Study Of Immuno-ablative Regime Followed By Immune Tolerance Inducing Therapy For PM Animal Model

Background and Objective:Autoimmune disease is a series of diseases and involves almost every system of human body. The patients usually go through repeatedly relapses and long deteriorating process.The possible mechanism of AID includes:1, Auto-reactive T lymphocyte avoid "colon losing", which is to avoid the process of "negative chose", auto-reactive T lymphocyte go into peripheral blood and response to auto-antigen, then cause AID.2, Mutation of lymphocyte causes abnormal recognition about antigen, then response to auto-antigen and cause AID.3, Lymphocyte activated by side path detour Th cells with tolerance and activate immuno-silent responding cells directly or indirectly, then cause AID.Till today, there are all kinds of drugs for AID treatment. That include single or combined use of corticosteroid, immunosuppressant drug which include cyclophosphamide(CTX), cyelosporine A(CsA),anti-malarias, mycophenolatemofeti (MMF), azathipporin(AZA), Tacrolimus(FK-506), lefluncmid(LEF), fludarabine, abetimus sodium, intravenous high dose Ig; Biochemical products include rituximab, epratuzumab(anti-CD22 monoclonal antibody), belimumab, CD40L monoclonal antibody, anti-CD11a antibody, anti-CD80/86 monoclonal antibody; Monoclonal antibody specifically designed for cytokines include anti-TNF-a monoclonal antibody(infliximab), anti-IL-10 monoclonal antibody,anti-IL-6R monoclonal antibody; Synthesized protein include human CDR1 synthesized peptide, atacicept, Type I INF neutralize antibody, IL-21R.Fc fusing protein; T lymphocyte vaccine, Complement suppressant; All the above drugs usually just achieve symptom control, diseases will eventually relapse sooner or later.Hematologic stem cells transplantation is an alternative option for more and more patients with refractory AID. The theory is to destroy patient's immune system by high dose chemotherapy or radiotherapy, the AID will achieve remission by deleting abnormal immune response. Then auto peripheral stem cells will be reintroduced to establish a new normal immune system, and immune modulate medicines will be administered to prevent auto-antibodies'production, or induce the tolerance for auto-antibodies. However, there are still quite a few problems due to infection, relapse, post transplantation complications and death, so there is still exploring work need to do.The reason of AID's repeatedly reoccurrence include: pathological changes at stem cell level which will cause the reoccurrence after auto-immune system's rebuilding; Mutated immunologic cells were not eliminated thoroughly or reintroduced via auto stem cell transplantation cause the reoccurrence; Existence of mutated somatic cells (exp. Virus infected somatic cell) are attacked by newly generated immunologic cells will cause the reoccurrence.Pathological changes at the stem cell level can only be cured by allogeneic stem cell transplantation. Intensified immune abrasive treatment without reintroducing auto stem cells will resolve the problem caused by incompletely immunologic cells elimination and auto stem cell transplantation. It leaves the problem caused by consistently existence of mutated somatic cells which can only be treated by inducing immune tolerance.Based on the above information and combined our present clinical experience, we shaped the theory of immune abrasive regime followed by immune tolerance inducing therapy. The key element of this therapy is to induce tolerance of mutated somatic cell. The graft-versus-host-disease(GVHD) caused by allogeneic stem cell transplantation is a classical model of AID.Early administration of CsA during the related HLA-matched donor HSCT process can prevent most of GVHD after related total matched HST. During the process of HLA-unmatched donor HSCT, completely elimination of mature T lymphocyte followed by CsA administration can also prevent most of GVHD. This particular fact suggest that drugs such as CsA probably has the ability to induce tolerance of newly generated immunocyte which differentiate from stem cell to somatic cell.Therefore, we presume that somatic cell's mutation exist permanently in AID, and these mutated somatic cells are the reason of disease, might be companied by mutation of immunocytes. If the immune abrasive regime followed by immune tolerance inducing therapy can has the newly generated immune system tolerate mutated somatic cell, AID which is not the result of stem cell level pathology will be cured. Using different AID models to study the immune abrasive regime followed immune tolerance inducing therapy will facilitate the research about the pathophysiology of all kinds of AIDS. We chose the immune polymeositis(PM) animal model to do the study in this research.Immune polymeositis(PM) is a syndrome caused by multi factors and has the characters of inflammatory damage in skeletal muscle and degeneration of muscle fibers. Its pathological changes include wild range muscle fibers' lesion and infiltration of monocyte and lymphocyte, the clinical manifestation includes amyosthenia and myoma. The research about mechanism of PM has been making progress. It was reported that its mechanism has certain relationships with damage of myocyte which was mediated by toxicity of T lymphocyte, micrangium damage which mediated by complement, and the existence of auto-antibodies in patients' system. All these above facts suggest that PM is a inflammatory muscle disease caused by auto-immune responding, and that PM mainly related with cellular immunity. Same as most of AIDs, PM has the long deteriorating, repeatedly relapses process and has been one of the major problem of clinical work.There are 3 theories about the etiology of PM: immunity, infection and hereditary. Most of present research consider PM as an auto-immune disease, and mainly related with cellular immunity. Though there research found the existence of perivasculitis in patients' muscle, suggest that humoral immunity also play an important role in PM's nosogenesis. Also there is research found out that Tumor Necrosis Factor, adhesion factor, chemotactic factor and toxin of lymphocyte ext. have related with PM's pathophysiology. All of these suggest that PM is a AID.Method1, Build immune polymyositis animal model.Made rabbit muscle tissue homogenate, diluted the homogenate to protein concentration 45 mg/ml, thoroughly mixed CFA and homogenate by the ratio 2:1. 36 healthy guinea pigs were chose to make the model. Mixture of CFA and homogenate was hypodermic injected behind the neck, once a week, totally 6 weeks. Blood samples were taken before and after the model creation, muscle tissue pathologic examination was conducted during the process. After 6 weeks, serologic index data was analyzed with SPSS 13.0 statistic software to define the success of model creation.2, Study the effect of immune abrasive regime followed by immune tolerance inducing therapy which applied to immune polymeositis animal models.28 animal models were chose and divided into 4 groups randomly, 7 animals form a group. Data of all the index of all the animals was analyzed and there is no significant difference among all groups. Every animal was given intraperitoneal injection at day zero, animals of intensified group accept busulfan 1mg/kg (guinea pig 5.5 mg/kg), once every 12 hours, total 8 shots; followed by CTX 40 mg/kg (guinea pigs 220mg/kg), once daily, total 4 shots, then followed by CsA 3 mg/kg (guinea pigs 16.5 mg/kg), once daily and continued to the end of experiment. High dose group accept bulsulfan 0.8mg/kg, CTX30mg/kg, CsA 3mg/kg, total shots and schedule are same as intensified group. Regular dose CTX group accept CTX 10mg/kg (guinea pig 55 mg/kg), once daily and 3 shots in all. And control group has no treatment.All the animals were weight and blood was taken to test for CK, AST and LDH, FBC before injection, the 7th day of injection and at the end of experiment. Dead animal's blood was taken and muscle tissue pathology examination was conducted right after death. Statistic analyze strategy:All the data collected from this experiment was analyzed with SPSS 13.0 statistic software, one way ANOVA, general linear model of repeated measures, Kaplan-Meier, Log-rank, Breslow and Tarone-Ware analysis were used, and graph of survival plots was drawed. P≤0.05 was used to define the significance of difference.Resulte:1, There is significant difference between the data of serologic index before and after the model creation. Injection of mixture of rabbit muscle tissue homogenate and CFA successfully created immune polymyositis animal models. 28 animal models were chose and divided randomly into 4 groups, there is no significant difference among 4 groups' general information. CK and AST of intensified dose group and high dose group have obvious improvement after the treatment, FBC showed significant decrease, especially count of peripheral lymphocyte and neutrophile granulocyte. There is no significant change in CK, AST and LDH of regular dose CTX group and control group, anyhow, there is significant decrease in FBC of regular dose CTX group. Data showed the estimate surviving proportion, mean survival time and median survival time of intensified dose group are obviously lower than the other groups, there is no significant difference between regular dose CTX group and control group, and the index of high dose group is significantly better than the other groups.Conclusion:1, Guinea pigs accepted weekly injection of mixture of rabbit muscle tissue homogenate and CFA evolved immune polymyositis after 6 weeks.2, Animal models were divided randomly into 4 groups and given different treatment regimens, intensified dosage of busulfan and CTX achieved abrasive effect on bone marrow, and the index of polymyositis improved significantly after the therapy, but intensified chemotherapy caused animal models severe damage beyond repairing and the survival proportion of this group decreased.3, Regular dose CTX caused animal's peripheral blood cell count changed significantly, but had no influence on CK, AST and LDH, the survival proportion of regular dose CTX group had no difference with control group, therefore, the effect of regular dose CTX in treating immune polymyositis animal models need further study.4, High dose chemotherapy can achieve immune system deletion also, the index of immune polymyositis also improved significantly after injection, furthermore, the survival period of this group is exceptionally longer than the other groups. This result suggests that immune abrasive regimen followed by immune tolerance inducing therapy is a preferable alternative treatment for AID.5, In conclusion: after high dose immune-abrasive and tolerance induce therapy and immune-abrasive and tolerance induce therapy, index of animal model of PM improved significantly, and animal received immune-abrasive and tolerance induce therapy has significantly longer survive period than all the other groups animals.