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

Recent reports have clearly demonstrated that bone marrow cells can be differentiated into neurons, suggesting the existence of cells with the differentiation capacity in the bone marrow cell population. It is well known that hematopoietic stem cells as well as mesenchymal stem cells (MSCs) can be transplanted and therefore, alternative of them might contribute to the process. In the present study it was addressed whether marrow MSCs could be coaxed into neuron-specific antigen bearing cells and if so, whether the differentiated cells possess the cytochemical features seen in neurons. The report here showed that high concentration of 2-mercaptoethanol (2-ME) could induce some of the MSCs into neuron-like cells expressing neurofilament (NF) and neuron specific enolase (NSE). The neuron-like cells were alkaline phosphotase positive while the others MSCs were kept negative. Cells treated with 2-ME were positive for alpha-naphthylacetate esterase and glycogen and negative for acetylchonlinesterase, which were similar with the results seen in untreated cells. Furthermore, Nissel body was not observed in treated cells shown by toluidine blue staining. Therefore, it is likely that the cells described here seem not belong to the neuronal lineage. These findings, however, reveal that human MSCs could alter their committed fates under some circumstances.

To address the question whether there exists mesenchymal stem cells in adult mouse skeletal muscle, mononuclear cells from muscle were obtained by digestion and density gradient centrifugation and plated in alpha-MEM/F12 medium containing 10% fetal bovine serum. Cell biological properties including morphology, cytochemistry, growth pattern and phenotypes as well were evaluated. Likewise, the osteogenesis of cultured cells was also observed. The results showed that adherent cells homogenous in shape proliferated quickly in the culture system. The phenotypes of the cells were unique, which were positive for CD29 and Sca-1, and negative for CD34 and CD45. Cytochemistry evaluation showed that they were homogeneously positive for acid alpha-naphthl acetate esterase (ANAE) and acid phosphatase (ACP), and negative for alkaline phosphatase (ALP) and that, around 5% of them were positive for glycogen (periodic acid-Schiff reaction, PAS). Cells became ALP-positive after the induction by ascorbic acid, beta-phosphoglycerol and dexamethasone. It is concluded that mesenchymal stem cells exist in murine skeletal muscle and compose the complex heterogenous population of stem cells in muscle.
Animals ; Cell Cycle ; Cell Division ; Cell Separation ; methods ; Cells, Cultured ; Female ; Male ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal ; cytology

Stem cell growth factor (SCGF) is an early-acting hematopoitic cytokine that has two isoforms including hSCGF with full length molecules and hSCGFbeta, 78 amino acids of which lost in the conserved calcium-dependent carbohydrate-recognition domain (CRD). It has been demonstrated that hSCGFbeta is strictly species-specific in regulating he-matopoiesis. This study was aimed to explore whether human SCGF can exert synergistic stimulatory effect on heterogenous murine CFU-GM progenitor. Firstly, hSCGF cDNA was amplified from human fetal liver cDNA library by using two-step PCR. The hSCGF mature peptide coding sequence was subsequently placed at downstream of glutathione S-transferase (GST) sequence in GST gene fusion expression vector. The results indicated that there existed an additional 60 kD protein compared with mock BL21 when the cells hosting recombinant plasmid were induced with IPTG at 37 degrees C. SDS-PAGE analysis demonstrated that the GST-hSCGF fusion protein mainly existed in insoluble form. When induced at low temperature (28 degrees C), the recombinant protein was mostly soluble. The GST-fusion recombinant protein was subsequently purified by using affinity chromatography. The clonogenic assay revealed that, unlike hSCGFbeta, hSCGF had the granulocyte/macrophage promoting activity (GPA) for murine bone marrow GM progenitor. It is concluded that, in contrast to human SCGFbeta, the intact molecular hSCGF may have no species specificity, implying that CRD domain in human SCGFbeta does not directly bind to corresponding SCGF receptor, but may have certain biological function.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; isolation & purification ; Hematopoiesis ; genetics ; Humans ; Species Specificity ; Stem Cell Factor ; biosynthesis ; genetics ; isolation & purification

To investigate the mechanisms underlying the issue that bone marrow mesenchymal stem cells (MSC) could trigger low responsiveness of allogeneic T lymphocytes against alloantigens, phenotypes of T cells were analysed with flow cytometry before and after coculture of allogeneic T lymphocytes with MSC. Further, expression of cytokines including IL-4, IFN-gamma and TGF-beta1 was evaluated by RT-PCR and ELISA as well. The results showed that CD8(+) subpopulation was increased and CD25(+) subset was decreased in proportion after coculture of allogeneic T lymphocytes with MSC for 2 weeks. RT-PCR assay showed that MSC expressed TGF-beta1 but not IL-4 and IFN-gamma, and the result was further proved by ELISA assay showing that the secretion of TGF-beta1 reached to 1 ng/ml in 72 hours. It was concluded that allogeneic MSC suppressed T cells activation by alteration of T cell subpopulations. The suppressive effect was at least in part due to the secretion of TGF-beta1. The results indicate that MSC pretreatment might be useful in the prevention of GVHD in HLA-mismatched bone marrow transplantation and further donors for hematopoietic stem cells could be selected from greater potentials.
Bone Marrow Cells ; physiology ; Bone Marrow Transplantation ; immunology ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Humans ; Isoantigens ; immunology ; Lymphocyte Culture Test, Mixed ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; physiology ; secretion ; T-Lymphocytes ; immunology ; Transforming Growth Factor beta ; physiology ; Transforming Growth Factor beta1

Six naphthalimide polyamine conjugates were synthesized and their structures were confirmed by elemental analysis, 1H NMR, 13C NMR and MS. Antitumor activities were evaluated in vitro using MTT assay on Leukemia cells (K562), human breast cancer cells (MB-231) and prostate cancer cells (Ln cap cell). The results showed that most of the six compounds were superior to the control (amonafide), 6d, 6e, and 6f exhibited nice selectivity in a screen of hepatoma cells (BEL-7402) and human normal hepatocytes (QSG-7701).
Antineoplastic Agents ; chemical synthesis ; pharmacology ; Cell Line, Tumor ; drug effects ; Humans ; Male ; Molecular Structure ; Naphthalimides ; chemical synthesis ; pharmacology ; Polyamines ; chemical synthesis ; pharmacology

Objective] To investigate the relationship between hepatitis B virus( HBV) genotype and their mutations on the development of hepatocellular carcinoma ( HCC ) . [ Methods ] A cohort study on patients with chronic HBV infection was followed up.HBV genotypes were identified by nested multiplex PCR and multiplex PCR.And HBV mutations in the basic core promoter region were sequencing by PCR amplification. [ Results] The patients infected with genotype B were followed up for an average of 8.52 years (IQR:6.67-10.75), of whom the incidence of HCC was 6.55/1 000 person-years.After follow up with an average of 8.87 years (IQR:6.85-11.33), the incidence of HCC was 11.63/1 000 person-years for the patients infected with genotype C, which were significantly higher than those infected with genotype B (P=0.006).In genotype B HBV infected patients, age (≥60 years), cirrhosis can in-crease the risk of HCC, and in genotype C patients, male, age (≥40 years), cirrhosis, C1653T, T1753V, A1762T/G1764A mutation as well.Interferon therapy can reduce the risk of HCC.In genotype C group, interferon treatment reduced HCC risk in patients carrying A1762T/G1764A mutation (HR=0.21, P=0.008) and in those without T1753V ( HR=0.08, P=0.012) and C1653T mutation ( HR=0.17, P=0.013). [Conclusion] HBV genotypes and mutation are closely associated with HCC.Patients infected with genotype C, carrying 1762T/G1764A mutation should be given priority of receiving antiviral treatments in order to prevent HCC;those carrying C1653T or T1753V mutation should be monitored closely to detect early HCC and receive timely surgical resection.

OBJECTIVETo determine the prevalence of urolithiasis in young children fed infant formula (IF) contaminated with melamine, and the association between IF consumption and urolithiasis.

DESIGNA total of 2 733 children < or = 3 years of age on September 1, 2008 in two townships of Gansu Province, China were studied. Cases of urolithiasis were diagnosed by ultrasonography. Milk product consumption was determined by their caregivers. Remaining IF samples were tested for melamine and cyanuric acid.

RESULTSOf 2 733 eligible children in the two townships, 2 186 (80%) were enrolled in our study. Overall, 16.6% (362) of 2 186 children had urolithiasis. The prevalence was 24.6% in children exclusively fed Sanlu brand IF, 9.7% in those fed other IF, and 8.5% in those fed exclusively on other milk products. For children exclusively breast-fed, no urolithiasis was found (P < 0.05). The prevalence of urolithiasis was 11.4% in children fed 400 g of Sanlu IF, rising to 37.5% in children fed over 25 600 g. Of 48 Sanlu IF samples, 91.7% contained melamine (median = 1 800 ppm; range = 45-4 700) and 66.7% contained cyanuric acid (median = 1.2 ppm; range = 0.4-6.3). Melamine was also detected in 22.2% of 36 other brand IF (median = 27.5 ppm, range = 4-50).

CONCLUSIONSUrolithiasis was associated with melamine-contaminated IF. Although one product caused most morbidity, other milk products may have also contributed to the outbreak.

Child, Preschool ; Data Collection ; Food Contamination ; Humans ; Infant Food ; analysis ; Triazines ; toxicity ; Urolithiasis ; chemically induced

Aim To analyze the main components of effective fractions of Inula cappa in rat plasma with UHPLC/Q-TOF/MS technology and serum pharmacochemistry theory.Methods After gavage administration with Inula cappa extracts,blood was collected from abdominal aorta,and the protein in plasma sample was precipitated by methanol.Eclipse Plus C18 RRHD (2.1 mm × 100 mm,1.8 μm) was used,with 0.1％ formic acid agueous solution (A)-0.1％ formic acid and acetonitrile (B) as the mobile phase for gradient elution,and the flow rate was 0.3 mL ·min-1 Detected at negative ion mode,and with the help of Metabolite Tools software from Bruker Corporation,components in plasma were defined.Results A total of 16 compounds were identified,including 9 prototypes and 7 metabolites.Main metabolites were isomerization,methylation,glucuronidation and recombination of caffeoylquinic acid.Conclusions Therefore,our study has comprehensively expounded Inula cappa extracts' constituents migrating to rat plasma,and provided a reference for further studies for in vivo metabolic process and effective material base of Inula cappa.