Islet regeneration during the reversal of autoimmune diabetes in NOD mice

Nonobese diabetic (NOD) mice are a model for type 1 diabetes in humans. Treatment of NOD mice with end-stage disease by injection of donor splenocytes and complete Freund's adjuvant eliminates autoimmunity and permanently restores normoglycemia. The return of endogenous insulin secretion is accompanied by the reappearance of pancreatic beta cells. We now show that live donor male or labeled splenocytes administered to diabetic NOD females contain cells that rapidly differentiate into islet and ductal epithelial cells within the pancreas. Treatment with irradiated splenocytes is also followed by islet regeneration, but at a slower rate. The islets generated in both instances are persistent, functional, and apparent in all NOD hosts with permanent disease reversal.     

Reversal of diabetes in non-obese diabetic mice without spleen cell-derived beta cell regeneration

Autoimmune destruction of beta cells is the predominant cause of type 1 diabetes mellitus (T1DM) in humans and is modeled in non-obese diabetic (NOD) mice. Many therapeutic interventions prevent the development of T1DM in NOD mice, but few can induce its reversal once established. Intervention with Freund's complete adjuvant, semi-allogeneic splenocytes, and temporary islet transplantation has been reported to cure NOD mice of established T1DM. Using the same approach, we report here that this treatment cured 32% of NOD mice of established diabetes (>340 milligrams per deciliter blood glucose), although beta cells in these mice were not derived from donor splenocytes.     

Comment on papers by Chong et al., Nishio et al., and Suri et al. on diabetes reversal in NOD mice

Chong et al., Nishio et al., and Suri et al. (Reports, 24 March 2006, pp. 1774, 1775, and 1778) confirmed that treating nonobese diabetic (NOD) mice with an immune adjuvant and semisyngenic spleen cells can reverse the disease but found that spleen cells did not contribute to the observed recovery of pancreatic islets. We show that islet regeneration predominately originates from endogenous cells but that introduced spleen cells can also contribute to islet recovery.     

Immunological reversal of autoimmune diabetes without hematopoietic replacement of beta cells

Type 1 diabetes mellitus results from the autoimmune destruction of the beta cells of the pancreatic islets of Langerhans and is recapitulated in the nonobese diabetic strain of mice. In an attempt to rescue islet loss, diabetic mice were made normoglycemic by islet transplantation and immunization with Freund's complete adjuvant along with multiple injections of allogeneic male splenocytes. This treatment allowed for survival of transplanted islets and recovery of endogenous beta cell function in a proportion of mice, but with no evidence for allogeneic splenocyte-derived differentiation of new islet beta cells. Control of the autoimmune disease at a crucial time in diabetogenesis can result in recovery of beta cell function.     

Prevention of type I diabetes in nonobese diabetic mice by virus infection

The nonobese diabetic (NOD) mouse is an animal model of type I diabetes and develops a characteristic autoimmune lesion in the islets of Langerhans with lymphocytic infiltration and destruction of pancreatic beta cells. The result is hypoinsulinemia, hyperglycemia, ketoacidosis, and death. Diabetes usually begins by the sixth month of age but can occur earlier when young NOD mice are infused with lymphocytes from older NOD donors. When newborn or adult NOD mice were infected with a lymphotropic virus they did not become diabetic. The interaction between viruses and lymphocytes is pivotal in aborting diabetes, as established by experiments in which lymphocytes from virus-infected donors failed to transfer diabetes. In contrast, lymphocytes from age- and sex-matched uninfected donors caused disease. Therefore, viruses and, presumably, their products can be developed to be beneficial and may have potential as a component for treatment of human diseases. Further, these results point to the utility of viruses as probes for dissecting the pathogenesis of a nonviral disease.     

Nod2 mutation in Crohn's disease potentiates NF-kappaB activity and IL-1beta processing

Variants of NOD2, an intracellular sensor of bacteria-derived muramyl dipeptide (MDP), increase susceptibility to Crohn's disease (CD). These variants are thought to be defective in activation of nuclear factor kappaB (NF-kappaB) and antibacterial defenses, but CD clinical specimens display elevated NF-kappaB activity. To illuminate the pathophysiological function of NOD2, we introduced such a variant to the mouse Nod2 locus. Mutant mice exhibited elevated NF-kappaB activation in response to MDP and more efficient processing and secretion of the cytokine interleukin-1beta (IL-1beta). These effects are linked to increased susceptibility to bacterial-induced intestinal inflammation and identify NOD2 as a positive regulator of NF-kappaB activation and IL-1beta secretion.     

Localization of the pancreatic beta cell glucose transporter to specific plasma membrane domains

Immunocytochemical techniques revealed that the "liver-type" glucose transporter is present in the insulin-producing beta cells of rat pancreatic islets but not in other islet endocrine cells. Ultrastructural analysis of the transporter by the protein A-gold technique showed that it is restricted to certain domains of the plasma membrane, its density being sixfold higher in microvilli facing adjacent endocrine cells than in the flat regions of the plasma membrane. These results support a possible role for this glucose transporter in glucose sensing by beta cells and provide evidence that these cells are polarized.     

Virus persists in beta cells of islets of Langerhans and is associated with chemical manifestations of diabetes

Molecular hybridization, monoclonal antibody, and electron microscopic analyses showed lymphocytic choriomeningitis virus (strains Armstrong and WE) persistently infecting cells of the islets of Langerhans in BALB/WEHI mice. When monoclonal or monospecific antibody conjugated with two different fluorochrome dyes was used to mark insulin-containing beta cells or viral antigens, viral nucleoprotein was identified predominantly in beta cells. Electron microscopy confirmed these findings by showing virions budding from the beta cells. Persistent infection was associated with chemical evidence of diabetes (hyperglycemia, abnormal glucose tolerance, and normal or low-normal concentrations of insulin). Concentrations of cortisol and insulin-like growth factor in blood were normal, as was the level of growth hormone in the pituitary gland. The virus-infected islet cells showed normal anatomy and cytomorphology. Neither cell lysis nor inflammatory infiltrates were routinely seen. Thus a virus may persistently infect islet cells and provide a biochemical and morphological picture comparable to that of early adult-onset diabetes mellitus in humans.     

Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells

Insulin-expressing beta cells, found in pancreatic islets, are capable of generating more beta cells even in the adult. We show that fibroblast-like cells derived from adult human islets donated postmortem proliferate readily in vitro. These mesenchymal-type cells, which exhibit no hormone expression, can then be induced to differentiate into hormone-expressing islet-like cell aggregates, which reestablishes the epithelial character typical of islet cells. Immunohistochemistry, in situ hybridization, and messenger RNA measurements in single cells and cell populations establish the transition of epithelial cells within islets to mesenchymal cells in culture and then to insulin-expressing epithelial cells.     

Effects of cyclosporine A on T cell development and clonal deletion

Cyclosporine A (CsA) is an important immunosuppressive drug that is widely used in transplantation medicine. Many of its suppressive effects on T cells appear to be related to the inhibition of T cell receptor (TCR)-mediated activation events. Paradoxically, in certain situations CsA is responsible for the induction of a T cell-mediated autoimmunity. The effects of CsA on T cell development in the thymus were investigated to elucidate the physiologic events underlying this phenomenon. Two major effects were revealed: (i) CsA inhibits the development of mature single positive (CD4+8- or CD4-8+) TCR-alpha beta+ thymocytes without discernibly affecting CD4-8- TCR-gamma delta+ thymocytes and (ii) CsA interferes with the deletion of cells bearing self-reactive TCRs in the population of single positive thymocytes that do develop. This suggests a direct mechanism for CsA-induced autoimmunity and may have implications for the relative contribution of TCR-mediated signaling events in the development of the various T cell lineages.     

Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes

The role of defective glucose transport in the pathogenesis of noninsulin-dependent diabetes (NIDDM) was examined in Zucker diabetic fatty rats, a model of NIDDM. As in human NIDDM, insulin secretion was unresponsive to 20 mM glucose. Uptake of 3-O-methylglucose by islet cells was less than 19% of controls. The beta cell glucose transporter (GLUT-2) immunoreactivity and amount of GLUT-2 messenger RNA were profoundly reduced. Whenever fewer than 60% of beta cells were GLUT-2-positive, the response to glucose was absent and hyperglycemia exceeded 11 mM plasma glucose. We conclude that in NIDDM underexpression of GLUT-2 messenger RNA lowers high Km glucose transport in beta cells, and thereby impairs glucose-stimulated insulin secretion and prevents correction of hyperglycemia.     

Recognition by human V gamma 9/V delta 2 T cells of a GroEL homolog on Daudi Burkitt's lymphoma cells

All human gamma delta T cells coexpressing the products of the variable (V) region T cell receptor (TCR) gene segments V gamma 9 and V delta 2 recognize antigens from mycobacterial extracts and Daudi cells. Exogenous and endogenous ligands on the cell surface, homologous to the groEL heat shock family, induced reactivities that resembled superantigen responses in this major subset of human peripheral blood gamma delta T cells. Stimulation of human V gamma 9/V delta 2 T cells is not restricted by human leukocyte antigens (HLA), including nonpolymorphic beta 2-microglobulin (beta 2M)-associated class Ib molecules. These data may be important for understanding the role of gamma delta T cells in autoimmunity and in responses to microorganisms and tumors.     

大口黑鲈胰腺的组织学观察

采用组织切片和免疫组化技术对大口黑鲈的胰腺进行了组织学观察。经H-E染色和G-醛复红染色了的切片显示:大口黑鲈腹腔中10多个肉眼可见的黄白色颗粒物其实是被一层结缔组织和外分泌胰腺包裹着的胰岛结构,其中最大的一个即为主岛;还有许多肉眼不可见的胰岛结构被埋藏于总胆管的管壁中;肝脏中有外分泌胰腺的存在与分布。过氧化物酶标记的链霉卵白素免疫组化切片和图像分析显示:B细胞主要集中胰岛的中央区;A细胞分布于胰岛的全部。本研究的结论为:大口黑鲈的内分泌胰腺主要分布于总胆管周围,外分泌胰腺除了包裹于胰岛之外也分布于肝脏中,胰腺的分布属于弥散型。     

Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans

Activated mononuclear cells appear to be important effector cells in autoimmune beta cell destruction leading to insulin-dependent (type 1) diabetes mellitus. Conditioned medium from activated mononuclear cells (from human blood) is cytotoxic to isolated rat and human islets of Langerhans. This cytotoxic activity was eliminated from crude cytokine preparations by adsorption with immobilized, purified antibody to interleukin-1 (IL-1). The islet-inhibitory activity and the IL-1 activity (determined by its comitogenic effect on thymocytes) were recovered by acid wash. Purified natural IL-1 and recombinant IL-1 derived from the predominant pI 7 form of human IL-1, consistently inhibited the insulin response. The pI 6 and pI 5 forms of natural IL-1 were ineffective. Natural and recombinant IL-1 exhibited similar dose responses in their islet-inhibitory effect and their thymocyte-stimulatory activity. Concentrations of IL-1 that inhibited islet activity were in the picomolar range. Hence, monocyte-derived pI 7 IL-1 may contribute to islet cell damage and therefore to the development of insulin-dependent diabetes mellitus.     

助剂对高效氯氰菊酯在甘蓝叶片表皮渗透性的影响

 研究了3类助剂（矿物油、高级脂肪酸、非离子表面活性剂）对非内吸性杀虫剂高效氯氰菊酯在甘蓝叶片表皮渗透性的影响,证明药液的渗透性在一定范围内与药剂中助剂含量和给药后的时间成正相关。助剂间一定比例的配合对该渗透性具有协同作用。通过添加助剂后药液的各种物理性状进行测试及助剂溶液改变甘蓝表面蜡质结构的电镜观察,初步讨论了各类助剂影响渗透性的作用机理。     

Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes

In the fruit fly Drosophila, four insulin genes are coexpressed in small clusters of cells [insulin-producing cells (IPCs)] in the brain. Here, we show that ablation of these IPCs causes developmental delay, growth retardation, and elevated carbohydrate levels in larval hemolymph. All of the defects were reversed by ectopic expression of a Drosophila insulin transgene. On the basis of these functional data and the observation that IPCs release insulin into the circulatory system, we conclude that brain IPCs are the main systemic supply of insulin during larval growth. We propose that IPCs and pancreatic islet beta cells are functionally analogous and may have evolved from a common ancestral insulin-producing neuron. Interestingly, the phenotype of flies lacking IPCs includes certain features of diabetes mellitus.     

Transgenic mice with I-A on islet cells are normoglycemic but immunologically intolerant

Insulin-dependent diabetes mellitus (IDDM) is caused by a specific loss of the insulin-producing beta cells from pancreatic Langerhans islets. It has been proposed that aberrant expression of major histocompatibility complex (MHC) class II molecules on these cells could be a triggering factor for their autoimmune destruction. This proposal was tested in transgenic mice that express allogeneic or syngeneic class II molecules on the surface of islet cells at a level comparable with that normally found on resting B lymphocytes. These animals do not develop diabetes, nor is lymphocyte infiltration of the islets observed. This immunological inactivity does not result from tolerance to the "foreign" class II molecules.     

MHC-linked protection from diabetes dissociated from clonal deletion of T cells

The I-E molecule of the major histocompatibility complex (MHC) can prevent the spontaneous development of diabetes in nonobese diabetic (NOD) mice. The mechanism of this protection has been investigated by breeding wild-type and promoter-mutated E kappa alpha transgenes onto the NOD genetic background. Animals carrying the various mutated transgenes expressed I-E on different subsets of immunocompetent cells, and thus cells important for the I-E protective effect could be identified. Although the wild-type transgene prevented the infiltration of lymphocytes into pancreatic islets, none of the mutants did. However, all of the transgenes could mediate the intrathymic elimination of T cells bearing antigen receptors with variable regions that recognize I-E. Thus, the I-E molecule does not protect NOD mice from diabetes simply by inducing the deletion of self-reactive T cells.     

Reciprocal TH17 and regulatory T cell differentiation mediated by retinoic acid

The cytokine transforming growth factor-beta (TGF-beta) converts na?ve T cells into regulatory T (Treg) cells that prevent autoimmunity. However, in the presence of interleukin-6 (IL-6), TGF-beta has also been found to promote the differentiation of na?ve T lymphocytes into proinflammatory IL-17 cytokine-producing T helper 17 (T(H)17) cells, which promote autoimmunity and inflammation. This raises the question of how TGF-beta can generate such distinct outcomes. We identified the vitamin A metabolite retinoic acid as a key regulator of TGF-beta-dependent immune responses, capable of inhibiting the IL-6-driven induction of proinflammatory T(H)17 cells and promoting anti-inflammatory Treg cell differentiation. These findings indicate that a common metabolite can regulate the balance between pro- and anti-inflammatory immunity.     

NOD小鼠的遗传学特性和部分生物学特性观察

应用定期测定NOD小鼠的体重、血糖，统计繁殖特性，绘制生长曲线图，检测遗传位点，镜检病理学特征等方法，对NOD小鼠的遗传学特性和部分生物学特性进行了研究。结果显示：4－32周龄♀、♂小鼠有明显统计学差异（P＜0．01）。非糖尿病小鼠的受孕率为86．7％，而糖尿病小鼠的受孕率仅为53．3％，两组也差异显著（P＜0．05）。小鼠发病后体重随周龄的增长而急剧下降，至死亡时平均体重仅为发病前的60％。NOD（14♀）小鼠从发病到死亡的平均存活时间为22d。NOD小鼠只有在所有胰岛完全或近乎完全破坏时才发生糖尿病，若仍存有少量未萎缩的胰岛，其血糖水平维持于正常范围内。NOD小鼠在所检测的生化标志基因位点上，等位基因全部为纯合子，基因型一致，因某些位点不同程度地参与糖的代谢过程，可能导致糖尿病的发生。