| Rheumatoid arthritis(RA) continues to be a puzzling and often discouraging disease for the practicing physician but one which bears infinite interest to both clinican and investigator because little that is new or startling has emerged in the way of therapy for this disease. Recent clinical research in rheumatoid arthritis (RA) has led to significant advances in care using inhibitors of the circulating proinflammatory cytokines tumor necrosis factor (TNF)-αand interleukin-1. As all current therapies suppress proinflammatory processes instead of targeting the underlying cause of the disease, remission is induced in only a small subset of patients and cure is not achieved. In addition, adverse events such as serious infection, tumour also occurred. Therefore, searching for optimum treatment which could cure RA and induce sustained remission is necessary. The ideal treatment of RA is required to specifically target the cause of disease and prevent the progression of RA. Numerous studies demonstrated that RA is an autoimmune disease mediated by CD4+CD28+ T cells. In view of the relationship of immune system and symmetric inflammation of the synovial joints, drugs that regulate the body's immune response were utilized and the new strategies that target immune molecule were explored."gene and DNA vaccine"was one of the most important and useful strategy that devolped in 1990s and is extensively used in therapy of autoimmune disease.As B7:CD28/ CTLA-4 costimulatory signals play a central role in T activation, approaches targeting B7:CD28/ CTLA-4 costimulatory signals would be potentially useful in RA, in which the initial trigger for the autoimmune response remains unclear. Data have shown that the chimeric fusion protein CTLA-4Ig which blocked the interactions of CD28 with CD80 and CD86 act to inhibit immune responses. It is also the first immune-regulating drug to treat moderate and severe RA approved by FDA in USA. However, immune tolerance induced by the anti-B7 monoclonal/polyclonal antibodies, anti-CD28 monoclonal, anti-CD4 monoclonal and CTLA4-Ig theoretically does not last long due to reversion by IL-2 and other cytokines.Base on the previous studies, we constructed a novel exotoxin fusion gene vaccine B7-2-PE40 which consists of human B7-2 with the membrane-penetration and ADP-ribosylation domains of Pseudomonas aeruginosa exotoxin A in the present study. The B7-2 moiety replaces the native membrane binding domain of the bacterial toxin, thus specifically targeting the CD28-expressing cells. We investigated the selective cytotoxicity of the eukaryotic target toxin, evaluated the effects of B7-2-PE40 through gene transfer on CIA induction, the course and severity of established CIA, and examined the influence of B7-2-PE40 on cytokines and T subtypes in a rat model of CIA.B7-2-PE40 was amplified by PCR from pRSETA-B7-2-PE40KDEL and a eukaryotic expression vector pcDNA3.1/B7-2-PE40 was constructed by inserting the B7-2-PE40 cDNA into the vector pcDNA3.1/Zeo(+). After sequenced to confirm correct insertion of cDNA, the stable transfectants were generated by transducing pcDNA3.1/ B7-2-PE40 into RPE.40 cells using LipofectamineTM 2000 and ZeocinTM selection. 1919 bp of B7-2-PE40 mRNA was specifically amplified from the transduced cells using RT-PCR. Western blot showed that the fusion toxin were produced and secreted from stable transfected cells. The molecular mass of 90kDa was apparently larger than that expressed in E.coli cytosol (68 kDa ), indicating some posttranslational modifications in protein synthesis. By comparison with the standard curve of purified PEA activity, over 0.23μg/L PEA was produced at 24 hour per 1×106 cells ml-1. To determine the selective cytotoxicity, cell lines expressing different CD28 levels were co-cultivated with B7-2-PE40 stable-transfected RPE.40 cells. Cell killing was significant in human T-lymphoma Jurkat cells (92.87%) which expressed high level CD28 receptor and only a small proportion of the human Burkitt's lymphoma Raji cells (19.14%) expressing low levels of CD28 were killed in the co-cultures. The RPE.40 control had no significant effects on tumor cells expressing high or low levels of CD28. These results indicated that in vitro target toxin proteins were expressed and secreted by mammalian cells. Although modified after translation, B7-2-PE40 was specifically toxic to CD28-overexpressing cells.To confirm the expression of the B7-2-PE40 in vivo, 150μg plasmid DNA pcDNA3.1/ B7-2-PE40 were injected into the gastrocnemius muscle of CIA rats combined with electric pulses, then the mRNA extracted from untreated-side muscles was measured at appropriate time points after gene transfer by RT-PCR. Our results showed that a 1919-bp amplifier, full length of this recombinant molecule, could readily be detected by PCR analysis from all treated muscle samples on day 2 following i.m. injection of pcDNA3.1/ B7-2-PE40. On day 14, the expression reached the peak and gradually decreased thereafter. On 56 days after the genetic transfer, the expression level was very low, if any could be observed. CD28+ T cells depletion was monitored by FACS to evaluate the cytotoxicity of immunotoxin. Consistent with expression of B7-2-PE40, CD28+ T cells were severally depleted on day 2 and decreased to 34% on day 4 followed by a partial recovery ( 30%-40% ). A significant reduction in the percentage was detected again on day 21. Then CD28+ T cells gradually recovered to normal controls with the weakening expression of B7-2-PE40. An IgG response against the PEA portion of immunotoxin was detected by ELISA, which showed that few antibodies against exogenous PE40 were detected. Our in vivo studies suggested that B7-2-PE40 has potential therapeutic application.Then chicken collagenⅡwere applied to induce collagen-induced arthritis (CIA) in Wistar rats. The plasmid of B7-2-PE40 was electroporated on day 0 or 12 after collagen immunization, and the severity, histopathology were evaluated. Weak arthritis appeared 1 week after immunization in all B7-2-PE40 pretreated groups. With the expression of recombinant toxin, 200μg B7-2-PE40 plasmid resulted in the complete inhibition of the development of arthritis as compared with naked vector pretreated controls and CIA controls up to 10 weeks. Injection of 300 microgram of B7-2-PE40 only resulted in the partial inhibition and arthritis became evident from 3 weeks after immunization. Naked vector did not result in the any inhibition of arthritis, which is similar to untreated CIA control. The therapeutic effect of B7-2-PE40 on the development of arthritis was also examined. The results showed that it took effect 5 days after administration and the therapeutic effect was dose-dependent. When given 200μg or 300μg per rats, the development of arthritis was delayed significantly and the severity of arthritis was reduced up to 10 weeks. Few differences were observed between the 200μg and 300μg doses. When given 150μg per rats, the severity of arthritis was partially reduced as compared with CIA control. MTX undoubtedly had the best protection against CIA and the subsequent development of arthritis remained completely suppressed up to 10 weeks. When given naked vector, no therapeutic effect was detected. It should be noted that injection sites ulcerates occurred in 300μg B7-2-PE40 treated groups and not easily healed. Histopathologic analysis of joints on day 28 after immunization showed that joints obtained from CIA control rats exhibited typical arthritis lesions such as severely inflamed synovium, erosion and degeneration of cartilage cap, and pannus formation. 200μg pretreated joints exhibited inhibition of the development of arthritic lesions, thus showing only mild inflammation of synovium. However, severe arthritis lesions similar to those observed in control mice were detected in ankle joints obtained from 300 microgram of B7-2-PE40 injection. Histology of MTX pretreated groups showed neither synovial inflammation nor pannus formation. B7-2-PE40 treatment abrogated the pannus formation, cartilage destruction, bone erosion that are characteristic of CIA, only showing mild inflammation of synovial tissue. 200 microgram of B7-2-PE40 treated rats exhibited the minimal abnormalities, which is similar to MTX. We also investigated the levels of anti-CⅡin serum of rats and the production of anti-CⅡantibodies were significantly reduced in the 200μg pretreated group and MTX treated group (P<0.05). Although the levels of anti-CⅡantibodies in other groups were lower than CIA, they showed no statistically significant differences.We further evaluated the influence of B7-2-PE40 on immune system of CIA, and investigated its potential immunological mechanism using ELISA and flow cytometry. Our studies showed that B7-2-PE40 can inhibit the pathological activation by cytotoxicity to CD28+ T cells. At the same time, the percentages of Ts and Treg subsets were upregulated significantly and the ratios of Th1/Th2,CD4/CD8 were decreased. In addition, it could suppress the production of inflammatory cytokines TNFα和IFNγ(P<0.05), and enhance the synthesis of IL-10. There was no difference in IL-2 and TGF-β. IL-4 was not detected. We concluded that B7-2-PE40 could inhibit the inflammation response and relieve the symptom of CIA. The possible mechanism involves the induction of immunotolerance to collagen via T lymphocytes pathway.In present study, we first explored a novel exotoxin fusion gene vaccine B7-2-PE40 which could selectively block B7:CD28 costimulation pathway in vivo and effectively prevent the progression of collagen-induced arthritism. A wide spectrum of biological effects suggested that B7-2-PE40 not only induce T cell anergy via costimulation blockade, but also function as an immunomodulatory factor with capacity to develop the collagen-specific tolerance during the depletion and recovery of T cells, which need to be further studied.
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