Tim-3 In The Pathogenesis Of Immune Thrombocytopenia In A Preliminary Study

Background:Immune thrombocytopenia(ITP) is an acquired autoimmune hematologic disorder in which platelets are prematurely destroyed in the reticuloendothelial system by platelet autoantibodies.Although the pathogenesis of the disease is thought to be antibody-mediated,the production of these IgG anti-platelet autoantibodies is regulated by T-cells and thus T-cells indirectly might play a major pathogenetic role in the development of ITP.A variety of T-cell mediated abnormalities have been described in patients with ITP,including the activation of platelet-specific autoreactive T cells,decreased expression of Killer-cell immunoglobulin-like receptor(KIR) on cytotoxic lymphocytes(CTL) resulting in lysis of autologous platelets in ITP,and defective Tregs.TIM-3,a member of the novel TIM(T cell immunoglobulin and mucin domain) family of molecules,is the first molecule identified to be specifically expressed on CD4+ Th1 and not on Th2 cells.Up to now,it has also been found that TIM-3 is expressed on multiple cells of innate immune system,therefore it may play an important role in autoimmunity. Accumulating datas have shown that a high Th1/Th2 ratio was reported in patients with chronic ITP,suggesting that ITP is one of Th1-mediated autoimmune diseases. Whereas differentiated Th1 cells selectively express TIM-3,in the present study,we have detected TIM-3 expression in active and remission patients with ITP,relative to control subjects.Methods:TIM-3 expression of Peripheral Blood Mononuclear Cells(PBMCs) in ITP patients was detected by real-time PCR and flow cytometry.The correlation between TIM-3 expression levels and clinical parameters were further analyzed.We also evaluated the expression of TIM-3 in CD4+T cells,monocytes and dendritic cells (DCs) in peripheral blood of ITP patients.And the apoptosis of CD3/CD28 antibody-activated CD4+T cells was determined.Results:The data of real-time PCR and flow cytometry both showed that the TIM-3 expression of PBMCs of patients in active phase was significantly lower than that in healthy controls.The expression level of TIM-3 was significantly increased in patients in remission and had a positive correlation with the number of platelet.The TIM-3 expression was downregulated in CD4+T cells and monocytes in patients of active phase,but it was upregulated in DCs.In contrary,its level was upregulated in CD4+T cells and monocytes and was downregulated in DCs of patients in remission. The ratio of apoptosis in CD3/CD28 antibody-activated CD4+T cells was decreased in patients of active phase.Conclusion:The TIM-3 expression was downregulated in CD4+T cells and monocytes in patients of active phase,but it was upregulated in DCs.The apoptosis in activated CD4+T cells was decreased,suggesting that it has related to the downregulation of TIM-3.Taken together,TIM-3 may play an important role in the pathogenesis of ITP. Objective T cell immunoglobulin-and mucin-domain-containing molecules(TIMs) family have an important role in immune regulation.TIM-3 is a transmembrane protein preferentially expressed on terminally differentiated Th1 cells,which plays a role in Th1-mediated autoimmune disease.Idiopathic thrombocytopenic purpura(ITP) is an acquired organ-specific autoimmune disease with a polarization of Th1.The purpose of this study was to investigate whether the -1516G＞T,-574T＞G,4259G＞T single nucleotide polymorphisms within TIM-3 gene contribute to the genetic susceptibility to ITP.Method Genotyping of TIM-3 -1516G＞T,-574T＞G and 4259G＞T were performed in 187 patients with ITP and 123 healthy individuals by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) assay.Result No significant differences were found in genotype and allele distributions between the patients with ITP and the controls in all 3 sites.There was strong linkage disequilibrium(LD)(r~2=0.633) between -574T＞G and 4259G＞T,whereas -1516G＞T was not in LD with -574T＞G(r~2 = 0.007) or with 4259G＞T(r~2=0.002).Conclusion The -1516G＞T,-574T＞G and 4259G＞T of TIM-3 gene polymorphisms might not play an important role as a genetic risk factor in the pathophysiology of ITP. Background:Chemotherapy is the best treatment option for malignant leukemia patients.At present,high-dose combination chemotherapy and early regular intensive therapy have got a series of remarkable achievements in treating leukemia,but many patients died of chemotherapy failure.Multidrug resistance(MDR) is the major cause of failure of drug treatment modalities in leukemia.After developing resistance to a single drug or a class of drugs,cancer cells show cross-resistance to other functionally and structurally unrelated drugs.This phenomenon is known as cancer multidrug resistance.Accumulating data have shown that the overproduction of the 170-kDa, membranespanning P-glycoprotein(MDR1/P-gp,P-170,ABCB1) is the main underlying mechanism conferring this MDR phenotype.It is believed that inhibition of MDR1/P-gp function or of its expression may reverse the "classical" MDR phenotype and improve the effectiveness of chemotherapy.RNA interference(RNAi) is a conserved biologic response to double-stranded RNA that resuRs in the sequence-specific silencing of target gene expression.Double-stranded RNAs (dsRNA) are cleaved into 21-23nt small/short interference RNAs(siRNA).And then it guides the specific degradation of its cognate RNA.In recent years,RNA interference(RNAi) technique has enormous potential for the functional study of various genes and the development of novel gene therapeutic treatment strategies against cancer and viral infection.In this study,we used shRNA-encoding plasmid to transfect leukemia multidrug resistant cell line K562/A02 and detected the expression and the function of MDR1 gene and P-glycoprotein(P-gp) in vitro and vivo to select the most efficient vector.Methods:In order to design and screen short hairpin RNA(shRNA)molecules targeting multidrug resistance gene(MDR1),MDR1-shRNA expression vector was transfected into K562/A02 cells by lipofectamine2000,and G418 was added to screen and establish the stable expression cell strain.The expression of MDR1 was detected by real-time RT-PCR and western blot.The proliferation of cells was tested by CCK8 assay.The function of p-glycoprotein was determined by Rhodamine123 efflux experiment.And we selected the most efficient vector to determine whether this vector combined with transferrin-liposome could increase the inhibition of doxorubicin to K562/A02 cells xenografts in vivo.Results:The results showed that all of four mdr1-shRNA expression vector can significantly knockdown the expression of p-glycoprotein as compared with control vector,moreover,the vector targeting 508-526 sites of MDR1 gene is the best one.In vivo experiments demonstrated that this vector was able to downregulate the expression of P-glycoprotein(P-gp) on tumor cells and reverse the resistant phenotype.Conclusion:Our study show that the mdr1-shRNA expression vector by screening can significantly knockdown the expression of MDR1 gene and reverse leukemia drug resistance,which provide a new target site for MDR1 and pave the way for the future cancer gene therapy.