| Our immune system provides effective protection from infectious agents and foreign substances, however, this immune system is not perfect. Failure in the negative deletion of self-reactive T cells in the thymus, combined with the deficiency of regulatory T cell (Regulatory T cell, Treg) cells which can control the activity of self-reactive T cells, can result in autoimmune disease attacking the individuals own tissues. Recently, antigen-specific immunotherapy for the specific blockade of self-reactive T cells or induction of Treg cells has been viewed as a promising means of curing autoimmune diseases.Type 1 diabetes is characterized by the selective destruction of pancreatic isletβcells by self-reactive T lymphocytes recognizing a list of autoantigens. Many self-antigens have guided in designing therapeutic strategies, including insulin, glutamic acid decarboxylase antigen and islet cell antigen. There exists a sequential hierarchy in self-reactive T-cellular responses directed against these islet autoantigens, and the T-cellular response against insulin is viewed to be upstream of the responses to other self-antigen. Insulin-specific immunotherapy has shown some protective results in rodent models of Type 1 diabetes, however, insulin antigen-driven immunotherapy that could counter diabetes in the clinic has yet to be achieved successfully. These suggest that principles guiding the antigen-based immunotherapy are very complicated and theβcells-specific T cell reactivity in patients is largely ill defined. Perhaps, there exist some other unknown antigens in diabetic patients, and administration of these unknown autoantigens may be more effective for human application.Pancreatic and duodenal home box factor-1 (Pdx1) is a key transcription factor that plays an important role in pancreas development,βcells differentiation and functional maintenance ofβcells. In matureβcells, Pdxl is also involved in insulin gene expression. In our previously published paper, we reported that treatment of streptozotocin-induced diabetes with recombinant pancreatic duodenal homeobox 1 protein reverses diabetes by stimulatingβ-cell regeneration and liver cell reprogramming into insulin-producing cells. More existed, we found that Pdx1 is a novelβ-cell-specific autoantigen existing in the nonobese diabetic (NOD) mice. Autoantibodies against Pdx1 were observed in serum samples from both prediabetic and diabetic NOD mice and a subset of Type 1 diabetic patients. The NOD mouse is an ideal experimental model of autoimmune type 1 diabetes. In female mice, diabetes onset typically occurs at 12 to 14 weeks of ages, characterized by T cell-mediated inflammation of the pancreatic islets and the destruction of theβcells. In this study, we investigated whether Pdx1 protein (or M-Pdx1, with no biological function, but intact immunologically active component) could delay the onset of diabetes in NOD mice and the underlying mechanisms. Adoptive transfer assay of splenocytes, in vivo T cells proliferative assay, histology of pancreas, cell flow, real-time PCR assays and in vitro T cells proliferative assay were performed to investigate the underlying mechanism.OBJECTIVE- In this study, we investigated whether immunization with Pdx1 (or M-Pdx1) protein could delay the onset of diabetes in female NOD mice and the underlying mechanism.RESEARCH DESIGN AND METHODS- In this experiment, NOD mice at the age of seven or ten weeks were immunized with Pdx1 protein (or M-Pdx1 protein, control proteins). Because the therapeutic effects could be influenced by many factors, in our experiments we tried different route and form, as well as different dosage and timing of administration. For different route and form of administrations, NOD mice were treated with protein intraperitoneally, subcutaneously or orally; For different dosage and timing of administrations, NOD mice were chosen for experiment at the age of seven week or ten week, and treated with Pdx1 protein (or M-Pdx1 protein, control proteins) for different weeks. Blood glucose was measured weekly and mice with a blood glucose level>11.1 mmol/l for 2 consecutive days were considered diabetic.In order to investigate the underlying mechanism, we tried to do the following assays:(1) Spleen adoptive transfer experiment:Splenocytes from Pdx1 protein (or M-Pdx1 protein, control proteins) treated mice were isolated and subsequently transferred into nonobese diabetic-severe combined immunodeficient mice (NOD-SCID) mice, and the diabetic incidence of NOD-SCID mice was observed. (2) In vivo T cell proliferative assay:CD4+ T cells isolated from the spleen of NOD.BDC2.5 mice were marked with carboxyfluorescein diacetate succinimidyl ester (CFSE) and subsequently injected intravenously into the Pdx1-treated (or M-Pdx1 protein, control proteins) NOD mice. Five days later, pancreatic lymph node cells and inguinal lymph node cells were harvested from NOD mice and analyzed by cell flow cytometry aiming to evaluate CFSE labeled CD4+ T cell proliferation.(3) Histology of pancreas:Female NOD mice immunized by either M-Pdx1 protein or control proteins underwent pancreatic histological studies. Tissue section of the islet stained with hematoxylin and eosin or with anti-insulin antibodies. The slides were coded and an insulitis score was determined.(4) Cell flow:Female NOD mice were immunized by either M-Pdx1 protein or control proteins. Cell flow assays were performed to evaluate the ratio of CD4+/CD8+ T cells in spleen or pancreatic lymph nodes, including the changes of CD4+ FoxP3+ regular cells, IL-10 secreting cells and Th-17 cells. The expression of cytokines such as IL-4,IL-10,IFN-γwere determined.(5) Real-time PCR assays:The expression of cytokines such as IL-2,IL-4,IL-10,Th-17,IFN-γ,GF-α,TGF-βand FoxP3 were determined by Real time PCR to evaluate the effects of M-Pdxl protein (or control proteins) on NOD mice.(6) In vitro T cell proliferative assay:To observe the proliferation of spleen lymphocytes from NOD mice immunized by either M-Pdx1 protein or control proteins in presence of different antigens.RESULTS-Compared with control group, Pdxl protein (or M-Pdx1 protein) specific immunotherapy (intraperitoneal or subcutaneous administration) delayed the onset of diabetes in NOD mice, however, oral administrations of Pdx1 protein (or M-Pdx1 protein) did not prevent diabetes obviously.Spleen adoptive transfer experiment:NOD-SCID mice injected with splenocytes from PBS-treated NOD mice in control group showed no significant therapeutic effect, and most of mice become diabetic within 3 weeks of cell transfer. However, non-diabetic incidence analysis revealed a significant delay in the onset of diabetes among the NOD-SCID mice that received infusions of splenocytes from Pdxl or M-Pdxl immunized donors. This suggested the activation of immunoregulatory cells in NOD mice treated with Pdx1 protein (or M-Pdx1 protein), and protection from diabetes could be adoptively transferred to NOD-SCID recipients.In vivo T cell proliferative assay:In the inguinal lymph node of M-Pdx1 treated or control protein treated NOD mice, CFSE labeled NOD.BDC2.5 CD4+T cells barely proliferated (p>0.05). CFSE labeled NOD.BDC2.5 CD4+T cells proliferated obviously in the pancreatic lymph node of control mice, however, cell flow cytometry revealed a depressed T cells proliferation in the pancreatic lymph node of M-Pdx1 treated mice (p<0.05). These results suggested that M-Pdx1 maybe could increase the number of Treg cells in NOD mice, which displayed a depressing effect of autoreactive NOD.BDC2.5 CD4+T cells proliferation.Histology of pancreas:In control group, histological examination of pancreas revealed islets heavily infiltrated by leukocytes. The few islets with remaining insulin containingβ-cells were infiltrated by abundant leukocytes, and the number of glucagon-positive cells was increased compensatorily. In contrast, in M-Pdx1 protein treated mice, the extent of lymphocyte infiltration of the islets was reduced, and immunohistochemistry of pancreas revealed abundant insulin containingβ-cells. Insulitis severity scores in NOD mice treated with M-Pdx1 were much better than that in control group.Cell flow:The number of Foxp3+ regulatory T cells among the total CD4+ T cells in the spleen cells of M-Pdxl-treated NOD mice increased, as compared with controls. Similarly, the number of IL-10 secreting cells were also increased. Moreover, the result showed that CD4+ cells expressing Th17 were decreased in NOD mice treated with M-Pdx1. Clow cytometric analyses revealed no significant differences in the the ratio of CD4+/CD8+ in spleen cells. Besides, cells expressing IFN-γremained unchanged.Real-time PCR assays showed an increased expression of Th2 gene, such as IL-4, IL-10, Foxp3, TGF-αand TGF-β,in the spleen and pancreatic lymph node from NOD mice treated with M-Pdxl protein. Down regulation of Thl gene, including IL-2, IFN-γ, was noted in the spleen and pancreatic lymph node of the M-Pdx1-treated mice, Moreover, the expression of FoxP3 gene was up regulated in NOD mice immunized with M-Pdx1 protein.T cells isolated from NOD mice in control group exhibited a more efficient proliferation towards Pdx1 protein than cells from NOD mice treated with Pdx1 (p<0.05) These data indicated that the function of Pdxl-specific T cells were decreased by immunizing NOD mice with Pdxl protein. Moreover, T cells proliferation against insulin were also decreased by immunizing NOD mice with Pdx1 protein. In Pdx1 immunized NOD mice, IFN-γwas decreased and Th2 cytokine IL-10 were increased.CONCLUSIONS-Our results demonstrated that immunization with Pdx1 antigen (or M-Pdxl protein) could delay the onset of diabetes in NOD mice. The underlying mechanism includes induction of self-reactive T cell clearance or nonfunction, up-regulation of CD4+Foxp3+ regulatory T cells and IL-10 secreting cells, and down-regulation of Th-17 cells. Besides, immunization with Pdxl antigen can also change the ratio of Th1 cells to Th2 cells. This novel Pdx1-based protein therapy maybe can offer a promising way for the treatment of type 1 diabetes. OBJECTIVE- The coculture microfluidic chip was fabricated based on microfluidic technology to facilitate the coculture of bone marrow mesenchymal stem cells (BM-MSCs) together with INS-1 cells, and we tried to observe whether some cytokines secreted by BM-MSCs can ameliorate IL-1β/IFN-γ-induced apoptosis or dysfunction of INS-1 cells.RESEARCH DESIGN AND METHODS-A coculture microfluidic chip were designed and fabricated. BM-MSCs were obtained from diabetes mellitus patients and the representative cell surface antigen expression profiles were analyzed by flow cytometric analysis. BM-MSCs and INS-1 cells were seeded into two champers on the chip separately, permitting the medium to flow from the BM-MSCs culture area to the INS-1 cells culture area unidirectionally. INS-1 cells were cultured in medium supplemented with IL-1βand IFN-y on the chip, and INS-1 cells were cocultured with BM-MSCs on a microfluidic chip with persistent perfusion of medium. Annexin V/PI double staining analysis were performed to evaluate the depressive effects of bone marrow mesenchymal stem cells on IL-1β/IFN-γinduced INS-1 cell apoptosis. RIA analysis was performed to investigate whether bone marrow mesenchymal stem cells could ameliorate IL-1β/IFN-γ-induced dysfunction of INS-1 cells. Immunofluorescence and real time PCR assay were performed to study whether bone marrow mesenchymal stem cells could enhance the insulin content and gene expression.RESULTS- This microfluidic chip assures continuous and single-line perfusion from BM-MSCs to INS-1 cells without returning, which is a better platform for us to investigate the protective effect of BM-MSCs on INS-1 cells in vitro. In this experiment, exposure to the cytokine combination IL-1β/IFN-γresulted in a marked increase in the number of apoptotic INS-1 cells at different observing times. Some cytokines secreted by BM-MSCs partially rescued INS-1 cells from cytokine-induced apoptosis and dysfunction. Simultaneously, insulin content and the mRNA expression of insulin and were also markedly ameliorated. CONCLUSIONS- This microfluidic device is a better platform for us to investigate the relationship between stem cells and other cells in vitro. BM-MSCs may enhance the survival of INS-1 cells,insulin content and the expression of insulin gene through excreted cytokines such as anti-inflammatory, anti-apoptotic and nutrient factors.
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