CD40/CD40L interactions play a pivotal role in T cell activation, and take part in many physiologic and pathologic procedures and different levels. In this article, stable CHO transformants secreting human CD40-Ig fusion protein were established through transfection and selection with Lipofectamaine and G418, respectively. In order to obtain great valume of recombinant protein, big batch serum-free cultures of engineered CHO cells were performed in roller-bottle using CHO-II-SFM medium. After cultures, the cell-culture supernatants were harvested, concentrated through ultra-filtration, and finally purified by affinity choromatography with Protein G Sepharose Fast Flow. Human peripheral bloods were collected freshly and seperated with Ficoll, CFU-T was cultured in semi-solid culture system with peripheral blood mononuclear cells (PBMNC). Effect of human CD40-Ig fusion protein on the formation of CFU-T was observed in vitro. The results showed that the yield of human CD40-Ig fusion protein was 30 mg in total 3 liter CHO-II-SFM culture supernatant, and it supposed that the expression level of CD40-Ig in CHO cells was more than 10 micro g/ml. The purity of purified fusion protein is above 95%. Furthermore, compared with human IgG, human CD40-Ig fusion protein significantly inhibited the formation of CFU-T at dose 0.25, 1.0, 4.0, and 10 micro g/ml, it lays a good foundation to evaluate its potential functions in vivo.

Embryonic hematopoiesis in mammals is characterized by successive temporal and spatial changes. Previous investigations indicate that in vitro differentiation of embryonic stem cells (ES cells) derived from 129 mice can mimic embryonic hematopoiesis to some extent. To investigate the in vitro hematopoietic differentiation capacity of ES cells derived from C57BL/6 mice, the authors initially established the murine ES cell line with standard identification methods employed. Next, two-step culture system was utilized for embryoid bodies formation and the appearance of different hematopoietic precursors was confirmed by CFC assay, cellular chemical staining as well as RT-PCR. The results demonstrated that the ES cell line MES-1 fulfilled the criteria of ES cell line and its progeny after in vitro differentiation included primitive and definitive erythrocyte precursors, mixed colony-forming cells and granulocyte/macrophage colony-forming cells. RT-PCR analysis revealed the molecular consistence of transcription factors and hematopoietic markers with cellular event. In conclusion, MES-1 established from C57BL/6 mice was able to differentiate in vitro to a variety of hematopoietic precursors, thus could partly recapitulate embryonic hematopoiesis.
Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; genetics ; Cell Line ; Colony-Forming Units Assay ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; cytology ; Erythroblasts ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Erythroid-Specific DNA-Binding Factors ; Gene Expression ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; Time Factors ; Transcription Factors ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

OBJECTIVETo investigate the regulatory mechanism of the transcription of tumor metastasis suppressor gene TMSG-1.

METHODSLuciferase reporter assay and site-directed mutagenesis were used to analyze the regulatory region of TMSG-1. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were carried out to verify the interaction of KLF6 and Sp1 with the regulatory region of TMSG-1. Co-immunoprecipitation (CoIP) was performed to analyze the interaction between KLF6 and Sp1. TMSG-1 and wt-KLF6 mRNA expressions in cells with different metastatic capacities were quantitated by real-time PCR. Cell invasive capability was determined by Matrigel invasion assay.

RESULTSA 63 bp inducible regulatory region (+59 bp - +123 bp) in exon 1 was identified by luciferase assay using reporter plasmids with a series of TMSG-1 regulatory region deletions. Mutations in KLF6/Sp1 binding sites of this region resulted in a decrease of luciferase activity, while cotransfection with KLF6 or Sp1 expressing plasmids led to a remarkable increase of luciferase activity. EMSA and ChIP demonstrated that KLF6 as well as Sp1 interacted with this region. CoIP also indicated a possible interaction between KLF6 and Sp1 proteins. In the highly metastatic cell sublines, a low level of wild type KLF6 was associated synchronously with a low TMSG-1 level. Prostate carcinoma cells overexpressing KLF6 exhibited a higher TMSG-1 level and a lower invasive capability.

CONCLUSIONSTranscription factor complex of KLF6 and Sp1 may participate in the inducible transcriptional regulation of TMSG-1, and a decreased wild type KLF6 expression is likely associated with a low TMSG-1 level in the highly metastatic cell sublines.

Binding Sites ; genetics ; Cell Line, Tumor ; Electrophoretic Mobility Shift Assay ; Humans ; Immunoprecipitation ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mutagenesis, Site-Directed ; Mutation ; Neoplasm Invasiveness ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Sp1 Transcription Factor ; genetics ; metabolism ; Sphingosine N-Acyltransferase ; genetics ; metabolism ; Transcriptional Activation ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism

OBJECTIVETo investigate the apoptosis-promoting effect of PDCD5 on human prostate cancer cells PC-3M-1E8.

METHODSPCI-neo and PCI-neo-PDCD5 were transfected into PC-3M-1E8 cells by Lipofectamine 2000, the viability of the cells was analyzed by MTT assay 16 hours after removal of the serum, and the apoptosis was determined by in situ end-labeling and electron microscopy.

RESULTSThe viability and growing speed of the transfected cells were significantly decreased and their apoptotic indexes significantly increased as compared with the control group (P < 0.001).

CONCLUSIONPDCD5 may significantly inhibit the in vitro growth and promote the apoptosis of human prostate cancer cells PC-3M-1E8.

Apoptosis ; genetics ; physiology ; Apoptosis Regulatory Proteins ; genetics ; physiology ; Cell Line, Tumor ; Humans ; In Situ Nick-End Labeling ; Lipids ; chemistry ; Male ; Neoplasm Proteins ; genetics ; physiology ; Plasmids ; chemistry ; genetics ; Prostatic Neoplasms ; genetics ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; methods

OBJECTIVETo establish the quantitative methods for calycosin glycoside and formononetin in Radix Astragali, and the samples from different sources were analyzed, in order to supply the basis for the quality control of Radix Astragali.

METHODThe content of calycosin glycoside and formononetin in 59 samples of Radix Astragali from eight with different provinces was analyzed by HPLC-DAD.

RESULTThe contents of calycosin glycoside and formononetin in Radix Astragali from different sources, with different cultivating method or in different ages differed markedly, and the results showed that the quality of samples from Shannxi, Innermongolia and Shanxi were better than other sources, and the semi-wild samples were better than other cultiving samples, moreover the shorter age, the better quality.

CONCLUSIONThis simple, accurate and reproducible method could use to determine the contents of isoflavanoids in Radix Astragali.

Astragalus membranaceus ; chemistry ; growth & development ; China ; Chromatography, High Pressure Liquid ; methods ; Ecosystem ; Glucosides ; analysis ; standards ; Isoflavones ; analysis ; standards ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Reproducibility of Results

Adolescent ; Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Female ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Male ; Middle Aged ; Nucleosides ; therapeutic use ; Pyrimidinones ; therapeutic use ; Thymidine ; analogs & derivatives ; Young Adult

BACKGROUNDLocalization of sensory cortical areas during the operation is essential to preserve the sensory function. Intraoperative direct electrostimulation under awake anesthesia is the golden standard but time-consuming. We applied 3T high field blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to identify the relationship between glioma and cortical sensory areas preoperatively and to guide intraoperative direct electrostimulation for quick and precise localization.

METHODSFive glioma patients with sensory cortex involvement by or next to the lesion had preoperative BOLD fMRI to determine the spatial relationship of cortical sensory areas to the tumours. Bilateral hand opposite movement was performed by these patients for fMRI. Precentral and postcentral gyri were identified by electrical stimulation during the operation. Karnofsky Performance Status scores of the patients' pre- and postoperative and the role of BOLD fMRI were evaluated.

RESULTSThe cortical sensory areas were all activated in five glioma patients involving postcentral gyrus areas by BOLD fMRI with bilateral hand opposite movement. The detected activation areas corresponded with the results from cortical electrical stimulation.

CONCLUSIONSThe relationship between cortical sensory areas and tumour can be accurately shown by BOLD fMRI before operation. And the information used to make the tumour resection could obtain good clinical results.

Adult ; Female ; Glioma ; blood ; pathology ; Humans ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Oxygen ; blood ; Somatosensory Cortex ; physiology ; Young Adult

OBJECTIVETo study the methylation status of fragile histidine triad (FHIT) gene promoter in patients with myelodysplastic syndrome (MDS) and its clinical relevance.

METHODSMethylation-specific PCR (MSP) was used to detect FHIT promoter methylation in bone marrow samples from 54 MDS cases.

RESULTSHypermethylation of FHIT promoter was detected in 26 cases (48.1%). Association was not found between FHIT gene hypermethylation and sex, hematologic parameters and chromosomal abnormalities of MDS patients, but found between FHIT gene hypermethylation and age of the MDS cases. Although significant difference was not observed in the frequencies of FHIT gene hypermethylation among patients with refractory anemia/refractory anemia with ringed sideroblasts (RA/RAS) (1/6, 16.7%), refractory anemia/refractory anemia with ringed sideroblasts (RCMD) and refractory cytopenia with multilineage dysplasia with ringed blasts (RCMD-RS) (6/19, 31.6%), refractory anemia with excess blasts-1 (RAEB-1) (7/11, 63.6%), refractory anemia with excess blasts-2 (RAEB-2) (4/7, 57.1%) and refractory anemia with excess blasts in transformation/acute myeloid leukemia (RAEBt/AML) (8/11, 72.7%)(chi-square=8.417, P=0.077), it was observed in patients in early stages (RA/RAS and RCMD) (7/25, 28.0%), advanced stages (RAEB-1 and RAEB-2)(11/18, 61.1%) and RAEBt/AML (8/11, 72.7%) (chi-square=7.938, P=0.019). Furthermore, there was a positive correlation between the frequency of FHIT gene hypermethylation and different IPSS groups (chi-square=10.110, P=0.018).

CONCLUSIONFHIT gene hypermethylation might be one of the molecular events involved in the disease progression of MDS.

Acid Anhydride Hydrolases ; genetics ; Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; Base Sequence ; DNA Methylation ; Female ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Myelodysplastic Syndromes ; classification ; genetics ; pathology ; Neoplasm Proteins ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; genetics

OBJECTIVEThis review focuses on the state-of-the-art of CXCL12/CXCR4 signaling axis in pancreatic cancer and its role in tumor progression.

DATA SOURCESRelevant articles published in English were identified by searching in Pubmed from 1997 to 2013, with keywords "CXCL12", "CXCR4" and "pancreatic cancer". Important references from selected articles were also retrieved.

STUDY SELECTIONArticles about CXCL12/CXCR4 signaling axis in pancreatic cancer and relevant mechanisms were selected.

RESULTSPancreatic cancer has been one of the most lethal human malignancies, with median survival less than one year and overall 5-year survival only 6%. Tumor cells from pancreatic cancer express high level of CXCR4. CXCL12, the ligand for CXCR4, is extensively secreted by neighboring stromal cells and other distant organs. CXCL12 primarily binds to CXCR4, induces intracellular signaling through several divergent pathways, which are involved in progression and metastasis of pancreatic cancer.

CONCLUSIONSCXCL12/CXCR4 signaling axis may play an important role in the communication between pancreatic cancer cells and their microenvironment, which may have effect on tumor proliferation, invasion, angiogenesis, metastasis and chemoresistance. CXCL12/CXCR4 signaling axis may serves as a novel therapeutic target for pancreatic cancer.

Chemokine CXCL12 ; genetics ; metabolism ; Humans ; Pancreatic Neoplasms ; genetics ; metabolism ; Receptors, CXCR4 ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology

OBJECTIVETo study the impact of an agonist anti-CD(40) monoclonal antibody 5C11 on the induction and biological characteristics of leukemic dendritic cells.

METHODSCombinations of 5C11 and different cytokines were used to induce differentiation of leukemic blasts into dendritic cells. Morphology was observed by light microscopy. Surface antigens of the induced cells were analyzed by fluorescence-activated cell sorting (FACS), the yields of dendritic cell by cell counting, the levels of IL-6 and IL-12 by ELISA, T cell proliferating activity by allo-mixed lymphocyte reaction (MLR) in vitro. Allogeneic T cells were stimulated with leukemic dendritic cells and T-cell cytotoxicity was measured by MTT assay.

RESULTSWhen cultured with combinations of 5C11 and different cytokines, the leukemic cells isolated from the patients could differentiate into dendritic cells. The morphology showed typical features of dendritic cells, which expressed high levels of CD(40), CD(80) and CD(86). In comparison with the original leukemia cells, the leukemic dendritic cells secreted less IL-6 but more IL-12 (P < 0.05). The leukemic dendritic cells were potent to stimulate the proliferation of allogeneic T cells, and the latter was able to lyse the original leukemia cells.

CONCLUSIONLeukemic blasts could be induced to differentiate into functional dendritic cells. It may be of great value in the adoptive immunologic therapy of leukemia.

Antibodies, Monoclonal ; immunology ; CD40 Antigens ; physiology ; Cell Differentiation ; Dendritic Cells ; immunology ; Humans ; Immunophenotyping ; Immunotherapy ; Interleukin-12 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Leukemia ; immunology ; pathology ; therapy