B cell activation factor (BAFF) is a novel member of the TNF ligand superfamily, mainly produced by myeloid cells. BAFF has been shown to participate in B-cell survival and B- and T-cell maturation. BAFF expression in adipocytes has been recently demonstrated. In the current study, we verified that BAFF expression is increased during adipocyte differentiation. BAFF expression was augmented by TNF-alpha treatment and was decreased by rosiglitazone treatment. BAFF secretion in lean and in ob/ob mice sera were compared and smaller amount of BAFF was secreted in ob/ob mice. mRNA and protein expression were different between epididymal and visceral adipose tissue. BAFF expression was also increased in ob/ob mouse adipose tissue. We sought to identify known BAFF receptors (BAFF-R, BCMA, and TACI) in adipocytes, and determined that all three were present and upregulated during adipocyte differentiation. However, the expression of TACI was distinct from that of BAFF-R and BCMA under TNF-alpha and BAFF ligand treatment. BAFF-R and BCMA expression levels were upregulated under pro-inflammatory conditions, but TACI was reduced. Conversely, BAFF-R and BCMA expression levels were downregulated by rosiglitazone treatment, but TACI was increased. Taken together, our results suggest that BAFF may be a new adipokine, representing a link between obesity and inflammation.
Adipocytes/cytology ; Adipokines/biosynthesis/*physiology ; Animals ; B-Cell Activating Factor/biosynthesis/*physiology ; B-Cell Activation Factor Receptor/metabolism ; Cell Differentiation ; Hypoglycemic Agents/pharmacology ; Inflammation/*metabolism ; Mice ; Obesity/*metabolism ; Thiazolidinediones/pharmacology ; Tumor Necrosis Factor-alpha/pharmacology

The prokaryotic expression plasmid pET-30a(+)/sBLyS was constructed and transformed into E. coli BL21 (lambda DE3). The recombinant protein was found to be highly expressed by the plight of soluble part and inclusion body. For the sake of enhancing the proportion of the soluble part, inducement at 16 degrees C for 12 h was ascertained. The expressing product was then purified by Ni2+ affinity chromatography gel. PI of the recombinant human sBLyS(rhsBLyS) is about 7.1-7.3 and it assembles into a homotrimer. The effect of rhsBLyS on B lymphocytes by MTT method told us the B lymphocytes' proliferating capacity dose depended on concentration and also stimulating time of the rhsBLys. With rhsBLyS(2 micrograms/mL) stimulating 3 days, B lymphocytes can proliferate the most.
B-Cell Activating Factor ; B-Lymphocytes ; drug effects ; Escherichia coli ; genetics ; Humans ; Isoelectric Point ; Lymphocyte Activation ; drug effects ; Membrane Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Recombinant Proteins ; biosynthesis ; Temperature ; Tumor Necrosis Factor-alpha ; biosynthesis ; isolation & purification ; pharmacology

BCMA is one of the transmembrane receptors belonging to BAFF and APRIL. In order to identify the feasibility of sBCMA as decoy receptor and obtain active sBCMA for its structural and functional research, full length of hBCMA was amplified with total RNA from Raji cell line by RT-PCR, and the cDNA encoding the extracelluar soluble domain of hBCMA was inserted into pET43.1a(+) vector. The recombinant vector pET43.1a(+)-sBCMA was transformed into E. coli Origami B(DE3) pLyS which is helpful for disulfide bond construction of expression proteins. After IPTG induction, the recombinant protein was expressed as soluble fusion protein, sBCMA-NusA-His6, and identified by western blotting. Then the target protein was purified by Ni(+)-chelating Sepharose Fast Flow. The binding activity between recombinant sBCMA and BAFF was detected by ELISA. Also, Recombinant sBCMA inhibited proliferation of mouse B cell stimulating by rhsBAFF. It was proved that recombinant sBCMA has good bioactivity and the method to express those proteins rich in disulfide bond is feasible and effectual.
B-Cell Activating Factor ; chemistry ; B-Cell Maturation Antigen ; biosynthesis ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Disulfides ; chemistry ; Escherichia coli ; genetics ; metabolism ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Solubility ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; chemistry

Mouse colon cancer cells CT26 were transfected with constructed plasmid expressing mouse soluble B lymphocyte stimulator (msBlyS). Single cell clones were selected with 100 microg/ml Zeosin and subcloned by serial limiting dilution. Eight resistant transfectants were isolated and expanded, and five of them displayed the desirable msBlyS cDNA band amplified by semi-quantitative RT-PCR assay. Western blot analysis showed that only msBlyS molecules of the expected size were detected in the cell lysates from transfectants. The supernatant of transfectants could costimulate B cell proliferation in standard costimulation assay. Thus we have successfully screened the stable transfectants expressing high levels of msBlyS in CT26 cells, which could be used as cancer vaccines for further anti-tumor immunotherapy.
Animals ; Antibodies, Monoclonal ; B-Cell Activating Factor ; B-Lymphocytes ; immunology ; Cancer Vaccines ; biosynthesis ; genetics ; Cloning, Molecular ; Colonic Neoplasms ; pathology ; DNA, Complementary ; biosynthesis ; genetics ; Epitopes, B-Lymphocyte ; genetics ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Transfection ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

<b>OBJECTIVEb>The purpose of this study was to clone the soluble form of the mouse BlyS (msBlyS) and insert it into a eukaryotic expression vector pSecTag2B in order to further elucidat the antitumor activity induced by msBlyS expressed by the recombined plasmid pSecTag2B-msBlyS.

<b>METHODSb>Full length cDNA of mouse soluble BlyS (msBlyS) was amplified by reverse transcription-PCR from total RNA of mouse spleen. The PCR product was ligated directly with linearized vector pCR2.1 supplied in the TA cloning kit. The recombined plasmid pCR2.1-msBlyS which was selected and identified using blue-white screening method and restriction map analysis and the purified original plasmid pSecTag2B were both cut by HindIII and EcoR I. The digested fragments were extracted and purified from low-melting temperature agarose and ligated by T4DNA ligase. The recombined plasmid pSecTag2B-msBlyS were isolated and identified by restricted endonuclease cutting and Sanger dideoxy DNA sequencing.

<b>RESULTSb>The sequencing data indicated that inserted msBlyS gene had correct DNA sequence and orientation.

<b>CONCLUSIONb>Eukaryotic expression vector pSecTag2B. Expressing mouse BlyS have successfully been cloned. This will provide us an opportunity to do further research work on BlyS.

Animals ; B-Cell Activating Factor ; Cloning, Molecular ; Epitopes, B-Lymphocyte ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis, DNA ; Spleen ; cytology ; immunology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

Asthma was induced by the sensitization and challenge with ovalbumin (OVA) in mice. B-cell activating factor (BAFF) plays a role in mature B cell generation and maintenance. Here, we investigated whether, BAFF expression was changed in OVA-induced mice and whether the control of BAFF expression level alleviates the symptom of bronchial asthma. BAFF expression was detected in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues. BAFF protein was also increased in OVA-induced mouse serum. The increased BAFF transcripts was detected in OVA-induced mouse splenocytes. OVA-induced asthma was associated with the increased number of eosinophils in bronchoalveolar lavage fluid (BALF). When TACI:Fc scavenging soluble BAFF was injected to OVA-induced mice, a significant inhibition was detected in the thickness of airway smooth muscle and glycol-containing cellular elements in airway that were visualized by hematoxylin/eosin Y and periodic acid-Schiff staining, respectively. In addition, when mice were treated with TACI:Fc protein, BAFF protein level was decreased in alveolar-associated cells surrounding bronchi of OVA-induced mouse lung tissues compared to control mice. When compared to OVA-induced control, TACI:Fc treatment reduced the percentage of non-lymphoid cells and no changes were detected in lymphoid cell population. Hypodiploid cell formation in BALF was decreased by OVA-challenge but it was recovered by TACI:Fc treatment. Collectively, data suggest that asthmatic symptom could be alleviated by scavenging BAFF and then BAFF could be a novel target for the develpoment of anti-asthmatic agents.
Animals ; Apoptosis ; Asthma/chemically induced/*drug therapy/immunology ; B-Cell Activating Factor/*biosynthesis ; Bronchi/metabolism/pathology ; Bronchoalveolar Lavage Fluid/cytology ; Eosinophils/pathology ; Female ; Humans ; Immunoglobulin Fc Fragments/*genetics ; Immunoglobulin G/*genetics ; Lymphocytes/pathology ; Mice ; Mice, Inbred BALB C ; *Ovalbumin ; Pulmonary Alveoli/metabolism ; Recombinant Fusion Proteins/genetics/*therapeutic use ; Spleen/metabolism ; Transmembrane Activator and CAML Interactor Protein/*genetics

<b>AIMb>To investigate the effect of ginkgolide B on the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL or PAF. In addition, to analyze whether the effect of oxLDL is mediated through PAF receptor.

<b>METHODSb>Using 3H-Tdr incorporation assay, the proliferation of VSMC was measured. The protein and mRNA level of MCP-1 and IL-8 in U937 cells were determined by RT-PCR and ELISA. Using Western blotting the p65 and IkappaB was quantified. The binding of oxLDL to U937 cell was measured by a radio-ligand binding assay of 3H-PAF.

<b>RESULTSb>Ginkgolide B inhibited, in dose-dependent manner, the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL, and inhibited the oxLDL-induced p65 activation and depletion of IKappaB. oxLDL inhibited PAF binding to U937 cells.

<b>CONCLUSIONb>Ginkgolide B, as a PAF antagonist, possesses the effect of inhibiting the proliferation of VSMC and the secretion of chemokines by U937 cells stimulated by oxLDL in vitro. The effect of oxLDL is, at least in part, mediated through PAF receptor.

Animals ; Aorta, Thoracic ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chemokine CCL2 ; biosynthesis ; genetics ; Diterpenes ; isolation & purification ; pharmacology ; Dose-Response Relationship, Drug ; Ginkgo biloba ; chemistry ; Ginkgolides ; Humans ; I-kappa B Proteins ; metabolism ; Interleukin-8 ; biosynthesis ; genetics ; Lactones ; isolation & purification ; pharmacology ; Lipoproteins, LDL ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Plants, Medicinal ; chemistry ; Platelet Activating Factor ; antagonists & inhibitors ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Synaptotagmin I ; metabolism ; U937 Cells

Wnt5a is a ligand that activates the noncanonical Wnt signaling pathways (beta-catenin-independent pathways). Human neutrophils expressed several Wnt5a receptors, such as Frizzled 2, 5 and 8. Stimulation of human neutrophils with Wnt5a caused chemotactic migration and the production of two important chemokines, CXCL8 and CCL2. CCL2 production by Wnt5a was mediated by a pertussis toxin-sensitive G-protein-dependent pathway. Wnt5a also stimulated the phosphorylation of three mitogen-activated protein kinases (MAPKs: ERK, p38 MAPK and JNK) and Akt. Inhibition of ERK, p38 MAPK or JNK by specific inhibitors induced a dramatic reduction in Wnt5a-induced CCL2 production. Supernatant collected from lipopolysaccharide-stimulated macrophages induced neutrophil chemotaxis, which was significantly inhibited by anti-Wnt5a antibody. Our results suggested that Wnt5a may contribute to neutrophil recruitment, mediating the inflammation response.
Activating Transcription Factor 2/metabolism ; Animals ; Cell Separation ; Chemokines/*biosynthesis ; Chemotaxis/*drug effects ; Culture Media, Conditioned/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; GTP-Binding Proteins/metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases/metabolism ; Lipopolysaccharides/pharmacology ; Macrophages/drug effects/metabolism ; Mice ; NF-kappa B/metabolism ; Neutrophils/*cytology/drug effects/enzymology/*metabolism ; Pertussis Toxin/pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Receptors, Wnt/metabolism ; Type C Phospholipases/metabolism ; Wnt Proteins/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism