OBJECTIVETo prepare a new targeted liposome ultrasonic contrast agent with anti-KDR antibody that binds specifically with KDR as the main receptor of VEGF and evaluate its physical characteristics, biological activity and specific binding capability in vitro.

METHODSA sonicator was used to prepare the biotinylated lipid micro-bubbles (MB-B), and biotin-avidin-mediated technique was used for attachment of anti-mouse KDR monoclonal antibody to the micro-bubble shell to generate MB-BAB-KDR. MB-BAB-KDR was incubated with fluorescent second antibody to assess the link condition, and the control groups were the MB-B and micro-bubbles with the antibody alone (MB-B-KDR). A parallel plate flow chamber system was used to characterize micro-bubbles attachment efficiency to KDR Fc.

RESULTSThe surface of the micro-bubbles could carry KDR antibody through the biotin-avidin bridge and MB-BAB-KDR were spherical and well-distributed. After incubation with the second antibody, MB-BAB-KDR could be observed to emit bright green fluorescence (Grade II) as compared with little or weak fluorescence in the control MB-B group (Grade 0) and MB-B-KDR group (Grade I). Targeted micro-bubbles bound to KDR Fc increased as the KDR Fc concentration increased (P<0.05).

CONCLUSIONThe targeted liposome contrast agent linked with KDR antibody by biotin-avidin bridge we prepared shows an increased binding number as the KDR Fc concentration increases, which provides a novel approach to molecular imaging study of endometrial receptivity.

Animals ; Antibodies, Monoclonal ; Contrast Media ; chemistry ; Endometrium ; physiology ; Female ; Liposomes ; chemistry ; Mice ; Microbubbles ; Molecular Imaging ; Sonication ; Ultrasonography ; Vascular Endothelial Growth Factor Receptor-2 ; immunology

Aptamers are synthetic, relatively short (e.g., 20-80 bases) RNA or ssDNA oligonucleotides that can bind targets with high affinity and specificity, similar to antibodies, because they can fold into unique, three-dimensional shapes. For use in various assays and experiments, aptamers have been conjugated with biotin or digoxigenin to form complexes with avidin or anti-digoxigenin antibodies, respectively. In this study, we developed a method to label the 5' ends of aptamers with cotinine, which allows formation of a stable complex with anti-cotinine antibodies for the purpose of providing another affinity unit for the application in biological assays using aptamers. To demonstrate the functionality of this affinity unit in biological assays, we utilized two well-known aptamers: AS1411, which binds nucleolin, and pegaptanib, which binds vascular endothelial growth factor. Cotinine-conjugated AS1411/anti-cotinine antibody complexes were successfully applied to immunoblot, immunoprecipitation, and flow cytometric analyses, and cotinine-conjugated pegaptanib/anti-cotinine antibody complexes were used successfully in enzyme immunoassays. Our results show that cotinine-conjugated aptamer/anti-cotinine antibody complexes are an effective alternative and complementary technique for aptamer use in multiple assays and experiments.
Animals ; Antibodies, Anti-Idiotypic/immunology/metabolism ; *Aptamers, Nucleotide/chemistry/immunology ; Biological Assay ; *Cotinine/administration & dosage/chemistry ; Flow Cytometry ; Hep G2 Cells ; Humans ; Mice ; NIH 3T3 Cells ; Phosphoproteins/*chemistry/immunology ; Protein Binding ; RNA-Binding Proteins/*chemistry/immunology ; *Vascular Endothelial Growth Factor A/chemistry/immunology

OBJECTIVETo prepare (99m)Tc-labeled Anti-VEGF mAb 5-FU loaded polylactic acid nanoparticles ((99m)Tc-Ab-5-FU-NPs) and investigate its biodistribution in human gastric carcinoma xenografts.

METHODS(99m)Tc-Ab-5-FU-NPs were prepared by labeling Ab-5-FU-NPs with (99m)Tc using improved Schwarz method. After isolation of (99m)Tc-Ab-5-FU-NPs using SephadexG250 column, the labeling ratio and radiochemical purity were determined using chromatography. The immunocompetence of (99m)Tc- Ab-5-FU-NPs was detected by ELISA and immunohistochemistry. (99m)Tc-Ab-5-FU-NPs were then injected via the tail vein into SCID mice bearing human gastric carcinoma, and (99m)Tc labeled mice-derived monoclonal IgG loaded polylactic acid nanoparticles were used as the control, followed by radioimmunoscintigraphic imaging at 2 and 6 h. The radioactive count and radioactive ratio of the tumor and non-tumor tissue (T/NT) in the animal models were calculated using ROI technique. After imaging at 24 h, SCID mice were sacrificed and the radioactive distribution, the %ID/g, as well as the T/NT radioactive ratio were examined, respectively. The concentrations of 5-FU in the tumor and blood were also detected using HPLC method.

RESULTSThe labeling ratio of (99m)Tc-Ab-5-FU-NPs was 90%-95%. (99m)Tc-Ab-5-FU-NPs were detected in the tumor tissues by radioimmunoimaging 2 h after the injection. ID%/g in the tumor tissues at 2 and 6 h were both significantly higher than that of the control group. Both the ID%/g in tumor tissues and radioactive ratio of tumor and blood at 6 h were higher than those at 2 h, and the concentration of 5-FU in experimental group increased continuously with time and was significantly higher than that in control group.

CONCLUSIONS(99m)Tc-Ab-5-FU-NPs prepared in this study can meet the demands of radioimmunoimaging, and the anti-VEGF monoclonal antibody possesses reliable immune targeting ability. Six hours after injection, (99m)Tc-Ab-5-FU-NPs can specifically accumulate in the tumor tissues in human gastric carcinoma xenografts at high concentration.

Animals ; Antibodies, Monoclonal ; chemistry ; immunology ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; Female ; Fluorouracil ; blood ; chemistry ; pharmacokinetics ; Humans ; Lactic Acid ; chemistry ; Male ; Mice ; Mice, SCID ; Nanoparticles ; Polyesters ; Polymers ; chemistry ; Radioimmunotherapy ; Stomach Neoplasms ; blood ; metabolism ; pathology ; radiotherapy ; Technetium ; chemistry ; Vascular Endothelial Growth Factor A ; immunology

OBJECTIVETo explore the effect of Yishen capsule on the serum vascular endothelial growth factor (VEGF), the cell immunity and the theraphic.

METHODSerum VEGF and T cell subsets were studied in 30 normal subjects and 83 patients before and after treatment.

RESULTCompare with normal subjects, CD3, CD4, CD4/CD8 were decreased, CD8 and serum VEGF were increased obviously (P <0. 05 or P <0. 01). After three months treatment with YiShen capsule, CD4/CD8 was increased, CD8 and serum VEGF were decreased significantly (P <0.05 or P <0.01).

CONCLUSIONYishen capsule can reduce the proteinuria, increase the function of immunity and improve the clinical symptom of patients with chronic glomerulonephritis, achieved the effects of allevating chronic glomerular sclerosis ultimately.

Adolescent ; Adult ; CD3 Complex ; blood ; CD4 Antigens ; blood ; CD4-CD8 Ratio ; Capsules ; Child ; Chronic Disease ; Drug Combinations ; Drugs, Chinese Herbal ; administration & dosage ; isolation & purification ; therapeutic use ; Female ; Glomerulonephritis ; blood ; drug therapy ; immunology ; Humans ; Male ; Middle Aged ; Phytotherapy ; Plants, Medicinal ; chemistry ; T-Lymphocyte Subsets ; drug effects ; immunology ; Treatment Outcome ; Vascular Endothelial Growth Factor A ; blood ; Young Adult

Oxidative stress plays critical roles in airway inflammation that is usually accompanied by increased vascular permeability and plasma exudation. VEGF increases vascular permeability and leads to airway inflammation. In addition, VEGF has been shown to enhance receptor activator of NF-kappaB (RANK) expression in endothelial cells. An aim of the study was to determine the potential role of antioxidant in the regulation of RANK expression in murine model of asthma. We have used a C57BL/6 mouse model of allergic asthma to evaluate the effect of L-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, which acts as an antioxidant, and VEGF receptor inhibitor on RANK mRNA expression. The mice develop the following pathophysiological features of asthma in the lungs: increased expression of RANK mRNA, increased number of inflammatory cells of the airways, increased vascular permeability, and increased levels of VEGF. Administration of OTC and VEGF receptor inhibitor markedly reduced plasma extravasation and VEGF levels in allergen-induced asthmatic lungs. We also showed that the increased RANK mRNA expression at 72 h after ovalbumin inhalation were reduced by the administration of OTC or VEGF receptor inhibitor. The results indicate that OTC and VEGF receptor inhibitor which inhibit up-regulation of VEGF expression modulate RANK expression that may be in association with the regulation of vascular permeability, and suggest that VEGF may regulate the RANK expression. These findings provide a crucial molecular mechanism for the potential use of antioxidants to prevent and/or treat asthma and other airway inflammatory disorders.
Vascular Endothelial Growth Factor A/analysis/antagonists & inhibitors/metabolism ; Thiazolidines ; Thiazoles/*pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors ; Receptors, Tumor Necrosis Factor/genetics/*metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Reactive Oxygen Species/metabolism ; RNA, Messenger/genetics/metabolism ; Pyrrolidonecarboxylic Acid ; Proto-Oncogene Proteins c-akt/metabolism ; Protein Kinase Inhibitors/pharmacology ; Prodrugs/pharmacology ; Phosphorylation/drug effects ; Ovalbumin/immunology ; Osteoprotegerin ; Mice, Inbred C57BL ; Mice ; Immunohistochemistry ; Glycoproteins/genetics/*metabolism ; Gene Expression/drug effects ; Female ; Capillary Permeability/drug effects ; Bronchoalveolar Lavage Fluid/chemistry/cytology ; Blotting, Western ; Asthma/*drug therapy/immunology/metabolism ; Antioxidants/*pharmacology ; Animals