<b>OBJECTIVEb>To detect the B cell activating factor (BAFF) and explore its significance in patients with warm autoimmune hemolytic anemia (WAIHA).

<b>METHODSb>The levels of serum soluble BAFF (sBAFF) and BAFF mRNA in peripheral blood mononuclear cells (PBMCs) in 30 healthy volunteers (control group) and 43 patients with WAIHA were measured by ELISA and real-time quantitative polymerase chain reaction (RT-qPCR) respectively.

<b>RESULTSb>The levels of serum sBAFF and BAFF mRNA in PBMCs in pretreatment group \[2311 (825 approximately 6523) ng/L and 884 (463 approximately 2346) ng/L\] was significanly higher than those in posttreatment group\[1205(358 approximately 5014) ng/L and 446(138 approximately 2699) ng/L\] and control group\[1128 (590 approximately 3201) ng/L and 341 (102 approximately 965) ng/L\] (both P < 0.01), the difference between the posttreatment group and control group was not statistically significant. There was no significant difference between therapy responsive and nonresponsive groups before treatment. There was a significant difference between the pre- and post-treatment resuets in responsive group (P < 0.01), but not in nonresponsive group (P > 0.05). The serum levels of sBAFF was positively correlated with the levels of the BAFF mRNA in PBMCs both in pre- and post therapy group (both P < 0.05).

<b>CONCLUSIONb>The levels of serum sBAFF and BAFF mRNA in PBMCs are increased in patients with WAIHA, their dynamic alterations may contribute to the development of WAIHA.

Anemia, Hemolytic, Autoimmune ; B-Cell Activating Factor ; genetics ; Humans ; Interleukin-4 ; Leukocytes, Mononuclear ; RNA, Messenger ; genetics

<b>OBJECTIVEb>To analyze the polymorphisms of B cell activating factor (BAFF) gene and the plasma levels of BAFF in patients with idiopathic thrombocytopenic purpura (ITP), and to investigate their roles in the pathogenesis of ITP.

<b>METHODSb>Alleles specific polymerase chain reaction (AS-PCR) and agarose gel electrophoresis were used to identify polymorphisms -871C/T of BAFF promotor in 133 ITP patients and 117 healthy controls. The plasma levels of BAFF were assayed by ELISA.

<b>RESULTSb>In ITP group, the frequency of C/C, C/T and T/T was 33.1%, 42.1% and 24.8%, respectively, the corresponding frequency in control group was 55.6%, 33.3% and 11.1%, respectively. The allele frequency of T in ITP and control groups was 45.9% and 27.4%, respectively. There was a significant difference in the BAFF -871C/T genotypic frequency between the ITP and control groups (P < 0.05). BAFF antigen in untreated ITP, treated patients and controls was 875.86 pg/ml, 502.59 pg/ml and 736.88 pg/ml, respectively, being also a significant difference among the three groups (P < 0.05). BAFF antigen in homozygous T/T was higher than that in homozygous C/C and heterozygous C/T, but the difference was not statistically significant (P > 0.05).

<b>CONCLUSIONSb>Over expression of BAFF may be a risk factor for ITP patients. There is a correlation of the BAFF promotor polymorphism -871C/T with ITP, but the polymorphism does not affect the expression of BAFF.

B-Cell Activating Factor ; genetics ; Gene Frequency ; Humans ; Interleukin-4 ; Polymorphism, Genetic ; Purpura, Thrombocytopenic, Idiopathic ; immunology

<b>OBJECTIVEb>To detect potential mutation of the WAS gene in a Chinese family affected with Wiskott-Aldrich syndrome.

<b>METHODSb>Peripheral blood samples were collected from the proband and his family members. All exons and flanking regions of the WAS gene were subjected to PCR amplification - Sanger sequencing as well as restriction endonuclease analysis. Plasma level of B-cell activating factor (BAFF) was also determined for all family members.

<b>RESULTSb>A hemizygous mutation (c.257G>A) of the WAS gene was identified in all patients from the family, for which the patient's mother was heterozygous. The same mutation was not found among healthy members of the family. Compared with unaffected members, all patients had a higher level of BAFF.

<b>CONCLUSIONb>The c.257G>A mutation of the WAS gene probably underlies the Wiskott-Aldrich syndrome in this family.

B-Cell Activating Factor ; blood ; Child, Preschool ; Heterozygote ; Humans ; Male ; Mutation ; Wiskott-Aldrich Syndrome ; genetics ; Wiskott-Aldrich Syndrome Protein ; genetics

<b>OBJECTIVEb>To investigate the expression of B lymphocyte stimulator (BlyS) and its receptors in multiple myeloma (MM) cells, and to explore the relationship between BLyS and the development of human multiple myeloma.

<b>METHODSb>Flow cytometry, RT-PCR and Western blot were used to examine the expression of BLyS and its receptors in MM (KM3 and CZ1) cells. Fluorescence immunocytochemical method and confocal laser scanning technique were applied to the localization of BLyS in KM3 cell. WST proliferation assay was used to examine the effect of BLyS on MM cells growth and survival. Linear correlation analysis was used to detect LDH and beta 2-microglobulin (beta2M) levels with BLyS protein and mRNA expressions in MM patients.

<b>RESULTSb>(1) BLyS and its receptors were expressed in MM cells. (2) BLyS protein was localized on the KM3 plasma membrane. (3) BLyS promoted survival and proliferation of MM cells. (4) MM patients had significantly higher expression levels of BLyS [77.42% (24/31)] BLyS mRNA [93.55% (29/31)], which were significantly correlated with the levels of LDH and beta 2-microglobulin (beta2M).

<b>CONCLUSIONb>BLyS and its receptors in MM cell lines and MM patient bone marrow might have a potential role in the growth and survival of malignant plasma cells.

Aged ; B-Cell Activating Factor ; genetics ; metabolism ; B-Cell Activation Factor Receptor ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Female ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; metabolism ; pathology ; RNA, Messenger ; genetics ; beta 2-Microglobulin ; metabolism

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

This study was aimed to explore the expression of B cell-activating factor of TNF family (BAFF) and B cell lymphoma/leukemia-2 (BCL-2) in bone marrow mononuclear cells (BMMNC) of multiple myeloma (MM) and the significance of BAFF and BCL-2 for occurrence, development and prognosis of MM. The bone marrow of 40 cases of MM and 10 healthy persons was collected, the mononuclear cells (MNC) were isolated, the expression of BAFF and BCL-2 mRNA in BMMNC was detected by real-time PCR; the plasma was simultaneously collected and the β2-MG level was determined; the clinical staging of MM patients was performed according to Durie-Salmon (D-S) staging criterion. The results indicated that the expression level of BAFF and BCL-2 mRNA in MM patients increased, as compared with normal controls, the difference was statistical significant (p < 0.05); the expression level of BAFF and BCL-2 mRNA in plateau stage after treatment obviously decreased. The expression level of BAFF and BCL-2 mRNA in relapsed/refractory MM patients was significantly higher than that in normal controls and patients reached plateau stage (p < 0.05), there was no statistically significant difference between newly diagnosed and relapsed/refractory MM patients (p > 0.05). The expression of BAFF and BCL-2 mRNA related with D-S staging and β2-MG level. It is concluded that the expression levels of BAFF and BCL-2 mRNA increase, moreover the expression levels of BAFF and BCL-2 mRNA in newly diagnosed and relapsed/refractory MM patients are higher than those in patients reached plateau stage, which suggest the BAFF and BCL-2 may be involved in occurrence and development of MM; the relation of expression level of BAFF and BCL-2 mRNA to MM load is positive, which indicates the expression level of BAFF and BCL-2 mRNA may be a new indicator for evaluating the prognosis of MM patients.
Adult ; Aged ; B-Cell Activating Factor ; genetics ; metabolism ; Case-Control Studies ; Female ; Humans ; Lymphoma, B-Cell ; genetics ; Male ; Middle Aged ; Multiple Myeloma ; diagnosis ; genetics ; Prognosis ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; genetics

<b>OBJECTIVEb>B cell multiplication plays a key role in infections mononucleosis. The present study was designed to detect the expression of B-lymphocyte stimulator (BLyS) mRNA in peripheral blood using real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) in children with infectious mononucleosis in order to explore the role of BLys in this disorder.

<b>METHODSb>Specific primers and TaqMan probes of BLyS were designed, and fluorescence of the PCR products were detected continuously during amplification. According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples were calculated using Stata Software version 8.0, and the results were presented as the ratio of copies of target gene mRNA to beta2 microglobulin (beta2M) mRNA copies. BLyS mRNA expression in peripheral blood was measured by RFQ-PCR in 18 children with infectious mononucleosis and the results were compared with those measured in 15 healthy controls.

<b>RESULTSb>The range of target gene mRNA detected by REQ-PCR was from 109 ng/L to 101 ng/L. The coefficient of variation for intra-experimental and inter-experimental reproducibility ranged from 1.88% to 5.89% and 6.32% to 12.34%, respectively. BLyS mRNA expression in peripheral blood in children with infectious mononucleosis were significantly higher than that in controls (1.65+/-0.10 vs 0.56+/-0.08; P < 0.01).

<b>CONCLUSIONSb>RFQ-PCR has a high sensitivity and reproducibility for the measurement of BLyS mRNA expression. BLyS may be involved in the development of infectious mononucleosis.

B-Cell Activating Factor ; genetics ; Child ; Child, Preschool ; Female ; Fluorescence ; Humans ; Infectious Mononucleosis ; etiology ; metabolism ; Male ; Polymerase Chain Reaction ; methods ; RNA, Messenger ; analysis

The study was purposed to detect BAFF/APRIL gene expression changes in bone marrow mononuclear cells (BMMNC) and myeloma cell line U266 after interference with glucocorticoid and bortezomib. After separation of BMMNC from 7 patients with multiple myeloma, BAFF/APRIL mRNA expression in BMMNC and U266 cell line was detected by real-time PCR after treated with dexamethasone 100, 200 µg/ml, methylprednisolone 100, 200 µg/ml, bortezomib 0.1 µg/ml alone and dexamethasone or methylprednisolone combined with bortezomib respectively for 48 hours. The results showed that U266 cells and BMMNC of untreated MM patients highly expressed BAFF/APRIL genes. When dexamethasone, methylprednisolone or bortezomib was added to U266 cells or BMMNC alone, BAFF/APRIL gene expression decreased as compared with the blank control (p < 0.01). The inhibiting effect of bortezomib to BAFF/APRIL expression was obviously strong(p < 0.05). When dexamethasone or methylprednisolone combined with bortezomib, the BAFF/APRIL gene expression further decreased compared with dexamethasone or methylprednisolone alone (p < 0.01). As compared with the group of methylprednisolone combined with bortezomib, BAFF/APRIL gene expression decreased in dexamethasone combined with bortezomib with a statistically significant difference (p < 0.05). It is concluded that the expression of BAFF/APRIL gene is down-regulated after bing treated with glucocorticoids and bortezomib, which suggests that besides the glucocorticoid receptor and proteasomes targets, BAFF/APRIL and their receptor sites may be new targets of glucocorticoids and bortezomib.
B-Cell Activating Factor ; genetics ; metabolism ; Boronic Acids ; pharmacology ; Bortezomib ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; drug effects ; Glucocorticoids ; pharmacology ; Humans ; Multiple Myeloma ; metabolism ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; Tumor Necrosis Factor Ligand Superfamily Member 13 ; genetics ; metabolism

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

<b>OBJECTIVEb>To explore the regulating effect of miR-202 on B cell-activating factor, and check whether the regulation influences the growth of multiple myeloma cells.

<b>METHODSb>The potential binding sites of BAFF for miR-202 were predicted using bioinformatics software. Luciferase reporter gene analysis was used to evaluate the regulatory effect of miR-202 on BAFF. Human multiple myeloma U266 cells were transfected with has-miR-202-mimics, has-miR-202-inhibitor, siBAFF and their negative controls, respectively. After above treatments, BAFF mRNA and protein levels were detected by real-time PCR and Western blot analysis, and the proliferation and apoptosis in the multiple myeloma (MM) cells were examined by WST-1 and annexin V-FLUOS assay, respectively.

<b>RESULTSb>The BAFF mRNA expression levels in the untransfected group, has-miR-202-3P-mimics transfected group, has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 1.040 ± 0.057, 0.573 ± 0.073, 1.205 ± 0.097 and 0.368 ± 0.052, respectively. BAFF mRNA expressions in U266 cells transfected with has-miR-202-3P-mimics and siBAFF were significantly decreased compared with that in the untransfected group (P < 0.05). The BAFF protein expression level of each group was consistent with the mRNA assay result. The absorbance value in 450 nm of the untransfected group, has-miR-202-3P-mimics transfected group, has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 1.063 ± 0.052, 0.714 ± 0.045, 0.936 ± 0.066 and 0.764 ± 0.053, respectively. In comparison with the untransfected group, the absorbance value at 450 nm of has-miR-202-3P-mimics and siBAFF transfected groups was significantly reduced (P < 0.05). The cell apoptosis rates of untransfected group, has-miR-202-3P-mimics transfected group, has-miR-202-3P-inhibitor transfected group and siBAFF transfected group were 26.2%, 49.6%, 21.1% and 30.7%, respectively. Therefore, the cell apoptosis rate of has-miR-202-3P-mimics transfected group was significantly increased than that of the untransfected group (P < 0.05). p-JNK protein expression level was decreased in the has-miR-202-3P-mimics transfected cells.

<b>CONCLUSIONSb>MiR-202 can inhibit the proliferation and induce apoptosis in MM cells via regulating BAFF. JNK/SAPK signaling pathway is involved in the regulation of BAFF by miR-202.

Apoptosis ; B-Cell Activating Factor ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; HEK293 Cells ; Humans ; Luciferases ; metabolism ; MAP Kinase Signaling System ; MicroRNAs ; genetics ; metabolism ; Multiple Myeloma ; metabolism ; pathology ; Plasmids ; RNA, Messenger ; metabolism ; Transfection