在实验动物水平通过恢复和改善胰岛β细胞功能的糖尿病治疗新进展

糖尿病是一个世界性问题,在中国就有超过1.3亿人患有糖尿病.糖尿病主要分为1型糖尿病和2型糖尿病.遗憾的是,糖尿病至今尚未达成有效的预防和治愈手段.但是,近年来的一系列突破性的研究成果,让我们看到了治愈糖尿病的希望.在这里我们回顾了目前关于1型糖尿病和2型糖尿病可能的治愈途径的研究进展.这些途径包括胰岛移植和1型糖尿病...     

TNFR2 signaling modulates immunity after allogeneic hematopoietic cell transplantation

Tumor necrosis factor-α (TNF-α) signaling through TNF receptor 2 (TNFR2) plays a complex immune regulatory role in allogeneic hematopoietic cell transplantation (HCT). TNF-α is rapidly released in the circulation after the conditioning regimen with chemotherapy and/or radiotherapy. It activates the function of donor alloreactive T cells and donor Natural Killer cells and promotes graft versus tumor effects. However, donor alloreactive T cells also attack host tissues and cause graft versus host disease (GVHD), a life-threatening complication of HCT. Indeed, anti-TNF-α therapy has been used to treat steroid-refractory GVHD. Recent studies have highlighted another role for TNFR2 signaling, as it enhances the function of immune cells with suppressive properties, in particular CD4⁺Foxp3⁺ regulatory T cells (Tregs). Various clinical trials are employing Treg-based treatments to prevent or treat GVHD. The present review will discuss the effects of TNFR2 signaling in the setting of allogeneic HCT, the implications for the use of anti-TNF-α therapy to treat GVHD and the clinical perspectives of strategies that specifically target this pathway.     

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing β cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2rγ^null mice. The selective destruction of pancreatic islet β cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total β-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the β cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet β cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4⁺ T cell infiltration and clonal expansion, and the mouse islet β-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet β cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.     

Biomaterials as carrier,barrier and reactor for cell-based regenerative medicine

Cell therapy has achieved tremendous success in regenerative medicine in the past several decades. However, challenges such as cell loss, death and immune-rejection after transplantation still persist. Biomaterials have been designed as carriers to deliver cells to desirable region for local tissue regeneration; as barriers to protect transplanted cells from host immune attack; or as reactors to stimulate host cell recruitment, homing and differentiation. With the assistance of biomaterials, improvement in treatment efficiency has been demonstrated in numerous animal models of degenerative diseases compared with routine free cell-based therapy. Emerging clinical applications of biomaterial assisted cell therapies further highlight their great promise in regenerative therapy and even cure for complex diseases, which have been failed to realize by conventional therapeutic approaches.     

Importance of the stem cell microenvironment for ophthalmological cell-based therapy

Cell therapy is a promising treatment for diseases that are caused by cell degeneration or death. The cells for clinical transplantation are usually obtained by culturing healthy allogeneic or exogenous tissue invitro. However, for diseases of the eye, obtaining the adequate number of cells for clinical transplantation is difficult due to the small size of tissue donors and the frequent needs of long-term amplification of cells in vitro, which results in low cell viability after transplantation. In addition, the transplanted cells often develop fibrosis or degrade and have very low survival. Embryonic stem cells(ESCs) and induced pluripotent stem cells(i PS) are also promising candidates for cell therapy. Unfortunately, the differentiation of ESCs can bring immune rejection, tumorigenicity and undesired differentiated cells, limiting its clinical application. Although i PS cells can avoid the risk of immune rejection caused by ES cell differentiation post-transplantation, the low conversion rate, the risk of tumor formation and the potentially unpredictable biological changes that could occur through genetic manipulation hinder its clinical application. Thus, the desired clinical effect of cell therapy is impaired by these factors. Recent research findings recognize that the reason for low survival of the implanted cells not only depends on the seeded cells, but also on the cell microenvironment, which determines the cell survival, proliferation and even reverse differentiation. When used for cell therapy, the transplanted cells need a specific three-dimensional structure to anchor and specific extra cellular matrix components in addition to relevant cytokine signaling to transfer the required information to support their growth. These structures present in the matrix in which the stem cells reside are known as the stem cell microenvironment. The microenvironment interaction with the stem cells provides the necessary homeostasis for cell maintenance and growth. A large number of studies suggest that to explore how to reconstruct the stem cell microenvironment and strengthen its combination with the transplanted cells are key steps to successful cell therapy. In this review, we will describe the interactions of the stem cell microenvironment with the stem cells, discuss the importance of the stem cell microenvironment for cell-based therapy in ocular diseases, and introduce the progress of stem cell-basedtherapy for ocular diseases.     

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)‐silenced islet cell clusters (ICC). Single‐cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2‐microglobulin or class II transactivator, respectively. SLA‐silenced ICCs‐derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs‐derived cells. Xenogeneic T cell immune responses, NK cell and antibody‐mediated cellular‐dependent immune responses were significantly decreased in SLA‐silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single‐cell engineering and post‐transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.     

细胞微囊化免疫隔离技术在移植医学中的应用

作为一种十分有效的免疫隔离技术,细胞微囊化可排除细胞移植中出现的宿主与移植物之间的双向排斥作用,从而使能分泌生物活性物质的细胞在移植后得以存活。目前报道的多种微囊材料中,以海藻酸钠一聚赖氨酸一海藻酸钠的应用最为广泛,可通过提高其生物相容性来减弱免疫排斥反应。细胞微囊化在医学治疗上正在发挥越来越大的作用,特别是基因修饰细胞日益成为研究的焦点。尽管该技术尚需改进,但它在异体和异种组织或细胞移植等方面有着广阔的应用前景。     

Current status and perspectives of regulatory T cell-based therapy

The CD4⁺FOXP3⁺ regulatory T (Treg) cells are essential for maintaining immune homeostasis in healthy individuals. Results from clinical trials of Treg cell-based therapies in patients with graft versus host disease (GVHD), type 1 diabetes (T1D), liver transplantation, and kidney transplantation have demonstrated that adoptive transfer of Treg cells is emerging as a promising strategy to promote immune tolerance. Here we provide an overview of recent progresses and current challenges of Treg cell-based therapies. We summarize the completed and ongoing clinical trials with human Treg cells. Notably, a few of the chimeric antigen receptor (CAR)-Treg cell therapies are currently undergoing clinical trials. Meanwhile, we describe the new strategies for engineering Treg cells used in preclinical studies. Finally, we envision that the use of novel synthetic receptors, metabolic regulators, combined therapies, and in vivo generated antigen-specific or engineered Treg cells through the delivery of modified mRNA and CRISPR-based gene editing will further promote the advances of next-generation Treg cell therapies.     

Roles of cytokines in the pathogenesis and therapy of type 1 diabetes

Type 1 diabetes (T1D) results from autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans by autoreactive T helper 1 (Th1) cells characterized by their cytokine secretory products, interleukin-2 (IL-2) and interferon γ (IFNγ). Th1-type cytokines (IL-2 and IFNγ) correlate with T1D, whereas Th2 (IL-4 and IL-10), Th3 (transforming growth factor beta [TGFβ]), and T regulatory cell-type cytokines (IL-10 and TGFβ) correlate with protection from T1D. Paradoxically, however, administrations of Th1-type cytokines (IL-2 and IFNγ) and immunotherapies that induce Th1-type cytokine responses actually prevent T1D, at least in animal models. Therefore, immunotherapies that inhibit IL-2 production/action will block Th1 cell/cytokine-driven effector mechanisms of pancreatic islet β-cell destruction; however, anti-IL-2 therapy will not allow immune tolerance to be established. In contrast, immunotherapies that increase IL-2 production/action may correct an immunodeficiency in IL-2 production that appears to underlie the autoimmunity of T1D, thereby restoring immune tolerance to islet β-cells and prevention of T1D.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

Cellular therapies for type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is a disease that results from the selective autoimmune destruction of insulin-producing beta-cells. This disease process lends itself to cellular therapy because of the single cell nature of insulin production. Murine models have provided opportunities for the study of cellular therapies for the treatment of diabetes, including the investigation of islet transplantation, and also the possibility of stem cell therapies and islet regeneration. Studies in islet transplantation have included both allo- and xeno-transplantation and have allowed for the study of new approaches for the reversal of autoimmunity and achieving immune tolerance. Stem cells from hematopoietic sources such as bone marrow and fetal cord blood, as well as from the pancreas, intestine, liver, and spleen promise either new sources of islets or may function as stimulators of islet regeneration. This review will summarize the various cellular interventions investigated as potential treatments of T1DM.     

Immunotherapy of cancer for the elderly patient: does allogeneic bone marrow transplantation after nonmyeloablative conditioning provide a new option?

The critical role of antigen-specific T cells in cancer immunotherapy has been amply demonstrated. Though success of clinical trials still remains far behind expectations, the continuous improvement in our understanding of the biology of the immune response will provide the basis for optimized cancer vaccines. This review focuses on active therapeutic vaccination after allogeneic bone marrow cell transplantation with nonmyeloablative conditioning. This approach could provide a major breakthrough in cancer immunotherapy, particularly of elderly patients. The senescent immune system, mainly the T-cell compartment, displays reduced responsiveness, and this has to be overcome if therapeutic vaccination is to be of benefit for the patient. Although the defects are quite well characterized, the inducing factors and ways to overcome them are still to be explored in more detail. Many questions also remain to be answered in the field of allogeneic bone marrow transplantation after nonmyeloablative conditioning to optimize this therapeutic setting in cancer immunotherapy. Current considerations to improve engraftment and to reduce graft-versus-host disease while strengthening graft-versus-tumor reactivity will be briefly reviewed. Finally, I will discuss whether tumor-reactive T cells can be naturally maintained during the process of T-cell maturation in the allogeneic host. Provided this hypothesis can be substantiated, a T-cell vaccine will meet a pool of virgin T cells in the allogeneically reconstituted host, which are tolerant toward the host but not anergized toward tumor antigens presented by MHC molecules of the host. Inevitably, the problem of the aged immune system would be circumvented.Abbreviations APC antigen-presenting cell - BMCT bone marrow cell transplantation - CTL cytotoxic T cell - DC dendritic cell - GvHD graft-versus-host disease - GvT graft versus tumor - HvG host-versus-graft - LAK lymphokine-activated killer cell - mAB monoclonal antibody - MHC major histocompatibility complex - TCR T-cell receptor - TH helper T cell - TIL tumor-infiltrating leukocyteM. Zöller was supported by the Tumorzentrum Heidelberg/Mannheim, the Mildred-Scheel-Stiftung für Krebsforschung, the José Carreras Leukemia Foundation, and a German-Israel Joint Program.     

A novel technique for the in vivo imaging of autoimmune diabetes development in the pancreas by two-photon microscopy

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of beta cells in the pancreas. Little is known about the in vivo dynamic interactions between T cells and beta cells or the kinetic behavior of other immune cell subsets in the pancreatic islets. Utilizing multiphoton microscopy we have designed a technique that allows for the real-time visualization of diabetogenic T cells and dendritic cells in pancreatic islets in a live animal, including their interplay with beta cells and the vasculature. Using a custom designed stage, the pancreas was surgically exposed under live conditions so that imaging of islets under intact blood pressure and oxygen supply became possible. We demonstrate here that this approach allows for the tracking of diabetogenic leukocytes as well as vascularization phenotype of islets and accumulation of dendritic cells in islets during diabetes pathogenesis. This technique should be useful in mapping crucial kinetic events in T1D pathogenesis and in testing the impact of immune based interventions on T cell migration, extravasation and islet destruction.     

Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.     

Extrapancreatic Autoantibody Profiles in Type I Diabetes

Type I diabetes (T1D) is an autoimmune disease characterized by destruction of insulin-producing β-cells in the pancreas. Although several islet cell autoantigens are known, the breadth and spectrum of autoantibody targets has not been fully explored. Here the luciferase immunoprecipitation systems (LIPS) antibody profiling technology was used to study islet and other organ-specific autoantibody responses in parallel. Examination of an initial cohort of 93 controls and 50 T1D subjects revealed that 16% of the diabetic subjects showed anti-gastric ATPase autoantibodies which did not correlate with autoantibodies against GAD65, IA2, or IA2-β. A more detailed study of a second cohort with 18 potential autoantibody targets revealed marked heterogeneity in autoantibody responses against islet cell autoantigens including two polymorphic variants of ZnT8. A subset of T1D subjects exhibited autoantibodies against several organ-specific targets including gastric ATPase (11%), thyroid peroxidase (14%), and anti-IgA autoantibodies against tissue transglutaminase (12%). Although a few T1D subjects showed autoantibodies against a lung-associated protein KCNRG (6%) and S100-β (8%), no statistically significant autoantibodies were detected against several cytokines. Analysis of the overall autoantibody profiles using a heatmap revealed two major subgroups of approximately similar numbers, consisting of T1D subjects with and without organ-specific autoantibodies. Within the organ-specific subgroup, there was minimal overlap among anti-gastric ATPase, anti-thyroid peroxidase, and anti-transglutaminase seropositivity, and these autoantibodies did not correlate with islet cell autoantibodies. Examination of a third cohort, comprising prospectively collected longitudinal samples from high-risk individuals, revealed that anti-gastric ATPase autoantibodies were present in several individuals prior to detection of islet autoantibodies and before clinical onset of T1D. Taken together, these results suggest that autoantibody portraits derived from islet and organ-specific targets will likely be useful for enhancing the clinical management of T1D.     

Construction of glomerular epithelial cells expressing both immune tolerance and GFP genes and application to cell therapy by cell transplantation

Cell therapy applied to wound healing or tissue regeneration presents a revolutionary realm to which principles of gene engineering and delivery may be applied. One promising application is the transplantation of cells into the wounded tissue to help the tissue repair. However, when cells are transplanted fromin vitro toin vivo, immune rejection occurs due to the immune response triggered by the activation of T-cell, and the transplanted cells are destroyed by the attack of activated T-cell and lose their function. Immune suppressant such as FK506 is commonly used to suppress immune rejection during transplantation. However, such kind of immune suppressants not only suppresses immune rejection in the periphery of transplanted cells but also suppresses whole immune response system against pathogenic infection. In order to solve this problem, we developed a method to protect the desired cells from immune rejection without impairing whole immune system during cell transplantation. Previously, we reported the success of constructing glomerular epithelial cells for removal of immune complex, in which complement receptor of type 1 (CR1) was over-expressed on the membrane of renal glomerular epithelial cells and could bind immune complex of DNA/anti-DNA-antibody to remove immune complex through phagocytosis [1]. Attempting to apply the CR1-expressing cells to cell therapy and evade immune rejection during cell transplantation, we constructed three plasmids containing genes encoding a soluble fusion protein of cytolytic T lymphocyte associated antigen-4 (CTLA4Ig) and an enhanced green fluorescent protein (EGFP). The plasmids were transfected to the above-mentioned glomerular epithelial cells to express both genes simultaneously. Using the clone cells for cell transplantation showed that mice with autoimmune disease prolonged their life significantly as compared with the control mice, and two injections of the cells at the beginning of two weeks resulted in remarkable survivability, whereas it requires half a year and 50 administrations of proteins purified from the same amount of cells to achieve the same effect.     

Adiponectin gene therapy prevents islet loss after transplantation

Significant pancreatic islet dysfunction and loss shortly after transplantation to the liver limit the widespread implementation of this procedure in the clinic. Nonimmune factors such as reactive oxygen species and inflammation have been considered as the primary driving force for graft failure. The adipokine adiponectin plays potent roles against inflammation and oxidative stress. Previous studies have demonstrated that systemic administration of adiponectin significantly prevented islet loss and enhanced islet function at post‐transplantation period. In vitro studies indicate that adiponectin protects islets from hypoxia/reoxygenation injury, oxidative stress as well as TNF‐α‐induced injury. By applying adenovirus mediated transfection, we now engineered islet cells to express exogenous adiponectin gene prior to islet transplantation. Adenovirus‐mediated adiponectin transfer to a syngeneic suboptimal islet graft transplanted under kidney capsule markedly prevented inflammation, preserved islet graft mass and improved islet transplant outcomes. These results suggest that adenovirus‐mediated adiponectin gene therapy would be a beneficial clinical engineering approach for islet preservation in islet transplantation.     

Permanent survival of fully MHC-mismatched islet allografts by targeting a single chemokine receptor pathway

Chemokine receptor blockade can diminish the recruitment of host effector cells and prolong allograft survival, but little is known of the role of chemokine receptors in promoting host sensitization. We engrafted fully allogeneic islets into streptozotocin-treated normal mice or mice with the autosomal recessive paucity of lymph node T cell (plt) mutation; the latter lack secondary lymphoid expression of the CCR7 ligands, secondary lymphoid organ chemokine (CCL21) and EBV-induced molecule-1 ligand chemokine (CCL19). plt mice showed permanent survival of islets engrafted under the kidney capsule, whereas controls rejected islet allografts in 12 days (p < 0.001), and consistent with this, plt mice had normal allogeneic T cell responses, but deficient migration of donor dendritic cell to draining lymph nodes. Peritransplant i.v. injection of donor splenocytes caused plt recipients to reject their allografts by 12 days, and sensitization at 60 days posttransplant of plt mice with well-functioning allografts restored acute rejection. Finally, islet allografts transplanted intrahepatically in plt mice were rejected approximately 12 days posttransplant, like controls, as were primarily revascularized cardiac allografts. These data show that the chemokine-directed homing of donor dendritic cell to secondary lymphoid tissues is essential for host sensitization and allograft rejection. Interruption of such homing can prevent T cell priming and islet allograft rejection despite normal T and B cell functions of the recipient, with potential clinical implications.     

Nanotechnology as an adjunct tool for transplanting engineered cells and tissues

Laboratory and clinical studies have provided evidence of feasibility, safety and efficacy of cell transplantation to treat a wide variety of diseases characterized by tissue and cell dysfunction ranging from diabetes to spinal cord injury. However, major hurdles remain and limit pursuing large clinical trials, including the availability of a universal cell source that can be differentiated into specific cellular phenotypes, methods to protect the transplanted allogeneic or xenogeneic cells from rejection by the host immune system, techniques to enhance cellular integration of the transplant within the host tissue, strategies for in vivo detection and monitoring of the cellular implants, and new techniques to deliver genes to cells without eliciting a host immune response. Finding ways to circumvent these obstacles will benefit considerably from being able to understand, visualize, and control cellular interactions at a sub-micron level. Cutting-edge discoveries in the multidisciplinary field of nanotechnology have provided us a platform to manipulate materials, tissues, cells, and DNA at the level of and within the individual cell. Clearly, the scientific innovations achieved with nanotechnology are a welcome strategy for enhancing the generally encouraging results already achieved in cell transplantation. This review article discusses recent progress in the field of nanotechnology as a tool for tissue engineering, gene therapy, cell immunoisolation, and cell imaging, highlighting its direct applications in cell transplantation therapy.