Background and purpose:Bispecific antibody is a special pattern of genetic engineering antibody.It possesses 2 antigen binding sites, one directed at the effecter cells and the other at the target cells which are always the tumor cells.This enables the effecter cells to recruit to the targets efficiently and exert cytotoxic effect.The experiment was aimed to construct the expression vector of anti-CD3?anti-CD19 bispecific antibody, purification and analyzing the activity of the diabody.Methods:Applyed PCR and overlap PCR to construct the expression vector of anti-CD3?anti-CD19 bispecific antibodies, transformed it into E.coli 16C9 for expression.Purified the diabody by His-tag affinity chromatography and confirmed by 12% SDS-PAGE and Western blot.Antigen binding activity of the diabody was analyzed by FACS.Results:The DNA sequence of the expression vector containing anti-CD3?anti-CD19 fragment was comfirmed.The yield of purified diabody was up to 5 mg/L.The molecular weight of diabody was correctly indicated by SDS-PAGE and Western blot.The diabody could specifically bind to CD19+ Raji cells and CD3+ Jurkat cells while competitively inhibit binding of HIT3a and HIT19a to the cells.Conclusion:Successfully constructed the expression vector of anti-CD3?anti-CD19 bispecific antibodies, the diabody acquired by the method could be produced with high level expression and also had high specific affinity with CD19+ Raji cells and CD3+ Jurkat cells.

Animals ; Carbon Radioisotopes ; Diabetes Mellitus, Experimental ; genetics ; metabolism ; Diabetes Mellitus, Type 1 ; genetics ; metabolism ; Gene Expression Regulation ; Glucose ; administration & dosage ; metabolism ; Lipid Metabolism ; Liver ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Transcriptome

OBJECTIVE:To investigate the differences of the active components of different parts of rhubarb(the head,the body and the end part of the root)from genuine medicinal materials in Gansu province so as to provide theoretical basis for its quality control.METHODS:A comparative study of the fingerprints of different parts of rhubarb extract were conducted by HPLC,in which,the C18 was used as the chromatographic column under room temperature,the mobile phase consisted of menthol-0.1%H3PO4(70∶30)with a flow rate of 1.0ml/min,the column's temperature of room temperature and detection wavel_ ength of 280nm.RESULTS:Significant differences were noted in the HPLC fingerprints of different parts of rhubarb,there were significant differences in the contents of the active components such as emodin,chrysophanol,etc.,the contents decreased progressively from the head of root,the body part to the end part.CONCLUSION:In order to ensure the medical value and the quality of rhubarb medicinal material,different parts of which should be differentiated in the quality control.

Objective To investigate the clinical and mammographic features of plasma cell mastitis.Methods Twenty-five patients(28 lesions)with histologically confirmed plasma cell mastitis, aged from 26 to 70 years(mean age 41 years),were examined with X-ray mammography.The clinical manifestations and imaging features were retrospectively reviewed.Results No case was in lactation.The painful irregular masses,ranged from 1.3 to 8cm in size,were found in 22 patients,while 3 patients with acute episode.Recurrent episodes of breast masses were noted in 4 patients.Based on the mammographic appearances,the plasma cell mastitis were classified as the following four types:inflammation-like type (2/28),ductal ectasia type(3/28),focal infiltration type(10/28)and nodular type(13/28).The valuable radiogyaphic signs:(1)An asymmetrically increased density along the lactiferous duct with a flame-like appearance,inhomogeneous low density tubular structures and scattered stick-shape calcifications.(2) Architectural distortion and oil cysts formation in adjacent area,(3)Subareolar ductal ectasia.Conclusions The clinical and mammographic characteristics of plasma cell mastitis are critical to avoiding unnecessary surgery.Histopathological result is needed for the diagnosis in patients highly suspected of malignancy.

OBJECTIVETo explore the influence of exogenous putrescine and cadaverine on pro-inflammatory factors in the peripheral blood of rabbits.

METHODSForty ordinary adult New Zealand rabbits were divided into saline, necrotic tissue homogenate (NTH), putrescine, and cadaverine groups according to the random number table, with 10 rabbits in each group. Saline, NTH, 10 g/L putrescine, and 10 g/L cadaverine were respectively peritoneally injected into rabbits of corresponding group in the amount of 1 mL/kg. The blood sample in the volume of 2 mL was collected from the central artery of rabbit ears before injection and at 2, 6, 12, 24, 30, 36, 48, 60 hours post injection (PIH). Contents of TNF-α, IL-1, and IL-6 in the serum were determined with enzyme-linked immunosorbent assay. Data were processed with repeated measurement data analysis of variance and Spearman correlation analysis, and cubic model curve was applied in curve fitting for the contents of inflammatory factors.

RESULTS(1) The serum contents of TNF-α, IL-1, and IL-6 were increased in NTH, putrescine, and cadaverine groups in different degrees at most post injection time points. There was no significant change in the concentrations of the three pro-inflammatory factors in saline group, and they were significantly lower than those of the other three groups at most post injection time points (with F values from 3.49 to 13.58, P values all below 0.05). The serum contents of TNF-α, IL-1, and IL-6 in putrescine group began to increase at PIH 2, 6, and 6, which was similar to the trend of NTH group, but the changes were delayed compared with those of cadaverine group(all at PIH 2). The peak values of TNF-α, IL-1, and IL-6 in putrescine group were respectively (339 ± 36), (518 ± 44), and (265.9 ± 33.5) pg/mL, which were significantly lower than those of cadaverine group [ (476 ± 86), (539 ± 22), and (309.4 ± 27.1) pg/mL], with F values respectively 5.11, 1.90, and 5.56, P values all below 0.05. (2) The period of time in which contents of TNF-α, IL-1, and IL-6 began to increase (PIH 3-4) and the peaking time of the three pro-inflammatory cytokines (PIH 18-30) in putrescine group appeared later than those of cadaverine group (PIH 2 and 12-30). The duration of peaking time of the three pro-inflammatory cytokines in putrescine group was shorter than that of cadaverine group (PIH 18-30 vs. PIH 12-30). The increasing period and the duration of peaking time of TNF-α, IL-1, and IL-6 in putrescine group were close to those of NTH group (PIH 3-5 and 18-30). The correlation coefficient test analysis showed that the trends of changes in contents of three pro-inflammatory cytokines in putrescine group were significantly correlated with those of NTH group (r(TNF-α) = 0.933, P < 0.01; r(IL-1) = 0.967, P < 0.01; r(IL-6) = 0.950, P < 0.01). The obvious correlation between cadaverine group and NTH group was only found in the contents of IL-1 and IL-6 (r(IL-1) = 0.913, P < 0.01; r(IL-6) = 0.883, P < 0.05).

CONCLUSIONSBoth exogenous putrescine and cadaverine can cause inflammatory reaction in rabbits. The trend of the inflammatory reaction induced by putrescine was similar with that by NTH, suggesting that putrescine may play a leading role in the inflammatory reaction induced by necrotic tissue decomposition.

Animals ; Cadaverine ; adverse effects ; Inflammation ; blood ; Interleukin-1 ; blood ; Interleukin-6 ; blood ; Necrosis ; blood ; Putrescine ; adverse effects ; Rabbits ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo investigate the effect of decreased expression of high mobility group Box-1 on the proliferation and apoptosis of HepG2 cells.

METHODSThree specific siRNAs of HMGB1 were designed and synthesized, and were transiently transfected into HepG2 cells by Lipofectamine 2000. The HMGB1 expression in HepG2 cells was detected by RT-PCR and Western blotting respectively. The proliferation activity in vitro was assessed by MTT assay. In situ apoptosis was evaluated by terminal deoxynucleotidyl transferase-deoxyuridine triphosphate nick end labeling (TUNEL) assay.

RESULTSAll of these specific HMGB1-siRNAs (1, 2, 3) efficiently and specifically inhibited the expression of the HMGB1 gene, and the levels of HMGB1 mRNA were 1.147+/-0.024, 1.014+/-0.042, 0.435+/-0.055, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in Lipofectamine 2000 alone group (1.411+/-0.065, P < 0.01). Correspondingly, all of these specific HMGB1-siRNAs (1, 2, 3) could efficiently and specifically inhibit the expression of the HMGB1 protein, and the levels of HMGB1 protein were 0.369+/-0.035, 0.340+/-0.028, 0.097+/-0.020, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in Lipofectamine 2000 alone group (0.553+/-0.051, P < 0.01). Of the 3 specific HMGB1-siRNAs, HMGB1-siRNA-3 (siRNAH3) had the highest inhibition rate (80%). The proliferation of HepG2 cells was markedly inhibited by siRNAH3 transfection. Compared to mock-transfection, siRNAH3 transfection dramatically suppressed the proliferation of HepG2 cells (P < 0.01). Moreover, siRNAH3 can induce apoptosis (P < 0.01).

CONCLUSIONsiRNA targeting HMGB1 mRNA can specifically reduce HMGB1 gene and protein expression. siRNAH3 can effectively suppress the proliferation and induce apoptosis of HepG2 cells.

Apoptosis ; Cell Proliferation ; HMGB1 Protein ; genetics ; Hep G2 Cells ; Humans ; RNA, Small Interfering

Objective To study the effect of intranasal(IN)delivery of nerve growth factor(NGF) on pyriform cortex of satin-poisoned rats.Methods Sprague-Dawley rats were treated with satin and atropine sulphate, pralidoxime to establish satin-poisoned rat model.Then NGF or saline was administered via the olfactory pathway.24 hours later, damaged and residual healthy neurons were estimated and quantified on pyriform cortex using hematoxylin-eosin(HE)staining and neuronal nuclei antigen(NeuN) immunohistochemistry.Results A massive quantity of degenerating neurons were seen in the pyriform cortex of rats with intranasal saline.And compared to the normal rats, the number of neurons of rats with intranasal saline was significantly reduced by 39.44% [(404.75?25.17)/mm~2].But the number of neurons in rats with intranasal NGF [(651.94?36.02)/mm~2] didn't change significantly compared to the normal rats.Conclusion Intranasal delivery of NGF, reducing the degenerating neurons on pyriform cortex of satin-exposure rats, is a potential treatment for satin intoxication.

OBJECTIVETo study the anti-tumor effects of all-trans retinoic acid (ATRA) and mechanisms of its action.

METHODSHuman esophageal carcinoma cell line EC9706 cells were treated with ATRA at different concentration. The proliferation inhibition was examined by MTT assay. Morphological examination, TUNEL method and flow cytometry were used to detect the apoptosis and changes of cell cycle. Immunohistochemical method was used to detect the expression of apoptosis-related genes caspase-3 and bcl-2. The semi-quantification of protein expression was analyzed by pathological image analysis.

RESULTSATRA inhibited the proliferation of EC9706 cells moderately. Apoptosis in EC9706 cells was induced by ATRA treatment. The morphology of EC9706 cells showed changes such as nuclear chromatin condensation and fragmentation. Sub-G1 peak was found by flow cytometry. The maximal apoptosis rate was 32.6%. The expression of caspase-3 gene was enhanced. The expression of bcl-2 gene was decreased. All these effects were presented in a dose-dependent and time-depend manner.

CONCLUSIONApoptosis is one of the key mechanisms of ATRA action on EC9706 cells.

Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tretinoin ; administration & dosage ; pharmacology

This paper is aimed to develop rapid, sensitive and convenient HPLC-MS/MS methods for the quantification of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma. According to the different natures of the compounds, two sets of liquid chromatography and ionization modes were used for determination the concentration of levosimendan and its metabolites OR-1855 and OR-1896 in human plasma, separately. Following protein precipitation with methanol, the levosimendan and internal standard (rosuvastatin) were separated on a Capcell MG III C18 column (35 mm x 2.0 mm ID, 3 microm) with the mobile phase consisted of methanol-15 mmol x L(-1) ammonium acetate-formic acid (55: 45: 0.02, v/v/v). A tandem mass spectrometer equipped with electrospray ionization source was used as the detector and operated in the negative ion mode. Its metabolites OR-1855, OR-1896 and internal standard doxofylline were extracted from plasma by liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Zorbax Extend C18 column (150 mm x 4.6 mm ID, 5 microm) with the mobile phase consisted of methanol-15 mmol x L(-1) ammonium acetate-formic acid (65 :35 :0.1, v/v/v). A tandem mass spectrometer equipped with electrospray ionization source was used as the detector and operated at the positive ion mode. The linear concentration ranges of the calibration curves for levosimendan and OR-1855 and OR-1896 were 0.10-50.0 ng x mL(-1), 0.20-100 ng x mL(-1), 0.20-100 ng x mL(-1), respectively. The lower limits of quantification of levosimendan and OR-1855 and OR-1896 were 0.10 ng x mL(-1), 0.20 ng x mL(-1), 0.20 ng x mL(-1), respectively. The methods proved to be sensitive, simple and rapid, and suitable for the pharmacokinetic study of levosimendan injection.
Acetamides ; blood ; Cardiotonic Agents ; blood ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Humans ; Hydrazones ; blood ; metabolism ; Male ; Pyridazines ; blood ; metabolism ; Spectrometry, Mass, Electrospray Ionization ; methods ; Tandem Mass Spectrometry ; methods

OBJECTIVETo study the immunoactivity,biodistribution and metabolic pattern of (131)I-Herceptin in rabbits.

METHODSHerceptin was radiolabelled with (131)I and its radiochemicalpurity (RCP) measured by size-exclusion high-pressure liquid chromatography (HPLC). The binding rate to BT-474 cells was measured to evaluate the immunoactivity of (131)I-Herceptin. (131)I-herceptin (2.0 mCi/kg) was injected intravenously into New Zealand rabbits. Scintigraphy on emission computed tomography was performed at 3 h, 1, 3 and 5 days after injection, and the radiocounts of the heart, liver and kidney etc. were compared with that of the muscle to calculate the organ-to-muscle activity ratio (O/M). On the fifth day,the rabbits were killed and the blood, myocardium, lung and other organs were obtained for measuring the radiocounts on gamma-counter to calculate the uptake percentage per gram tissue (ID%/g).

RESULTSThe labeling rate of (131)I-herceptin was 93% with RCP of 95% and binding rate to BT-474 cells of 36.9%. After injection of (131)I-herceptin, the heart, lung and liver displayed dense radioactive regions but not the muscles and intestines. Three hours after injection, the O/M ratio of the heart was significantly higher than that of the lung, kidney and intestine (P<0.05), but decreased significantly one day after injection (t=10.817, P<0.001) with further decrement on days 3 and 5 (P<0.05). The O/M ratio of liver on day 1, 3, and 5 reduced significantly in comparison with that at 3 h (P<0.05). The uptake percentage was higher in the blood (11.3 ID/g%) than in the liver (2.8 ID/g%) and the myocardium (1.8 ID/g%).

CONCLUSIONS(131)I-herceptin possesses high immunoactivity which distributes mainly in the blood, liver and kidney, but with low uptake in the myocardium.

Animals ; Antibodies, Monoclonal ; administration & dosage ; metabolism ; pharmacokinetics ; Antibodies, Monoclonal, Humanized ; Antineoplastic Agents ; administration & dosage ; pharmacokinetics ; standards ; Binding, Competitive ; Cell Line, Tumor ; Chromatography, High Pressure Liquid ; Female ; Humans ; Injections, Intravenous ; Iodine Radioisotopes ; administration & dosage ; metabolism ; pharmacokinetics ; Male ; Quality Control ; Rabbits ; Time Factors ; Tissue Distribution ; Trastuzumab