (n=12) with Relative correction factor of 1.0. CONCLUSIONS: The method is sensitive and accurate,and can be used for content determination of dendrobine and its metabolites M-250 and M-280 in mice plasma.

Objective To label granulocytes in a state of differentiation in mouse bone marrow (BM) with EdU (5-ethynyl-2′-deoxyuridine) for further understanding the changes in granulocyte produc-tion at different stages of differentiation during inflammation. Methods C57BL/6 mice were intraperitoneal-ly (i.p.) injected with EdU and heat-inactivated Escherichia coli(HI E.coli). BM cells were harvested at different time points after HI E.coli injection and then stained with fluorescent-conjugated antibodies(Abs). Myeloblasts,promyelocytes,myelocytes, metamyelocytes and band and segmented neutrophils were identi-fied by fluorescence-activated cell sorting(FACS). The percentage of EdU-positive cells in each population was recorded. Results The percentage of EdU-positive myeloblasts in mice increased by 10.0% at 24 h af-ter intraperitoneal injection with HI E.coli,but decreased by 75.0% and 23.0% at 48 h and 72 h,respec-tively. The percentage of EdU-positive promyelocytes declined by 23.0%,54.5%,64.3% and 77.8% at 24 h,48 h,72 h and 96 h,respectively. The percentage of EdU-positive myelocytes increased by 60.0% and 10.0% at 24 h and 48 h,but decreased by 80.0% and 90.0% at 72 h and 96 h. The percentage of EdU-positive metamyelocytes increased by 50.0% at 24 h,but decreased by 33.3%,61.5% and 66.7% at 48 h,72 h and 96 h. The percentage of EdU-positive band and segmented cells increased by 14.0% at 24 h,but decreased by 50.0%, 77.8% and 88.0% at 48 h, 72 h and 96 h. Conclusion Emergency granulopoiesis occurred 24 h after the establishment of HI E.coli-induced model of acute peritonitis, which meant that the proliferation of myeloid precursor cells,especially that of myelocytes and metamyelocytes,was accelerated and resulted in increasing number of mature neutrophils immigrating to sites of inflammation.

Aim Toexamineliverdamagebyrifampi-cin and hepatic gene expression related to bile acid me-tabolisminmice.Methods Adultmalemicewere given rifampicin(180 mg·kg-1 ,po)daily for 30 days and(90 mg·kg-1 ,po)daily for 90 days,blood bio-chemistry,histopathology,and gene expression were examined.Results Rifampicinincreasedanimalliver index and serum enzyme activities. Histopathology showed steatosis and spotted feathery-like degenera-tion.Rifampicin increased the expression of CYP7A1 after 30 and 90 days of administration,along with in-creased FXR and SHP.Rifampicin reduced the expres-sion of BSEP after 30 days of high dose administration. Conclusion Repeatedadministrationofrifampicin may cause liver injury and intrahepatic cholestasis in mice,and these effects are associated with the altera-tion of gene expression related to bile acid metabolism.

OBJECTIVETo clarify the role of endothelial cells (ECs) injury induced by anti-endothelial cell antibody (AECA) in allogeneic hematopoietic stem cell transplantation (allo-HSCT).

METHODSSerum immunoglobulin (IgG) from allo-HSCT recipients were purified and incubated with human umbilical vein vascular endothelium (HUVEC) in vitro, then the functional changes and cell apoptosis were tested.

RESULTSAfter incubation with AECA positive IgG, soluble adhesion molecules significantly elevated in culture supernatant. When concentration of IgG was 160, 320, and 640 μg/ml, concentrations of soluble intercellular adhesion molecule-1 in supernatant were statistically higher in AECA positive groups [(117.10 ± 12.82) vs (78.17 ± 4.90) pg/ml, (151.30 ± 15.35) vs (89.46 ± 6.02) pg/ml, (239.00 ± 32.53) vs (127.80 ± 13.86) pg/ml, P<0.01)]. When concentration of IgG was 40, 80, 160, 320, and 640 μg/ml, concentrations of soluble vascular cell adhesion molecule-1 in supernatant were also statistically higher in AECA positive groups [(38.51 ± 3.76) vs (24.78 ± 2.59) pg/ml, (61.34 ± 6.99) vs (38.20 ± 3.17) pg/ml, (135.60 ± 24.46) vs (63.73 ± 5.08) pg/ml, (221.30 ± 29.40) vs (112.80 ± 8.91) pg/ml, (420.90 ± 31.70) vs (224.40 ± 20.79) pg/ml, P<0.01]. Clotting activity factors also elevated in culture supernatant after incubation with AECA positive IgG. When concentration of IgG was 80, 160, 320, and 640 μg/ml, concentrations of von Willebrand factor were statistically higher in AECA positive groups [(19.51 ± 0.72) vs (17.17 ± 0.60) ng/ml, P=0.0193; (22.97 ± 1.18) vs (18.27 ± 0.61) ng/ml, (26.40 ± 1.54) vs (19.53 ± 0.70) ng/ml, (34.35 ± 1.60) vs (23.81 ± 0.92) ng/ml, P<0.01]. When concentration of IgG was 320 and 640 μg/ml, concentrations of thrombomodulin were statistically higher in AECA positive groups [(57.50 ± 4.50) vs (40.31 ± 4.39) pg/ml, P=0.0132; (59.18 ± 4.11) vs (38.84 ± 5.16) pg/ml, P<0.01]. However, inflammatory factors (IL-1β, IL-6, IL-8 and ANG2) were not statistically different in AECA positive and negative groups (P>0.05). Moreover, IgG from AECA positive samples did not change the proliferation or cell apoptosis.

CONCLUSIONAECA from allo-HSCT recipients dysregulates ECs' function in vitro, but do not induce apoptosis, which is valuable in the pathophysiology of graft-versus-host disease (GVHD) and other complications after allo-HSCT.

Allografts ; Autoantibodies ; Endothelial Cells ; Endothelium, Vascular ; Graft vs Host Disease ; Hematopoietic Stem Cell Transplantation ; Humans ; Intercellular Adhesion Molecule-1 ; Interleukin-6 ; Umbilical Veins ; von Willebrand Factor

Objective Gitelman Syndrome is a disease caused by the mutation of Na-Cl cotransporter gene(SLC12A3).The article studied the significance of diagnosis and identification by genetic mutation. Methods We collected the clinical data, then we sequenced the SLC12A3 gene by the first sequencing technology and MLPA. Results SLC12A3 complicated heterozygotic mutation was observed.One of them showed c.1964G>A, p.(Arg655His) and exon 8 deletion mutation, the other showed c.2543A>T, p.(Asp848Val) and c.976delG, p.(Val326fs) mutation of SLC12A3 gene in children. Conclusion The final diagnosis depended on gene diagnosis. Pediatrician must recognize the manifestations to advoid misdiagnosis.

Objective To explore a novel and highly specific small-molecule TNFβinhibitor by using computer-aid?ed virtual screening and cell-based assays in vitro. Methods Computer-aided drug design and virtual screening were used to design and identify chemical compounds that targeted TNFβbased on the crystal structure of the TNFβ-TNFR1 com?plex. The effect of the small-molecule compound against TNFβ-induced cytotoxicity of L929 cells was detected by MTT as?say, and the efficacy of the compound to inhibit TNFβ-induced apoptosis of L929 cells was determined by flow cytometry as?say. The impact of the compound on L929 cell cycle was examined by Propidium Iodide (PI) staining and flow cytometry, and the influence of the compound on TNFβ-triggered signal pathway was analyzed by Western blot assay and Ultra VIEW VOX 3D Live Cell Imaging System. Results No.35 compound (named as C35 thereafter) could effectively inhibit TNFβ-induced cell death in a dose dependent manner, and the half-maximum inhibition concentration (IC50) was 8.19μmol/L. Furthermore, C35 had lower cytotoxicity and minimal effect on L929 proliferation. Here we further revealed that C35 could affect TNFβ-induced apoptotic pathway by blocking the activation of Caspase 3, and markedly reduce L929 cell apoptosis induced by TNFβ. Conclusion A novel TNFβsmall-molecule inhibitor was identified by combining computer-aided virtual screening with functional assays, and which could block TNFβ-triggered apoptotic pathway and efficiently inhibit the cell death in?duced by TNFβ.

Objective Mizoribine ( MZR) is a new immunosuppressant , however , little domestic research has been done on MZR for treatment of nephrotic syndrome in children .This study was to investigate curative effect and adverse reaction of MZR in the treatment of children with frequently relapsing nephrotic syndrome , using prospective controlled trials . Methods A total of 59 pa-tients with frequency relapsing nephrotic syndrome were randomly divided into two groups .29 patients of treatment group were treated with MZR +glucocorticoid , while 30 patients of control group were given Tripterygium wilfordii ( TW)+glucocorticoid treatment , and the course of treatment lasted for 12 months.24-hour urine protein, urinary N-acetyl β-glucosidase (NAG), serum albumin, serum cholesterol, serum creatinine, recurrence frequency, and average prednisone dosage were observed . Results At the end of treat-ment, Serum albumin in treatment group was higher than that in control group [(40.95 ±6.12)g/L vs (30.25 ±9.02)g/L], and Se-rum cholesterol ([5.45 ±0.82]mmol/L vs [7.53 ±2.74]mmol/L), urinary protein ([0.89 ±0.52]g/24 h vs [1.63 ±2.02]g/24 h), urinary NAG enzyme ([21.43 ±14.16]U/g· Cr vs [41.67 ±12.35]U/g· Cr) levels were lower compared with control group . There was significant difference between the two groups .In terms of mean recurrence times , no significant difference was found at 6th months of follow-up between the two groups, however, treatment group had lower recurrence rate than control group at 3rd month, 9th month, 12th month of follow-up, which was of significant difference .The average amounts of hormone of treatment group were lower than those of control group ([0.56 ±0.16] mg/kg· d vs [0.72 ± 0.34]mg/kg· d)、([0.64 ±0.35]mg/kg· d vs [0.67 ±0.52]mg/kg· d)、([0.53 ±0.41] mg/kg· d vs [0.83 ±0.37] mg/kg· d)、([0.34 ±0.15] mg/kg· d vs [0.54 ±0.26] mg/kg· d) at 3rd month, 6th month, 9th month, 12th month of follow-up, which was of significant difference . Conclusion Compared to Tripterygium wil-fordii combined with hormone therapy , MZR combined with prednisone therapy in children with recurrent NS frequency can reduce the relapse rate and dosage of corticosteroid to improve the clinical remission rate .

To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Solubility ; Umbilical Veins ; cytology ; Vascular Endothelial Growth Factor Receptor-1 ; genetics ; isolation & purification ; metabolism

We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.
ATP Binding Cassette Transporter, Sub-Family B ; immunology ; Antibodies, Bispecific ; biosynthesis ; chemistry ; genetics ; immunology ; Binding, Competitive ; CD3 Complex ; immunology ; Cell Line ; Disulfides ; chemistry ; Drug Stability ; Escherichia coli ; genetics ; metabolism ; Humans

ObjectiveTo investigate the clinical application of tacrulimus (TAC, FK506) in children with primary nephrotic syndrome (NS). MethodsSixty-five primary NS children received routine or decreased-dosage glucocorticosteroid according to clinical NS types after hospitalization. At the same time, TAC was given orally with the dosage of 0.1 to 0.15 mg/kg, once every 12 hours, for 6 to 24 months. And the serum concentration of TAC was monitored during the course. ResultsAfter the treatment of TAC for 1 to 2 months, 65 patients were recovered with gradually reduced urinary protein, rapidly increased serum albumin, and improvement of cholesterol and triglycerides. Total remission rate was 83.1% and onset time was 7 to 54 days. Twelve cases experienced recurrence. Increased CD4, as well as 3/3 or 3/1 TAC genotype, indicated higher remission rate. Various pathological types had different remission rates or ratio, which were as follows: minimal change nephropathy (96.4%), mesangial proliferative glomendonephritis (90.0%), membranous nephropathy (2/3), membranous proliferative glomerulonephritis (3/5), focal segmental glomerulosclerosis (4/9). The patients would recover in the course of treatment under the conditions of TAC initial dose as 0.1 to 0.15 mg /kg per 12 hours and controlled serum concentration as 5 to 10 g/L. During the treatment, 12 cases appeared gastrointestinal symptoms, mainly as anorexia, nausea and vomiting, 1 abdominal pain, 2 headache, 1 tremor, 1 paresthesia, 3 insomnia, 4 transient increased Scr, 8 slightly increased NAG, 6 increased C3 and α-2 macroglobulin. The symptoms disappeared within one week or after stopping TAC. ConclusionsTAC is effective in primary NS children, even with abnormal liver function or tuberculosis infection. TAC can also be a substitute to cyclosporine A.