Characterization of a PRISTANE-induced lupus-associated model in the non-human primate cynomolgus monkey.

BACKGROUND: Lupus is an autoimmune disease with complex syndrome. Rodent models have limitations for recapitulating the spectrum of the disease. A more powerful translational model is desirable. METHOD: Lupus-associated model in cynomolgus monkeys was induced by two intraperitoneal injections of 2, 6, 10, 14-tetramethylpentadecane (PRISTANE). Lupus-specific biomarkers and manifestations over a 246-day period were observed at multilevel. To visualize and quantify kidney function in real time, contrast-enhanced ultrasound was used. RESULTS: The indicative biomarkers and manifestations fulfilled major diagnosis criteria according to the "Criteria of Lupus" of the American College of Rheumatology. Significant changes in time-intensity curve parameters were observed, indicating impaired renal function and the method as a feasible, non-invasive diagnostic method in primate model. CONCLUSIONS: We successfully induced lupus-associated model with systemic lupus syndrome. This primate model can be a valuable translational model for further pathogenesis and symptomology studies and for exploring therapeutic candidates.

A monoclonal antibody against CD86 and its protection in a murine lupus nephritis model of chronic graft-versus-host disease.

CONTEXT: Lupus nephritis is the most common complication that causes the death of systemic lupus erythematosus patients. CD28/CTLA4 and their ligands CD80 or CD86 costimulatory pathway play a pivotal role in autoimmune disease and organ transplantation. OBJECTIVES: We generated a monoclonal antibody (clone 1D1) against human CD86 (1D1) that could recognize both human and mouse CD86, and blocked the CD86/CD28 costimulatory pathway with our mAb on a murine lupus nephritis model induced with chronic graft-versus-host disease (cGVHD). MATERIALS AND METHODS: Experimental lupus nephritis mice were induced with cGVHD, and splenocyte population were analyzed by flow cytometry. Autoantibodies and proteinuria were detected to evaluate the severity of lupus nephritis. The change of histopathology was observed by microscopy, fluorescence microscopy and electron microscopy. RESULTS: we successfully generated a monoclonal antibody against human CD86(1D1). 1D1 mAb could recognize not only human CD86, but also mouse CD86. 1D1 was applied to the cGVHD-induced experimental lupus nephritis model, and our study found the production of ANA and anti-dsDNA in the 1D1-treated group was lower than those in IgG-treated group after four weeks. The pathological injure of kidney in the 1D1-treated group was lighten than that in IgG-treated group. DISCUSSION AND CONCLUSIONS: Our data showed that blockade of CD86/CD28 with 1D1 induced a significant remission of proteinuria, production of autoantibodies, immune complex deposition and renal parenchyma lesions in experimental mice. Anti-CD86 Abs might be a potential method for immune therapy in autoimmune diseases and transplantation.

4-1BB-mediated signals confer protection against folic acid-induced nephrotoxicity.

BACKGROUND: The role of co-stimulatory molecules in renal diseases has been previously examined, however, little is known about the role of 4-1BB in the context of renal diseases resulting from nonimmune-mediated tubulointerstitial fibrosis. Folic acid induced Nephrotoxicity (FAN) in mice was used to explore the role of 4-1BB in this setting. METHODS: CD1 mice were treated with folic acid and kidneys subsequently examined using histochemistry, in addition to defining T cell profiles and evaluating renal function. Increased CD3+ and CD4+ T lymphocytes present in blood and spleen at day 3 suggested immunopathological reactions during the early stages of FAN and decreased CD3+ and CD4+ T lymphocytes on day 14 were characteristic of an immunocompromised state observed during the late stages of FAN. RESULTS: After 14 days of co-treatment with agonistic anti-4-1BB monoclonal antibodies, renal tubulointerstitial lesions were reduced. Renal function was improved, with Bun scores decreasing (p<0.01) and sCr levels decreasing (p<0.01). CD3+ and CD4+ T lymphocytes levels were increased during the early stages of disease in FA treated mice and reduced to the normal level in the 4-1BB-treated mice. CD3+ and CD4+ T lymphocytes levels were decreased in FA treated mice and returned to baseline in the 4-1BB-treated mice during later stages. CONCLUSIONS: Data presented in this report demonstrated that 4-1BB signals had immunoregulatory effects that attenuated early immune-mediated pathology and reversed the immunocompromised state observed during the later stages of disease.

Role of HLA-G and NCR in protection of umbilical cord blood haematopoietic stem cells from NK cell mediated cytotoxicity.

Allogeneic umbilical cord blood haematopoietic stem cells (UCB-HSCs) can be transplanted into a host with the intact innate immunity with limited immuno-reaction, although the mechanisms remain unclear. The present studies aimed at investigating potential mechanisms of allogeneic UCB-HSCs escape from the cytolysis of natural killer (NK) cells. We compared UCB-HSCs ability to protect from NK-mediated cytotoxicity with peripheral blood or bone marrow haematopoietic stem cells (PB-HSCs and BM-HSCs). HSCs expressed lower levels of natural cytotoxicity receptor ligands including NKp30L, NKp44L and NKp46L than monocytes. Blocking these ligands respectively or in combination could increase the resistance of HSCs against NK cell mediated cytotoxicity. High expression of HLA-G was noticed on UCB-HSCs, rather than PB-HSCs or BM-HSCs, whereas blockade of HLA-G significantly elevated NK cell mediated cytolysis to UCB-HSCs. Thus, we conclude that natural cytotoxicity receptors and HLA-G on HSCs may contribute to the escape from NK cells, and activate and inhibitory NK cell receptors and their ligands can be novel therapeutic targets in cell transplantation.

A novel approach to induce human DCs from monocytes by triggering 4-1BBL reverse signaling.

Dendritic cells (DCs) are responsible for the initiation of immune responses. Our study demonstrates a new pathway for generating a large quantity of stimulatory monocyte-derived DCs (Mo-DCs) from human monocytes using anti-4-1BB ligand (4-1BBL) mAb to trigger reverse signaling. The anti-4-1BBL-driven Mo-DCs (DCs(alpha-4-1BBL)) not only express higher levels of CD86, CD83 and HLA-DR, when compared with the Mo-DCs matured by tumor necrosis factor alpha, but also exhibit a unique phenotype that expresses lower levels of PD-L1. High levels of GM-CSF, M-CSF and Flt3 ligand (FL) were found in the anti-4-1BBL-differentiation culture. Neutralizing M-CSF, GM-CSF and FL inhibited Mo-DC proliferation stimulated by anti-4-1BBL mAb, suggesting that M-CSF, GM-CSF and FL are involved in cell proliferation stimulated by anti-4-1BBL. Further analysis of the DCs(alpha-4-1BBL) showed increased secretion of T(h)1-type cytokines IL-12 and IFN-gamma and decreased secretion of IL-10. DCs(alpha-4-1BBL) induced much stronger proliferative responses in the mixed lymphocyte reaction assay when compared with DCs derived by GM-CSF. Moreover, DCs(alpha-4-1BBL) preferentially induced T(h)1 responses. We have further demonstrated that anti-4-1BBL antibody stimulated nuclear translocation of NF-kappaB from the cytoplasm in monocytes, suggesting that reverse signaling by 4-1BBL is likely responsible for mediating DC differentiation. Collectively, we have found that reverse signaling of 4-1BBL promotes the differentiation of potent T(h)1-inducing DCs from human monocytes.

A new approach to the blocking of alloreactive T cell-mediated graft-versus-host disease by in vivo administration of anti-CXCR3 neutralizing antibody.

Chemokines and chemokine receptors play critical roles in directing the migration of alloreactive donor T cells into graft-vs-host disease (GVHD) target organs. However, blockade of GVHD by antagonist Ab against chemokine receptors remains an elusive goal. Using a mouse model of human GVHD, we demonstrate that in vivo administration of anti-CXCR3 Ab for 21 days (long-term), but not for 7 days (short-term), inhibits alloreactive CD8(+) T cell-mediated GVHD. During a graft-vs-host reaction, infused donor CD8(+) T cells generate two subsets of potent inducers of GVHD: CXCR3(+)CD8(+) and CXCR3(-)CD8(+) T cells. Compared with CXCR3(+)CD8(+) T cells, CXCR3(-)CD8(+) T cells produce less granzyme B, Fas ligand, IFN-gamma, and TNF-alpha. Interestingly, stimulation with either dendritic cells or IL-2 induces a dynamic conversion between CXCR3(+)CD8(+) and CXCR3(-)CD8(+) T cells. Short-term anti-CXCR3 Ab treatment inhibits only CXCR3(+)CD8(+) T cell-mediated GVHD, but not the disease induced by CXCR3(-)CD8(+) T cells. Prolonged in vivo administration of anti-CXCR3 Ab significantly reduces the infiltration of alloreactive CD8(+) T cells into GVHD target organs and inhibits GVHD mediated by either CXCR3(+)CD8(+) or CXCR3(-)CD8(+) T cells. Thus, we have established a novel and effective approach with the potential to give rise to new clinical methods for preventing and treating GVHD after allogeneic hematopoietic stem cell transplantation.

A novel monoclonal antibody against human B7-1 protects against chronic graft-vs.-host disease in a murine lupus nephritis model.

Lupus nephritis (LN) is the most common complication that causes mortality in patients with systemic lupus erythematosus. The B7-1/B7-2 and CD28/cytotoxic T-lymphocyte associated protein 4 co-stimulatory pathway serves a key role in autoimmune disease and organ transplantation. The aim of the present study was to generate and characterize a monoclonal antibody (mAb; clone 4E5) against human B7-1 and to investigate its potential use for the treatment of LN. The results demonstrated that the 4E5 mAb was successfully generated and able to recognize both human and mouse B7-1. After injection of this mAb into a mouse model with chronic graft-vs.-host disease (cGVHD)-induced lupus-like disease, the expression of CD21, CD23, CD80 and CD86 on B220+ B-cells in the spleen, and the concentrations of serum autoantibodies and urine protein, were decreased. Direct immunofluorescence analysis of the kidneys revealed that immunofluorescence of immune complex deposits was weaker in the 4E5-treated mice and electron microscopy analyses of renal tissues indicated that pathological injury of the kidneys of 4E5-treated mice was decreased compared with that in the model control mice. The results of the present study demonstrated that inhibition of the B7-1/CD28 co-stimulatory signaling pathway with the 4E5 mAb may represent a promising strategy to decelerate the progression of LN that is induced by cGVHD with potential for use in the treatment of other autoimmune diseases.

Effects of preemptive intravenous lornoxicam on the analgesic efficacy of epidural morphine and expression of chemokines in women undergoing hysterectomy.

BACKGROUND: It is believed that preemptive IV lornoxicam treatment can reduce the consumption of other analgesics, improve analgesic efficacy, and ameliorate immune function during patient-controlled IV analgesia. However, the effects of preemptive IV lornoxicam treatment on the analgesic efficacy of patient-controlled epidural analgesia (PCEA) with morphine and on chemokine expression remain unknown. OBJECTIVE: The aim of this prospective, randomized, controlled study was to observe the effects of preemptive IV lornoxicam treatment on the analgesic efficacy of PCEA with morphine and on the expression of monocyte chemotactic protein-1 (MCP-1) and stromal cell-derived factor-1α (SDF-1α) in women undergoing hysterectomy. METHODS: Patients undergoing elective hysterectomy with combined spinal and epidural anesthesia were randomized to 1 of 3 groups to receive IV lornoxicam 8 mg before anesthesia (group 1), lornoxicam 16-mg injection before anesthesia (group 2), or isotonic saline (control) before anesthesia. PCEA was used to treat postoperative pain, and a visual analog scale (VAS) and the Bruggemann Comfort Scale (BCS) were used to evaluate analgesic efficacy. Morphine consumption was recorded. To measure plasma concentrations of MCP-1 and SDF-1α via enzyme-linked immunosorbent assay, venous blood samples were obtained from patients at 4 separate times: before anesthesia (baseline); 0 (immediately after anesthesia administration); and 24 and 48 hours after surgery. RESULTS: Forty-five patients (mean [SD] age, 41 [5] years; mean [SD] weight, 54 [6] kg) undergoing elective hysterectomy were included in the study. There were no significant differences in VAS scores, BCS scores, or morphine consumption between the 3 groups. Compared with baseline values, MCP-1 and SDF-1α concentrations were increased significantly immediately after surgery in all 3 groups (all, P < 0.01) and returned to near-baseline values at 24 hours postsurgery in groups 1 and 2, and by 48 hours postsurgery in the control group. MCP-1 and SDF-1α concentrations in groups 1 and 2 were significantly lower than those in the control group immediately (all, P < 0.01) and 24 hours postsurgery (all, P < 0.05). CONCLUSION: Preemptive IV lornoxicam treatment was associated with attenuation of the plasma concentrations of MCP-1 and SDF-1α immediately after and 24 hours after hysterectomy and was associated with more rapid resolution to near-baseline concentrations of both cytokines in these patients compared with controls; however, it was not associated with significantly reducing epidural morphine consumption.

Therapeutic effects of anti-B7-1 antibody in an ovalbumin-induced mouse asthma model.

Allergic asthma is a chronic inflammatory disorder of airways, which is characterized by attacks provoked by exposure to so-called asthma triggers, such as pet dander, second-hand tobacco smoke, dust mites, and mold spores. B7-1 (CD80), perhaps one of the most studied co-stimulatory molecules involved in asthma, plays a key role in regulating allergen-induced T cell activation in asthma, probably through T cell recruitment and Th cell differentiation upon allergen provocation. The present study was designed to test the hypothesis that anti-B7-1 antibody has therapeutic effects in asthma by blocking B7-1/CD28 pathway. The asthma model was established by ovalbumin (OVA) sensitization and challenging in female Balb/c mice. One hour after the last induction, mice were sacrificed and whole lung lavage was conducted. Cell numbers in bronchoalveolar lavage fluid (BALF) were determined and the expression levels of IFN-gamma and IL-4 in supernatant were measured by an enzyme-linked immunosorbent assay method. Sedimental cells smears were stained with Wright's-Gimsa mixed coloring method. The B7-1 expression was detected by immunohistochemistry method with frozen tissue sections. The anti-B7-1 antibody treatment could alleviate asthmatic syndromes induced by OVA. The number of recoverable eosinophils in BALF in the anti-B7-1 antibody treated group was significantly lower than that in the control group (P<0.01) and the eosinophils peribronchial infiltration was remarkably reduced in anti-B7-1 treated asthmatic mice, based on histological evaluation. The treatment with the anti-B7-1 antibody induced IFN-gamma expression and decreased IL-4 expression, compared with the asthmatic control group (P<0.01). In conclusion, the anti-B7-1 antibody approach may provide a novel therapy for allergic asthma.

High expression of VRK1 is related to poor prognosis in glioma.

Vaccinia-related kinase 1 (VRK1) is a member of the vaccinia-related kinase (VRK) family of serine/threonine protein kinases, which phosphorylates several transcription factors and has been postulated to be involved in regulation of cell proliferation. However, it remains unclear whether aberrant expression of VRK1 is related to the development of glioma. In this study, we aimed to investigate the clinical significance of VRK1 expression in human glioma and its biological function in glioma cells. Western blot and immunohistochemical analysis revealed that VRK1 was highly expressed in glioma tissues and cell lines. In addition, the expression level of VRK1 was positively correlated with glioma pathological grade, as well as Ki-67 expression. Kaplan-Meier analysis revealed that patients with high VRK1 expression was associated with a poorer prognosis. To determine whether VRK1 could regulate the proliferation of glioma cells, we transfected glioma cells with interfering RNA target VRK1, then investigated cell proliferation with cell counting kit (CCK) -8, flow cytometry assays and colony formation analyses. Our results indicated that knockdown of VRK1 would inhibit the proliferation of glioma cells. Besides, reduced expression of VRK1 could induce the apoptosis of glioma cells. On the basis of these findings, we suggested that VRK1 might be a promising prognostic biomarker of glioma.

[Preparation of mouse monoclonal antibody against human B7-1 (CD80) and its inhibitory effect on tumor cells in vitro].

Objective To prepare a mouse anti-human B7-1 monoclonal antibody (mAb) and study its effect on growth and migration of tumor cells naturally expressing human B7-1 (CD80) molecular in vitro. Methods BALB/c mice were immunized with L929-B7-1 cells transfected with human B7-1 gene and mAbs were prepared by B lymphocyte hybridoma technology. Then, the recognition ability of mAb to tumor cells expressing membrane B7-1 was detected by flow cytometry. Different concentrations of mAbs (5, 10, 20, 40 µg/mL) were added into Daudi, Raji, and 8266 cells to investigate the anti-proliferation effect by MTT assay. TranswellTM assay was used to analyze the effect of mAb on tumor cell migration in vitro, and flow cytometry was used to analyze the induction effect of mAb on apoptosis of tumor cells. Results A hybridoma cell stably secreting mouse anti-human B7-1 mAb was successfully obtained and named 5G10. The binding rates to tumor cells Daudi, Raji, 8266, U266, Bel-7402 and MCF-7 were (96.3±2.12)%, (95.7±1.79)%, (96.8±2.48)%, (23.2±2.35)%, (1.68±0.35)%, (0.55±0.04)%, respectively. Compared with control group, 20 µg/mL 5G10 mAb significantly inhibited the proliferation of Daudi, Raji and 8266 cells, reduced their migration, and increased cell apoptosis rates remarkably. Conclusion The 5G10 mAb can specifically recognize and bind to membrane B7-1, inhibit the proliferation, migration and promotes apoptosis of tumor cells in vitro.

Recombination of CXCR4, VEGF, and MMP-9 predicting lymph node metastasis in human breast cancer.

We investigated the expression of CXC chemokine receptor 4 (CXCR4), vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 to elucidate whether these markers could predict lymph node metastasis in human breast cancer. Higher rates of CXCR4 (61%), VEGF (68%), and MMP-9 (63%) expression were found in breast cancer tissues than in normal and atypical hyperplasia tissues. The expression of these markers was significantly associated with primary tumor progression, histological grade, and lymph node status. We found there were significant correlations between the expressions of any two of the three markers (P<0.001). Furthermore, our studies indicated that concomitant expression of CXCR4/VEGF (P=0.007), CXCR4/MMP-9 (P<0.001) or VEGF/MMP-9 (P=0.003) had stronger correlation with lymph node metastasis than did each alone and that combined expression of all three makers strongly correlated with lymph node metastasis (P<0.001). Thus, simultaneously examining the expression of CXCR4, VEGF, and MMP-9 in cancer tissues of breast cancer will provide valuable prognostic diagnosis of lymph node metastasis.

Reducing progression of experimental lupus nephritis via inhibition of the B7/CD28 signaling pathway.

The aim of the present study was to evaluate the effects of the B7/cluster of differentiation (CD)28 signaling pathway on experimental lupus nephritis and examine the molecular mechanism involved by inhibiting the B7/CD28 signaling pathway. A lupus nephritis model in C57BL/6 J mice was induced via intraperitoneal injection of pristane. A recombinant B7­1 short hairpin RNA (shRNA) lentivirus vector was constructed by synthesis and splicing. A neutralizing mouse anti­human B7­1 antibody termed 4E5 was also prepared. The mouse model of lupus nephritis was treated with B7­1 shRNA and 4E5 via injection through the tail vein. The silencing effects of B7­1 shRNA lentiviral infection on target molecules were evaluated using immunofluorescence and flow cytometry. The levels of protein in the urine were detected using Albustix test paper each month over 10 months. The concentration of interleukin (IL)­4 and interferon­Î³ in the serum was determined using an ELISA. The immune complex (IC) deposits in the kidney were analyzed using direct immunofluorescence. The results demonstrated that the C57BL/6 J mouse lupus nephritis model was successfully constructed with immune cells activated in the spleen of the mice, increases in the concentration of anti­nuclear antibody (ANA) and anti­double stranded DNA antibodies as well as positive IC formation. Following B7­1 shRNA lentivirus or 4E5 treatment, CD11b+B7­1+, CD11c+B7­1+ and CD21+B7­1+ cells in the spleen of the mice were significantly reduced. The concentration of ANA and IL­4 in the serum was also decreased. The concentration of urine protein was reduced and it was at its lowest level in the 4E5 early intervention group. It was also revealed that the immunofluorescence intensity of the IC deposits was weak in the 4E5 early intervention group. In conclusion, inhibiting the B7­1/CD28 signaling pathway is able to alleviate experimental lupus nephritis and provides an experimental basis for the therapeutic use of blocking the B7­1/CD28 signaling pathway in human lupus nephritis and other autoimmune disorders.

[CXCR3 monoclonal antibody inhibits the proliferation and migration of MCF-7 cells and HepG2 cells in vitro].

OBJECTIVE: To study the effects of chemokine (C-X-C motif) receptor 3 (CXCR3) monoclonal antibody (mAb) on the proliferation and migration of MCF-7 and HepG2 cells in vitro. METHODS: Ascites of CXCR3 mAb was prepared at first. MCF-7 and HepG2 cells with high expressions of CXCR3 were screened by flow cytometry. MTT assay was used to detect the effects of CXCR3 mAb on the proliferation of MCF-7 and HepG2 cells in vitro in the absence/presence of interferon-inducible T-cell alpha chemoattractant (I-TAC). Transwell™ assay was performed to investigate the effects of CXCR3 mAb on the migration of MCF-7 and HepG2 cells in vitro in the absence/presence of I-TAC. RESULTS: The expression rate of CXCR3 on MCF-7 and HepG2 cells were 83.5% and 96.2%, respectively. 50 mg/mL CXCR3 mAb significantly inhibited the proliferation and migration of MCF-7 and HepG2 cells, and also inhibited the promoting effect of I-TAC on the proliferation and migration of MCF-7 and HepG2 cells in vitro. CONCLUSION: CXCR3 mAb can significantly inhibit the proliferation and migration of the tumor cells highly expressing CXCR3 in vitro.

[Gene cloning, expression and binding activity of anti-CD86 scFv].

OBJECTIVE: To construct and express the single chain variable fragment (scFv) against human CD86 in CHO cells, and observe its biological functions of binding with antigen. METHODS: The V(H); and V(L); genes were cloned by RT-PCR from a murine hybridoma cell line 1D1, which produced the monoclonal antibody (mAb) against human CD86. The CD86-scFv gene was integrated into eukaryotic expression vector to construct pIREST2-EGFP/scFv. CHO cells were transfected by pIREST2-EGFP/scFv plasmid with Lipofectamine(TM); 2000 and then were selected by G418. We used IMAC affinity chromatography to purify CD86-scFv and quantified its concentration by BCA method. Then the identification of CD86-scFv binding to membrane CD86 was performed through competitive inhibition assay. In addition, the growth inhibition effect of CD86-scFv on Raji cells was detected via MTT assay. RESULTS: One cell line stably expressing CD86-scFv was obtained. The CD86-scFv could identify CD86 molecules on L929-CD86, Raji and Daudi cells, and the positive rates were 67.0%, 72.3% and 80.5%, respectively. CD86-scFv showed the competitive binding to murine parent antibody 1D1. Moreover, CD86-scFv inhibited the growth of Raji cells and the inhibition rate was 28.3% 72 h after 20 µg/mL CD86-scFv was added into Raji cells. CONCLUSION: CD86-scFv has been successfully expressed in CHO cells (named SA-IV) and the antibody shows a good biological function of recognizing CD86 and inhibiting the growth of Raji cells.

Association between polymorphisms in PDCD1 gene and aplastic anemia in Chinese Han population.

Single nucleotide polymorphism (SNP) of programmed cell death 1 (PD-1, encoded by PDCD1) has been reported to be associated with several autoimmune diseases including rheumatoid arthritis (RA), Graves' disease and multiple sclerosis (MS). In order to study the correlation between PD-1 gene polymorphism and aplastic anemia in a Chinese Han population, two SNPs, PD-1.1 G/A (rs36084323) and PD-1.6 G/A (rs10204525), were genotyped in 166 patients with aplastic anemia and 144 healthy controls by direct sequencing. All genotype distributions in both patients and controls were in Hardy-Weinberg equilibrium. Associations of genotypes and alleles with aplastic anemia were analyzed. The results suggested that the G allele of PD-1.1 was associated with an increased risk for aplastic anemia, while SNP of PD-1.6 was not associated with aplastic anemia in a Chinese Han population.

[Construction of 3D model of CD28 chimeric antibody with its antigen docked].

AIM: To construct a 3D model of the chimeric antibodies (AntiCD28: ch-2F5) with corresponding antigen molecule docked to theoretically verify the rationality of the binding of antibody with its antigen and to provide a method of 3D identification between antigen and antibody and spatial structure analysis. METHODS: We analyzed the sequence by submitting it to http://www.ncbi.nlm.nih.gov/ and made a comparison using integratly the 3 databases of GenBank, Protein data bank and GENO-3D. The 3D model was constructed by Swiss-model homology modeling server and molecular docking online was performed by GRAMM-X Protein Docking Web Server. Chimeric heavy chain, light chain, heavy-light chain complex, heavy-light chain and antigen complex were displayed and photographed by the Chimera Software. Meanwhile, the spatial structures of heavy, light chains, variable region, constant region, CDR and frame area were marked by different colours respectively to exhibit the 3D structure on every side. RESULTS: The 3D structure of the heavy-light chain and antigen complex we constructed was consistent well with the theory of antigen binding to antibody molecules. CONCLUSION: The structure of the chimeric antibody we constructed with the bioinformatic method was in accordance with the general structure of antibody, and its antigen binding site was also consistent with the molecular theory. Thus, the model helps to analyze the 3D structure of antibody and antigen-antibody interaction.

[Effect of CD80-scFv on recognition of CD80 molecule and proliferation of different tumor cells in vitro].

AIM: To prepare CD80-scFv in CHO cells and investigate its effect on the recognition and proliferation of different tumor cells. METHODS: The CD80-scFv was purified from culture supernatant without fetal calf serum (FCS) by IMAC affinity chromatography, and then bound to CD80 molecule on the Daudi, U251, A375 and 8266 cells detected by flow cytometry. Different concentrations of CD80-scFv were added in the training system of different tumor cells, and we analyzed the influence on the proliferation of these cells by MTT assay. With 8266 cells which expressed CD80 highly and were treated by mitomycin firstly as the APC, and human PBMC as the effect cells, we analyzed the influence of the CD80-scFv on the proliferation of PBMC by blocking the co-stimulatory signal. RESULTS: The concentration of CD80-scFv purified from FCS was about 6.67 mg/L and the binding rate of CD80-scFv with Daudi, U251, A375, 8266 and U266 were 96.8%, 39.6%, 20.5%, 99.9% and 3.8%, respectively. CD80-scFv suppressed the proliferation of Daudi and 8266 cells which expressed CD80 highly, and the inhibition rate of Daudi and 8266 cells cultured with CD80-scFv (the final concentration was 25 µg/mL) was 24.04% and 24.16%, respectively. Additionally, the antibody suppressed the proliferation of PBMC by blocking the co-stimulatory signal mediated by CD80-CD28. CONCLUSION: CD80-scFv can recognize CD80 molecule and suppress the proliferation of tumor cells which express CD80 highly.

[Expression and antigen binding activity of anti-human CD86 diabody].

AIM: To construct a recombinant eukaryotic expression vector of anti-human CD86 diabody gene and express anti-CD86 diabody through Chinese hamster ovary (CHO) cells, then analyze the capability of the diabody to recognize the tumor cells expressing CD86 and its biological effect. METHODS: The antibody heavy and light chain viable region gene (V(H); and V(L);) were cloned from hybridoma cell 1D1 which secreted anti-human CD86 monoclonal antibody. Anti-human CD86 diabody gene V(H);-(GGGGS)-V(L); was constructed by SOE-PCR. Then we inserted it into eukaryotic expression vector to construct a recombinant vector pIRES2-EGFP/CD86-diabody. The recombinant vector was transfected into CHO cells with Lipofectamine(TM); 2000, and the cell clones secreting CD86 diabody were screened by G418. We used IMAC to purify CD86 diabody and quantified its concentration by BCA method. The capability of the diabody to recognize the CD86 expressed on Raji and Daudi was analyzed through flow cytometry. After Raji cells were treated with CD86 diabody for 72 h, its proliferation inhibiting effect was investigated by MTT assay. RESULTS: We have obtained one CHO cell line that stably secreted CD86 diabody. The concentration of CD86 diabody after purification was 5.24 mg/L. The positive rates of CD86 diabody to recognize Raji and Daudi were 77.2% and 70.6%, respectively. After CD86 diabody treatment for 72 h, the inhibition rate of Raji cells was 37%. CONCLUSION: The anti-human CD86 diabody which we obtained successfully could recognize CD86 expressed on tumor cells specifically, and inhibit the proliferation of these tumor cells effectively.

[Changes and effects of significant membrane molecules on thymic T cell of aging mice induced by D-galactose].

AIM: To establish subacute aging mice model by D-galactose and to explore the changes and effects of significant membrane molecules on thymic T cell. METHODS: Female Kunming mice of 8 weeks old were injected with D-galactose of 12.5 mL/(kg.d) by subcutaneous in scruff for 42 days. The animals' living conditions and biological behaviors were observed everyday.SOD activities and MDA content of serum were measured to determine whether the aging model was successfully established.On the basis of successfully establishing aging model, detect the significant membrane molecules of thymic T cell by Immunofluorescence technique and Flow Cytometer. RESULTS: During the 42 days, gradually, the model mice showed bending body, loose skin, slow action and so on.The activities of SOD in the serum were significantly decreased(P<0.01), and the content of MDA in the serum was significantly increased(P<0.01). The thymic naive T cell significant molecule, CD45RA was decreased(P<0.05). T cell activation-related molecules, CD28 and CD25 were both decreased(P<0.05), and PD-1 was significantly increased(P<0.01). The memory T cell significant molecule, CD196 was increased, but was not significantly compared to the control mice. CONCLUSION: The D-galactose subacute aging mice model was successfully established.The naive and active T cell were decreased and the memory T cell was increased in the thymic of the aging.