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Study of the Theraputic Effect of Anti-human B7-1 Antibody on Lupus-like Disease

Author: GaoZengYan
Tutor: QiuYuHua；ShiQin
School: Suzhou University
Course: Immunology
Keywords: lupus like disease C57BL/6J mice murine model Pristane anti-human B7-1 antibody
CLC: R593.242
Type: Master's thesis
Year: 2011
Downloads: 42
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Abstract


Systemic lupus erythematosus (SLE) is a common system autoimmune disease, characterized by T、B lymphocyte dysfunction, a variety of antoantibodies (the most important antibodies are the antinuclear antibody and anti double-stranded DNA antibody) and immune complexes. Also inflammation can affect the body, resulting in destruction and the irreversible loss of function in joints, skin, kidneys, heart, lungs, blood vessels and other tissue and organs. Lupus nephritis (LN) is one of the most common and serious complications of SLE, which is the main cause of death in patients with SLE.B7-1 molecule is a member of immunoglobulin superfamily, and its combining to receptors CD28/CTLA-4 can generate costimulatory signals, which can mediate T cell immune response. If there is no B7/CD28 cosimulatory signal, T cells will enter to a state of anergy or tolerance, or even death. It is well reported that application B7 antibody can block the interaction of B7 and CD28/CTLA-4 and thereby inhibit the transmission of costimulatory signal. These can result in hypo-response or non-response. So by blocking B7-CD28/CTLA-4 costimulatory pathway can be expected to provide a new treat way of autoimmune disease, transplant rejection tumor immunity and other diseases.In the present study, we established a lupus like disease model in mouse similar to humans with Pristane (2,6,10,14-tetramethylpentadecane). Based upon this model, the therapeutic effects of mouse anti-human B7-1 monoclonal antibody on SLE were observed and elucidated the pathogenesis of SLE, which could contribute some experimental and theoretical data for SLE treatment.ⅠPreparation and assay of mouse anti-human B7-1 antibodyObject: To obtain pure B7-1 antibody and analyze its titer and recognition of membrane B7-1 molecule. Methods: Using of the 4E5 cell line our institute successfully established, ascites were induced to produce the antibody from BALB/c mouse, and were purified with ProteinG affinity chromatography method. Flow cytometry assay was used to test the ability of mAb to recognize the B7-1 molecule on different cell membranes. Results: The positive rate of ascites formation in mice was about 85%. Ascites were induced to produce the mAb and 3 ml ascites was obtained from each BALB/c mouse on average. The purified 4E5 from ascites was about 3 mg per ml ascites with ProteinG affinity chromatography method. The 4E5 could specifically recognize membrane B7-1 on cells of Daudi、Raji、H1299、spleen cells of mouse with the positive of 88.6%、95.7%、20.6%、48.5%, respectively.ⅡDeveloping and charactering a lupus like disease model in mouseObject: To explore the pathogenic mechanism of a lupus like disease model in mice induced by Pristane. Methods: Thirty 8-week-aged female mice were devided into two groups randomly. The experimental group were injected with 0.5ml Pristane by intraperitoneal while the control group with 0.5ml 0.9%N.S..The activation of macrophage and dendritic cell in spleens and markers on B cells were measured by FCM at day 5 and 10 after injection, respectively. Antinuclear antibodies(ANA) and anti-double strand DNA antibodies (dsDNA) in serum were detected monthly after injection meanwhile proteinuria was checked accordingly. 7 months after injection, all mice were killed and kidneys were slided and stained with H&E or FITC-labeled IgG to observed the evidence of glomerulonephritis histopathologically. Results: After Pristane injection, the populations of macrophage and dendritic cell were increased significantly compared to the control ones(p<0.05). The expressions of CD21、CD86、MHC-II on B cells were also increased(p<0.05). Autoantibody ANA and dsDNA appeared in serum as early as 4 months after Pristane injection(p<0.05). 7 months later, the protein concentration of uria in most experimantal mice was≥1+ (300mg/L). Both light microscopy and imunoflorescence of kidney sections indicated typical evidence of glomerulonephritis. In the control group, proteinuria weren’t detected under the same experiamental condition. Meanwhile, there was no evidence of glomerulonephritis in control groups.ⅢTherapeutic effects of mouse anti-human B7-1 antibody on lupus like diseaseObject: To explore the therapeutic effect of mouse anti-human B7-1 antibody on lupus-like disease induced by Pristane in mice. Methods: Forty-five 8-week-old female mice were divided into three groups randomly. The control group were injected with 0.5ml 0.9%N.S. by intraperitoneal while the other two groups with 0.5ml pristine to induce lupus-like disease. One group with Pristane were intervented with mouse anti-human B7-1 antibody by caudal vein injection (the intervention group) and the other one with mouse isotype IgG(the model–making group). The activation of macrophage and dendritic cell in local lymph nodes and spleens and markers on B cells in spleens were measured by FCM, respectively. Antinuclear antibody (ANA) in serum and proteinuria were detected monthly after injection. 7 months after injection, all mice were killed and kidneys were slided and stained with H&E or FITC-labeled IgG to observe the evidence of glomerulonephritis histopathologically. Results: After 10 days, compared with the control group, the intervention group and the model–making group macrophage and dendritic cells in local lymph nodes and spleens had different levels of activation, the intervention group activation was significantly lower than the model-making group (p <0.05). The expressions of CD21, CD40, CD86 on spleen B cell of the intervention group increased lower than ones of the model group (p <0.05). The concentration of ANA in mice sera and protein in uria showed the same trend. 7 months later, the intervention group showed less histopathologically damages than the model-making group. Immunofluorescence intensity of immune complex (IC) in the intervention group mice was weaker than the model-making ones. The control group mice kidneys had no significant pathological changes and IC couldn’t be detected.Conclusion:⑴M ouse anti-human B7-1 antibody could specifically recognize membrane B7-1 expressed on cells line and spleen cells in mice.⑵Mouse model of systemic lupus erythematosus in female mice injected with 0.5ml Pristane by intraperitoneal is established successfully.⑶B locking B7-1/CD28 co-stimulatory signal pathway, mouse anti-human B7-1 antibody alleviate mouse lupus-like disease induced by Pristane.

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CLC: > Medicine, health > Internal Medicine > Systemic disease > Autoimmune diseases > Autoimmune diseases, connective tissue disease > Lupus erythematosus > Lupus nephritis
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