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Objective This study is aimed at determining whether anti-von Willebrand factor (VWF) autoantibodies are present in the plasma of idiopathic thrombotic thrombocytopenic purpura (TTP) patients with normal ADAMTS13 activity and undetectable anti-ADAMTS13 antibodies,and at examining whether murine monoclonal antibodies (mAbs) against human VWF decrease the susceptibility of VWF to ADAMTS13 in vitro.Methods Anti-VWF autoantibodies and ultralarge VWF (UL-VWF) multimers were measured in plasma samples of 53 adult patients with idiopathic TTP by enzyme-linked immunosorbent assay and sodium dodecylsulphate-agarose gel electrophoresis,respectively.Moreover,the effects of eight murine mAbs to different human VWF domains on VWF cleavage by ADAMTS13 were evaluated under fluid shear stress and static/denaturing conditions,respectively.Results Anti-VWF antibodies and UL-VWF multimers were detected in two TTP patients with normal ADAMTS13 activity and undetectable anti-ADAMTS13antibodies.The SZ34,an anti-VWF mAb,inhibited VWF proteolysis mediated by ADAMTS13 under flow,but not static conditions.Conclusion Anti-VWF antibody may be one of the causes of idiopathic TTP with normal ADAMTS13 activity and undetectable anti-ADAMTS13 antibodies.
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Objective To summarize and learn the biological properties and clinical features of interdigitating dendritic cell sarcomas (IDCS).Methods The first IDCS patient concurrent with acute myelomonocytic leukemia (AML-M4) described herein,to our knowledge,was studied and 62 IDCS cases reported previously in the literature were reviewed.Results The patient had a history of breast cancer as well as radiotherapy and chemotherapy of it,and the patient showed poor response to 4 cycles of sequential chemocherapy regimens.Based on the laboratory results,IDCS and AML-M4 in this patient were both of myelogenous origination.Furthermore,review of the 62 IDCS patients reported previously showed that as high as 17 ％ of the patients had malignant disease and received radiotherapy or chemotherapy before they got IDCS,and patients of this group had worse prognosis compared with counterpart.Conclusion IDCS has poor prognosis,and therapy-related type worse.Prophylactic measures and stringent screening of the second cancer in those who received chemoterapy or radiotherapy are appropriated and necessary.
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This study was to acquire recombinant protein of von Willebrand factor cleaving protease (ADAMTS13, a disintegrin and metalloprotease with a thromboSpondin type 1 motifs 13), for further studies on its biological function in thrombosis and hemostasis. We transfected the Hela cells with the plasmid pSecTag-ADAMTS13 by lipofectamine. A positive cell cloning was selected by hygromycin-B. The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blotting respectively. We also measured the enzymatic activity of recombinant protein (rADAMTS13) by GST-His two-site ELISA assay. The results showed that we successfully constructed Hela cells ADAMTS2-4 which expressed high level of rADAMTS13. We received about 5.8 mg recombinant protein in culture supernantants per liter purified with Ni-NTA column. The protein formed a main lane at the position of 190 kDa with SDS-PAGE and reacted with polyclonal antibody against ADAMTS13 by Western blotting. The amount of rADAMTS13 activity was 6.4 U/mL, according to the normal plasma defined as 1 U/mL. In conclusion, rADAMTS13 protein had high purity, immune activity and good enzymatic activity, which could establish the experimental foundation for further research on biological function and mechanism of this unique metalloprotease.
Subject(s)
Humans , ADAM Proteins , Genetics , Metabolism , ADAMTS13 Protein , HeLa Cells , Recombinant Proteins , Genetics , Metabolism , Transfection , von Willebrand Factor , MetabolismABSTRACT
Objective To explore the expression and clinical significance of hypoxia-inducible factor 1α(HIF-1α)and glucose transporter-1(GLUT-1)in human lung adenocarcinoma cell line A549 and in elderly primary lung cancer tissue under hypoxia. Methods MTT assay was used to test the effect of CoCl2-induced hypoxia on the growth activity of lung adenocarcinoma cell line A549.Reverse transcription-polymerase chain reaction analysis(RT-PCR)and Western blot were used to determine the changes of HIF-1α and GLUT-1 in lung adenocarcinoma cell line A549 under hypoxia.The expressions of HIF-1α and GLUT-1 were detected by using immunohistochemical method and RT-PCR in 60 specimens of lung carcinoma and 26 benign lung tissues in elderly patients with pulmonary diseases.Their interrelations and their relationship with clinicopathologicaI characteristics of the tumors were analyzed. Results The growth activity of lung adenocarcinoma cell line A549 was increased under hypoxia.RT-PCR analysis indicated that the mRNA expressions of HIF-1α and GLUT-1 were 0.69±0.08,0.65±0.09 after hypoxia exposure for 24 hours,and 0.99±0.16,0.83±0.11 after hypoxia exposure for 48 hours,which were higher than those in control group (0.47±0.11,0.40±0.08,P＜0.01).The results from Western blot showed that the protein expressions of HIF-1α and GLUT-1 were 1.57±0.29,1.66±0.28 after hypoxia exposure for 24 hours,and 2.31±0.46,2.22±0.34 after hypoxia exposure for 48 hours,which were higher than those in control group(0.93±0.07,1.06±0.17,P＜0.01).In specimens of lung carcinoma,the positive expression rates of mRNA and proteins of HIF-1α and GLUT-1 were higher than those in benign lung tissues(all P＜0.01).Compared with the mRNA and protein levels of HIF-1a and GLUT-1 in lung carcinoma tissues of Ⅰ and Ⅱ stage,there were marked increases in Ⅲ and Ⅳ stage (all P＜0.05).The expressions of HIF-1α and GLUT-1 in lymph node metastasis group were higher than those in group without lymph node metastasis(all P＜0.05).There were positive correlations between the mRNA and protein expressions of HIF-1α and between the mRNA and protein expressions of GLUT-1(r1=0.527,r2=0.408,respectively,both P＜0.01). Conclusions CoCl2 can up-regulate the expressions of HIF-1α and GLUT-1 in lung adenocarcinoma cell line A549 by inducing hypoxia.The hypoxia-mediated effects on them have the potential to make lung cancer cells further grow and promote hypoxie tolerance.The overexpressions of HIF-1α and GLUT-1 are closely correlated with the progression of lung carcinoma.Combined detection of HIF-1α and GLUT-1 may be helpful for the diagnosis and prognosis prediction of primary lung cancer in elderly patients.
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<p><b>OBJECTIVE</b>To prepare a single chain antibody (ScFv) of mAb SZ-21 against platelet GPIIIa for its future clinical application.</p><p><b>METHODS</b>The expression vector pET20b-SZ-21ScFv was constructed and the fusion protein was expressed in E. coli BL21 (DE3) PlysS. The activated fusion protein was obtained after a series of purification steps, including cell breakage, inclusion body solubilization, His-bind resin affinity chromatography and protein refolding.</p><p><b>RESULTS</b>The fusion protein yields were up to 21% of the total amount of bacteria protein. The ScFv fragment could inhibit ADP-induced platelets aggregation in a dose-dependent manner in vitro and the maximal inhibition rate was obtained at a concentration of 20 micro g/ml. It also reacted with endothelial cells as detected by flow cytometry. Moreover, the ScFv fragment was able to inhibit the binding of fibrinogen to platelet.</p><p><b>CONCLUSION</b>The SZ-21ScFv fragment had the activity to inhibit platelets aggregation and the binding of fibrinogen to platelet, being potentially useful for the treatment of thrombotic diseases.</p>
Subject(s)
Humans , Antibodies, Monoclonal , Pharmacology , Blood Platelets , Metabolism , Endothelium, Vascular , Cell Biology , Fibrinogen , Metabolism , Immunoglobulin Fragments , Pharmacology , Platelet Aggregation , Platelet Glycoprotein GPIIb-IIIa Complex , Allergy and Immunology , Recombinant Fusion Proteins , PharmacologyABSTRACT
AIM: To obtain a McAb that can inhibit the function of matrix metalloproteinase-2 (MMP-2), we expressed the fibronectin-like domain of MMP-2 (MFD) in vitro and prepared a McAb against MMP-2. METHODS: The purified MFD protein was used to immunize BALB/C mouse three times. Then the spleen of mouse was taken out and hybridized with hybridoma cells SP2/0. The positive cell clones were screened with ELISA method. The subtype and tissue specificity of the McAb were identified and its effect on endothelial cell migration and tube-formation was analyzed. RESULTS: After the spleen cells of the mouse and hybridoma cells SP2/0 were hybridized, a piece of cells that continuously secreted McAb against MMP-2 was obtained and named SZ-117. The titers of this McAb in culture supernatants and ascites were 2?10~-3 and 2?10~-5 , respectively. The heavy chain of the McAb belongs to IgG1 subclass. The McAb identified native MMP-2. MMP-2 existed in the stromal tissue of stomach, cholecystis, spleen, ovarian, prostate, salping and lymph node. It inhibited the invasion behavior of endothelial cells Eahy926 and pancreatic carcinoma cells 1990 and inhibited the tube-formation of Eahy926 cells. CONCLUSION: A useful tool for testing MMP-2 is obtained and it will be helpful to look for a kind of new anti-tumor material.
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AIM To investigate the actions of basic fibroblast growth factor (bFGF) on the proliferation and differentiation of the non-adherent stromal precursor (NASP) cells. METHODS The osteogenic potential of the bone marrow stromal cells (BMSC) isolated from Wistar rats were cultured in the absence or presence of bFGF. After ALP cytochemistry, the colonies were counted by image analysis. The cells cultured in petri dishes were stained by the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique for collagen type Ⅰ, proliferating cell nuclear antigen ( PCNA ) and double staining for calcium and osteocalcin. RESULTS Many stromal precursor cells are present in bone marrow in a non-adherent form. bFGF not only stimulated the proliferation of NASP cells, but also enhanced the differentiation of NASP cells into osteoblasts. CONCLUSIONS NASP cells are possibly the main targets of the anabolic action of bFGF which may play an important role in fracture healing and bone formation.
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AIM: To investigate the expression of tissue factor(TF) induced by oxidized high density lipoprotein(oxHDL) in human umbilical vein cell line,ECV304,and the related mechanisms.METHODS: Four main groups were designed: the negative,the positive(ECV304 with histamine),the HDL group and the oxHDL group.Quantitative real-time polymerase chain reaction(RQ-PCR) and Western blotting were used to detect the expression level of TF.The specific inhibitors of MAPKs,SP600125(c-jun terminal NH2 kinase,JNK),SB203580(p38 MAP kinase,p38 MAPK),PD98059(extracellular signal-regulated kinase,ERK1/2) were used to investigate the underlying mechanisms.RESULTS: The TF expression in normal ECV304 cell line was not detected.Histamine administration resulted in a significant expression of TF in ECV304 cell line,with strongest effect after 1 h co-incubation at concentration of 1?10-5 mol/L histamine(about 4.8-fold higher expression of TF compared with that of 1?10-9 mol/L histamine).Expression level of TF was detected after stimulated with oxHDL in dose-and time-dependent manners.The highest expression of TF mRNA was found at 20 mg/L oxHDL and 6 h co-incubation,with 1.8-fold and 5.3-fold increase in TF expression,respectively,compared with that at 10 mg/L oxHDL and 2 h co-incubation.20 mg/L oxHDL also caused an apparent augmentation of TF protein expression,about 1.5-fold higher compared with that stimulated by 40 mg/L oxHDL.HDL co-incubation did not cause a detectable expression of TF protein.The mRNA levels of TF in ECV304 cell line induced by oxHDL were decreased by 95.0%,81.0%,87.0%,respectively(all P