Study Of Immunoablative And Tolerance-inductive To Treat Type 1 Diabetes Mellitus (T1DM) Animal Model

BackgroundType 1 Diabetes Mellitus(T1DM) is a organ specific autoimmune disease conducted by T lymphocyte.Immune system is broken under the influence of virus infection,physical or chemical factors and patient lose tolerance of auto-antigen, begin to produce auto-antibodies or auto-allergic lymphocytes which cause pancreaticβcell's progressing damage.Then insufficient insulin secretion causes a series of symptoms.The exact pathogenesis of T1DM is not thoroughly understand,we think that it has a close relationship with priming auto-immune response and loss of auto-immune tolerance.We found the expression levels of CD4+CD25+T regulatory cells play an important role in the incidence and progression of T1DM.T regulatory cell with immune tolerance and immune suppression can inhibit the immune response,to maintenance the balance homeostasis of the body.Recent years,immune inhibited therapy is the mostly choose therapy for TIDM, as well as hematopoietic stem cell transplantation(HSCT) and/or combined pancreatic cell transplantation which includes autologous hematopoietic stem cell transplantation(auto-HSCT) and allogeneic hematopoietic stem cell transplantation (Allo-HSCT).Immuen inhibited therapy usually can just control the symptom and relapse after drug stopped;Although auto-HSCT has the advantage of better tolerance and patient has a period of stable and remission,but still some patients relapse after transplantation.Allo-HSCT can cure the disease,but it also has the disadvantages as expensive,limited resource,and post-transplantation complications such as GVHD,secondary AID which limit the clinical application of allo-HSCT.Reasons of relapse after auto-HSCT probably are: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 relapse.Pathological changes at the stem cell level have to be cured by allogeneic stem cell transplantation.Beside that,we hope that clearing out auto-allergic clone and inducing tolerance of auto-antigen will prevent the relapse. Talking about feasibility of inducing tolerance of auto-antigen,we have something to learn from allo-HSCT,GVHD is a perfect model for AID.We give patients CsA, MMF right after stem cell infusion,this method will induce newly built immune system tolerate auto somatic cell,no need for life long drug administration.Presently, the combination of ATG\ALG plus CsA can cure 70%severe aplastic anemia patients,while single drug therapy doesn't have the same prognosis.Most of the 70%patients have immunologic disorder,this result can prove synergism of immunoablative and tolerance-inductive.All these above information suggest that immune modulate drugs as CsA,MMF have the ability to induce newly immunologic cells to tolerant auto-somatic cells,same as "negative choose" happens in thymus gland.TIDM is a organ specific auto-immune disease,although its mechanism is not thoroughly understand,the one thing is sure that destroy the original auto-allergic immunologic cell,rebuild a normal immune system and induce a tolerance of auto-antigen is the key of treatment,it probably will cure TIDM.Based on this theory and facts,we propose to treat early stage TIDM with condition regime which is used on HSCT but without stem cell transfusion,then induce tolerance of auto somatic cell with immune modulate drugs,reenact the "negative choose" and cure the disease.The most important part of this treatment is the choice of immune inhibit drugs.We chose busulfan and cyclophosphomid,both drugs has less effect on stem cells and less sensitive to immune system.And we chose CsA for animal experiment based on the clinical experience we have on HSCT.The objection is to explore the feasibility of immnoablative and tolerance-inductive therapy,at the same time explore mechanism of TIDM and immune tolerance.ObjectiveWe treat the NOD mice by giving therapeutic regimen about Immunoablative and tolerance-inductive and observation its effect.We monitor the expression levels of CD4+CD25+T regulatory cells because the importance of Treg cells on T1DM,to explore the possible mechanism about tolerance-inductive.Methods:1,animal models:chose 18 NOD female mice randomly from 20,divided into 3 groups.Group A is control group,group B is lower dose CTX group,group C is conditioning dose CTX and BU group which will continue with CsA administration. During the experiment,if there is animal died or model failed,chose other NOD mouse to follow the same protocol.2,experiment procedure design:If blood sugar value of NOD mouse is higher than 11.3 mmol/L twice consecutively,it will be included in experiment as animal model of TIDM.There is no significant difference of peripheral WBC,lymphocyte, blood sugar 2 hours after meal,serum C peptide and weight among groups before treatment.Gourp A:early stage NOD mice were given saline injection,hypodermic insulin was given if the blood sugar value is higher than 30.0 mmol/L to prevent the animal die of high blood sugar.Group B:early stage NOD mice were given LD-CTX intraperitoneal injection (mice dose 90mg/kg.d,equals to human dose 10 mg/kg),once daily,total 3 days, during the injection and after the treatment we monitored the blood sugar value,if the blood sugar value is higher than 30.0 mmol/L,treated them as group A.Group C:early stage NOD mice were given intraperitoneal injection with HD-CTX and BU,day 1～3 CTX,day 4～6 BU.CTX 360 mg/kg.d(equals to human dose 40 mg/kg.d),once daily;Bu 7.2 mg/kg.d(equals human dose 0.8mg/kg/time), once every 12 hours,total 6 shots consecutively;followed with CsA since 7th day, dose 27 mg/kg.d(equals human dose 3 mg/kg.d),once daily.Monitor blood sugar value and when the value is higher than 30.0 mmol/L,same treatment as group A.Immunoabrasive:run the peripheral blood cell count after 7 days after the injection.Objection:lymphocyte count is zero.3,index:blood sugar value,serum C peptide concentration,peripheral CD4+CD25+ T lymphocyte's expression level were tested at the 1st and 14th day. WBC and lymphocyte count was tested at 1 st and 7th day.At the end of experiment of right after mice dead,took spleen and pancreas to observe the damage of pancreatic island,and test the spleen Treg cell's expression with flew cytometer.Statistic analysis:There are 18 NOD mice in our experiment,data of 18 mice was collected to run the statistic analysis.Data of our experiment was analyzed with SPSS 13.0 software,results showed as means+standard deviation(X±SD),analysis of covariance was used to analyze the data of Treg expression and 2 hours of C peptide after meal in peripheral blood, one-way ANOVA was used to analyze the data of peripheral blood cell counts and Treg expression.Data before and after injection was analyzed with paired-samples t test,P≤0.05 was defined as having statistic meaning of significant difference.Results:1,observe animal model1.1 before the eruption,all mice are very active,feed normally.1.2 mice of group A gradually deteriorated.Feeding,activity and skin condition got worse gradually.Death had happened from day 16 to 19.Mice of group B didn't have obvious change during the injection of LD-CTX.2 days after injection,feeding,activity and skin condition of them began to deteriorate.Death had happened from day 15 to 19.Mice of group C experienced deteriorating of feeding,weight loosing, fatigue,activity and skin condition during the injection of HD-CTX,they also less responsive to stimulation,and the situation had no change after being given CsA injection.During the process,3 mice were found having blood clot around anus. Death had happened from day 16 to 19.1.3 there was no blood sugar value higher than 30.0 mmol/L during the process, value range is 15 to 25 mmol/L.Mice of group B experienced consistent high blood sugar value after treatment.Mice of group C had volatile blood sugar value after treatment,but there was no significant difference compared with value before treatment.2 mice's blood sugar values were lower than normal before death.2,Effect of immunoabrasive treatment at 7th day.WBC of group C: (0.20±0.07)G/L;LYM(0.09±0.07) G/L.Immunoablasive result was achieved.3,After 2 weeks,the data of control group were collected.Mice have TIDM have lower C peptide,Treg expression and the ratio of Treg/CD4+ than the early stage of disease,but there is no statistic significant difference found.(P=0.144; P=0.221;P=0.430)4,Effect of LD-CTX on early stage NOD mice.4.1 2h-CP,expression of Treg and ratio of Treg/CD4+ of mice decreased significantly after being given LD-CTX injection(P=0.002;P=0.013;P=0.018).4.2 2 weeks after LD-CTX injection,2h-CP of group B are lower than control group,but there is no statistic significance(p=0.158).Treg expression between two groups has significant difference(P=0.015).And the ratio of Treg/CD4+ has no difference(P=0.098).4.3 there is no significant difference of Treg expression in spleen and ratio of Treg/CD4+ after LD-CTX injection(P=0.774;P=0.357),and the ratio of Treg/CD4+ of LD-CTX group(7.38±3.61) had slight decreasing compared with control group(9.06±4.87).5.effect of HD-CTX+Bu followed with CsA treatment on early stage NOD mice.5.1 after HD-CTX+Bu followed CsA treatment,2h-CP,Treg expression and ratio of Treg/CD4+ significantly increased compared with data before injection(P= 0.030;P=0.045;P=0.015)5.2 after 2 weeks treatment,2h-CP,Treg expression and ratio of Treg/CD4+ of group C are significantly higher than that of control group(P=0.001; P=0.002;P=0.019) and LD-CTX(1.55±0.76) group(P=0.000;P=0.000;P=0.011).5.3 Treg expression in spleen and ratio of Treg/CD4+ are also higher than control group significantly(P=0.000;P=0.005),and LD-CTX group(P=0.000; P=0.001).6 pathologic examination of NOD mice's pancreas((hematoxylin and eosin stain) The gland alveolus of group Aand B are nearly integrity,infiltrating of inflammatory cell and the necrosis of pancreas is fewer comparison with group B. We can find the erosion pancreas in group B.Conclusion:1,NOD mice is a very good TIDM animal model,conditioning dose of HD-CTX+Bu can achieve the immunoablative result.2,2h-CP,Treg expression are lower after LD-CTX injection,and the value decreased after treatment compared with control group.It suggests that LD-CTX has no positive effect on TIDM,on the contrary,it might speed the development of disease.The possible mechanism is that LD-CTX decrease Treg expression and activity to achieve its immunologic intensified effect.3,2h-CP,Treg expression are higher than before the HD-CTX +Bu followed by CsA treatment obviously,and higher than the control group obviously.It suggests that early stage NOD mice treated with immunoablasive and tolerance-inductive therapy have optimistic prognosis.TIDM progressing can be slowed.The probable mechanism is:first,conditioning dose of chemotherapy eliminated mutated cells clone;second,CsA modulate Treg's expression and activity by increasing CD4+CD25+ T cells,induce newly built immune system produce auto-tolerance to auto-antigen again.