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Objective·To eliminate the effects of intraperitoneal injection of transmembrane activator and calcium modulator and cyclophilin ligand interactor-Ig (TACI-Ig) on the opitc neuritis and the integrity of myelin sheath in mice.Methods·Twenty-four C57BL/6J mice were randomly divided into 4 groups,which were blank control group,saline control group,low-dose (0.4 mg/kg) TACI-Ig group and high-dose (4 mg/kg) TACI-Ig group,with 6 mice in each group.All groups were received intraperitoneal injection every other day.The saline control group received 0.2 mL saline injection in the same way,and the blank control group was not given any intervention.After 20 d of treatment,the eyeballs and optic nerve tissues were collected from each mouse under anaesthesia,embedded in paraffin and stained with hematoxylin-eosin (H-E) and Luxol fast blue (LFB),respectively.Results·H-E staining indicated that optic nerve fibers arranged closely both in blank and saline control groups and the staining of tissues was uniform.The optic nerve structure of low-dose TACI-Ig group was similar to blank and saline control groups,while in high-dose of TACI-Ig group,the infiltration of inflammatory cells was observed.The inflammatory cell infiltration scores were not significantly different in all groups (P=0.610 3).The retinal structures of all groups were clear and distinct to observe,and single ganglion cells arranged tightly with complete cell shape,visible nucleus and uniform distribution.There was no difference in the retina structure among 4 groups.LFB staining indicated that there was no significant loss of the optic nerve myelin in 4 groups by microscope observation (P=0.473 6).Conclusion·Low-dose (0.4 mg/kg) TACI-Ig injection wouldn't damage the normal structure of optic nerves and retinal ganglion cells,meanwhile high-dose (4 mg/kg) of TACI-Ig injection might cause minor infiltration of inflammatory cells into retina.
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Objective To discuss the effects of Paclitaxel(PTX) on levels of CD28 and CTLA-4,B lymphocyte stimulator(BAFF) in experimental autoimmune encephalomyelitis(EAE).Methods The 50 rats were divided into 5 groups by the random number table, 10 rats in each group,the doses of small group,Middle group, High group were 1 mg/kg,2 mg/kg,4 mg/kg by intraperitoneal injection for 10 consecutive days, the normal group and model group were injected 0.9% NS 2 mL,Using brain tissue score to estimate the neurological dysfunctions of rats.Using flow cytometry to detect the levels of CD28 and CTLA-4,using enzyme-linked immunosorbent (ELISA) to detect the levels of BAFF.Results The brain tissue score in PTX experimental groups were lower than model group,the comparative differences between groups were statistically significant(P < 0.01);The levels of CD28 in PTX groups were lower than EAE group,the comparative differences between groups were statistically significant(P < 0.01).The levels of CTLA-4 in PTX groups were higher than EAE group,the comparative differences between groups were statistically significant(P < 0.01);the content of BAFF in all PTX groups were lower than EAE control group.Conclusions PTX could decrease the brain tissue score,the mechanism may adjust the express of CD28、CTLA-4 in brain and the expression of BAFF.PTX may have preventive and therapeutic effects on EAE rats.
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In order to study the structure-function relationship in the protein which is incorporated with p-nitro-L-phenylalanine,the method of MD(Molecular Dynamics) simulation was established and successfully used in the analysis of protein which contains p-nitro-L-phenylalanine.The force field of CHARMM can only stimulate protein with natural amino acid in NAMD.Compared with phenylalanine,p-nitro-L-phenylalanine just has one more group of nitro.If the parameter of group of nitro was defined,the protein containing p-nitro-L-phenylalanine can be simulated.CGenFF-paramchem was used to calculate the energy and topological structure of p-nitro-L-phenylalanine' s new bonds (r),angles (θ),dihendrals (φ) and improper angle (ψ).And then the new defined parameter and topology information was input into the related parameter files and topology files in CHARMM.On the basis of correct parameter,NAMD can successfully simulate the modified BAFF(B lymphocyte stimulator) which contains p-nitro-L-phenylalanine.The changes in structure indicated that there might be new B cell epitopes.The temperature distribution of each frame in the process of dynamics stimulation was in accord with normal distribution,which proved the defined force field parameters was feasible.The RMSD of whole protein solution systemis 2.5.Calculate each resides' RMSF in BAFF,the RMSF of p-nitro-L-phenylalanine's residue is 3.7,which is obviously higher than that of the other residues in β-pleated sheet,and close to the loop rings,indicate that there might be variation in the area of p-nitro-L-phenylalanine residue and might produce new comformational epitopes.The results of MD stimulation will guide the immunogenicity experiments of p-nitro-L-phenylalanine modified proteins.
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ObjectiveTo investigate the activation of MAPK signal pathway in multiple myeloma and the regulation of BLyS expression levels through MAPK signal pathway; preliminarily study the role of MAPK signal pathway in the up-regulation of BLyS expression levels induced by IFN-γ.MethodsActivated MAPK pathway were detected by Western blot,while the expression of BLyS were detected with RT-PCR and Western blot,and Western blot investigated the effect of MAPK pathway on BLyS expression levels induced by IFN-γ.ResultsIn addition to the expression of ERK,JNK,p38,p-JNK was also expressed in MM cell lines,the MAPK pathway inhibitor targeting JNK SP600125 can down-regulate the expression of BLyS,and its activator anisomycin can up-regulate the expression of BLyS.SP600125 restrained the proliferation and survival of MM cells.ConclusionJNK/SAPK pathway was activated in MM cells; The activated degree of JNK/SAPK pathway and the expression level of BLyS was positively correlated.JNK/SAPK pathway play an important role in the up-regulation of BLyS expression levels induced by IFN-γ.
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Objective To investigate the regulation of B-lymphocyte stimulator(BLyS) levels in response to IFN-γand IL-6. Methods Flow cytometry, quantitative polymerase chain reaction, ELISA and Western blot were applied to examine the expression level of BLyS in response to IFN-γ and IL-6 . Results IFN-γand IL-6 induced BLyS expression in KM3 cells. After treated with BAY11-7082, an IkB-α phospho- rylation inhibitor, the up regulation of BI,yS induced by IFN-γ was completely inhibited. Inhibiting the nu-clear faetor-kB (NF-kB) and mitogen activated protein kinase(MAPK) activation in KM3 cells reduced BLyS protein and gene expression. Conclusion MAPK and NF-kB pathways are involved in the regulation of BLyS expression, which suggests that MAPK and NF-kB might be used for the treatment of multiple mye- loma.
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B cell maturation antigen (BCMA) is a receptor of B lymphocyte stimulator (BLyS). Human IgG1Fc fusion proteins with the extracellular domain of BCMA(eBCMA), also called decoy receptors, have beenused as a potential BLyS antagonists to block BLyS activities. In order to design novel BLyS antagonistpeptides, computer-aided homologue modeling was used to construct an eBCMA-Fc fusion protein based on thecrystal structures of BCMA and Fc fragmant. To ensure the activity of eBCMA not to be interfered by Fcfusion, the root mean square distance (RMSD) for eBCMA and Fc were calculated to be 0.036 nm and 0.064nm, respectively, based on molecular docking modeling. An eBCMA-Fc fusion gene was constructed andintroduced into E. coli for expression. As expected, the purified 36 kD eBCMA-Fc fusion protein was able tobind BLyS in vitro at a dosage-dependent manner and demonstrated an anti-proliferative activity induced byBLyS in Daudi cells. The results have provided useful information on the evaluation of computer modeling andthe in vitro biological activity for the design of potential BLyS antagonist peptides.
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Objective To investigate the expression and significance of B lymphocyte stimulator (Blys) and its receptor BAFF (BAFF-R) in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE). Methods The expression of Blys and BAFF-R was measured by flow cytometry in 90 pa-tients with SLE,which was compared with that of 45 healthy controls. The relationships between the expression of Blys, BAFF-R and other laboratory parameters as well as disease activity were analyzed. Results The expression of Blys and BAFF-R in PBMCs from patients with SLE was significantly elevated compared to healthy controls (P <0.001), so did the active group (P < 0.001) and inactive group (P < 0.001 and P < 0.01). The expression of Blys in PBMCs from active SLE patients was higher than that of inactive patients (P <0.05). However,there was no statisti-cal difference of BAFF-R between the two groups. The expression of Blys in PBMCs was positively related to SLEDAI (r =0.728,P <0.001) ,IgG and IgM(r=0.691,P<0.001 and r =0.453,P<0.01) ,but negatively related to C3 and CA (r = -0.510, P < 0.001 and r = -0.312, P < 0.05). The expression of Blys in dsDNA positive group was higher than those of dsDNA negative group (P < 0.01). The expression of Blys and BAFF-R in Cl qAb positive group was higher than those of ClqAb negative group as well (P <0.01). Conclusion The expression of Blys and its receptor BAFF-R in PBMCs from SLE is elevated ,which may reflect the disease activity and is related to the pro-duction of autoantibody. They might be involved in the pathogenesis of SLE.
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Objective:To investigate the prevalence of the antibodies against the B lymphocyte stimulator(BlyS),(BlyS-Ab) in patients with clinical diseases,and to explore the role of BlyS-Ab in the pathogenesis of autoimmune diseases(AID).Methods:By optimizing experimental conditions,we developed ELISA methods for serum levels of BlyS-Ab,followed by methodological and clinical application assessment and investigation of the relationship of serum BlyS-Ab levels with the titers of anti-nucleus antibodies(ANA) and other autoantibodies.Results:The intra-assay precision,namely the average coefficient values of BlyS-Ab was 7.8%,and the specific inhibition rate for BlyS-Ab by series concentration of BlyS was up to 77.3%.Clinical investigation revealed that the serum levels of BlyS-Ab was significantly higher in patients with autoimmune diseases(including SLE,RA,myasthenia gravis) and other medical cases(such as allergic purpurics,chronic nephritis,etc.) than in the normal control(P
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Objective To determine the expression of membrane-bound B lymphocyte stimulator (Blys) and Blys mRNA in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE), and investigate the relationship between Blys mRNA expression and SLE activity or lupus nephritis. Methods Measure the expression of Blys mRNA with reverse transcription-PCR (RT-PCR) in PBMCs from patients with SLE, who were separated into active group (n=26) and inactive group (n=20) according to the SLE disease activity index (SLEDAI) and 20 healthy volunteers; analyze the relationship among Blys mRNA and several clinical items; measure the expression of membrane-bound Blys with flow cytometry (FACs) in 20 patients with SLE and 16 healthy controls. Results The expression of Blys mRNA in PBMCs from patients with SLE was significantly elevated compared to healthy controls (P
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0.05).Conclusion High level of Blys could be observed in serum of patients with SLE and somewhat correlated with the lupus acitivity,although the level of Blys in serum can not reflect morbility and mortality of SLE patients.
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Objective To determine the expression of membrane-bound B lymphocyte stimulator (BLyS) protein and its mRNA in vitro of peripheral blood mononuclear cells (PBMCs) from individuals with systemic lupus erythematosus (SLE),and to investigate the role of interferon-?(IFN-?) on the expression of BLyS.Methods PBMCs were obtained from 25 SLE patients (mean age of 31+14) and 20 healthy volunteers (mean age of 28?10).They were randomized into IFN-?(5 ng/ml) group and control group.PBMCs were col- lected at 0,6,12 and 24 h for BLyS mRNA assessment using semi-quantitative reverse transcription-PCR (RT-PCR).PBMCs were also collected at 72 h for membrane-bound BLyS protein detection using flow cy- tometry (FACS) and direct immunofluorescence.Results①The expression of BLyS mRNA and membrane- bound protein in PBMCs was significantly higher in individuals with SLE compared with healthy controls (P＜0.05);②IFN-?enhanced BLyS mRNA expression in PBMCs in both healthy controls and SLE patients,with the greatest effect at 6 h (stimulated vs unstimulated,0.42?0.19 vs 0.25?0.14,P＜0.01;0.59?0.28 vs 0.44?0.21,P＜0.01 );③IFN-?also increased the expression of membrane-bound BLyS protein in both healthy con- trols and individuals with SLE (FACs,mean fluorescence intensity,4.5+3.0 vs 3.7~2.6,P
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AIM: To establish the monoclonal antibody against human B lymphocyte stimulator (hBLyS) by DNA immunization and analyse its characterization. METHODS: The 858 bp DNA fragment of hBLyS was cloned into pcDNA3 plasmids. The cloned insert was identified by both sequence analysis and double digestion of the recombinant plasmid with restriction enzymes Xho I and EcoR I . After the splenocytes from BALB/c mice immunized with the recombinant plasmid of pcDNA3/hBLyS were fused with myeloma cells SP2/0,the hybridoma which can produce monoclonal antibodies against hBLyS were obtained. The specificity of anti-BLyS monoclonal antibody from hybridoma was verified by ELISA, Western blot and flow cytometry. RESULTS: The recombinant mammalian cell expression vector of pcDNA3/hBLyS was constructed, the sequence of the insert gene was identified to be the sequence encoding hBLy S antigen. The culture supernatants of hybridoma 9c10 were tested to be the monoclonal antibody with specificity against hBLyS on human peripheral blood CD3 + T cell activated by hIFN-? by ELISA, Western blot and flow cytometry. CONCLUSION: The monoclonal antibodies against hBLyS with high activity and specificity have been established successfully, and will be an useful tool in the studies of relationship between hBLyS and human autoimmunity diseases.