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.     

Islet recovery and reversal of murine type 1 diabetes in the absence of any infused spleen cell contribution

A cure for type 1 diabetes will probably require the provision or elicitation of new pancreatic islet beta cells as well as the reestablishment of immunological tolerance. A 2003 study reported achievement of both advances in the NOD mouse model by coupling injection of Freund's complete adjuvant with infusion of allogeneic spleen cells. It was concluded that the adjuvant eliminated anti-islet autoimmunity and the donor splenocytes differentiated into insulin-producing (presumably beta) cells, culminating in islet regeneration. Here, we provide data indicating that the recovered islets were all of host origin, reflecting that the diabetic NOD mice actually retain substantial beta cell mass, which can be rejuvenated/regenerated to reverse disease upon adjuvant-dependent dampening of autoimmunity.     

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.     

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.     

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.     

Acceleration of diabetes in young NOD mice with a CD4+ islet-specific T cell clone

Nonobese diabetic (NOD) mice develop an autoimmune form of diabetes, becoming hyperglycemic after 3 months of age. This process was accelerated by injecting young NOD mice with CD4+ islet-specific T cell clones derived from NOD mice. Overt diabetes developed in 10 of 19 experimental animals by 7 weeks of age, with the remaining mice showing marked signs of the disease in progress. Control mice did not become diabetic and had no significant pancreatic infiltration. This work demonstrates that a CD4 T cell clone is sufficient to initiate the disease process in the diabetes-prone NOD mouse.     

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.     

微量元素和维生素对四氧嘧啶致糖尿病小鼠模型的影响

用含低微量元素、维生素的饲料(L)和含高适量微量元素和维生素的饲料(H)分别喂养小鼠4周,采用腹腔注射ALX建立DM小鼠模型。血糖仪检测血糖结果,并在光镜下观察两组鼠胰岛细胞的形态变化。结果表明L组喂养的小鼠血糖明显上升,胰岛内分泌细胞排列稀疏不均,胰岛数及岛内细胞数减少;H组小鼠血糖基本维持正常水平,胰岛为圆形、椭圆形细胞团,境界清楚,胰岛数及岛内细胞数较多,内各分泌细胞大小一致。总之,DM发生前,微量元素和维生素的联合生物学效应可拮抗ALX引起的胰岛损害,保护胰岛细胞,使血糖维持正常水平。     

The NOD mouse: recessive diabetogenic gene in the major histocompatibility complex

Examination of the histocompatibility region of the nonobese diabetic (NOD) mouse with antibodies against class II glycoproteins (products of immune response genes of the major histocompatibility complex I-A and I-E), hybrid T-cell clones, and mixed-lymphocyte cultures and analysis of restriction fragment length polymorphisms indicate that the NOD mouse has a unique class II major histocompatibility complex with no expression of surface I-E, no messenger RNA for I-E alpha, and an I-A not recognized by any monoclonal antibodies or hybrid T-cell clones studied. In crosses of NOD mice with control C3H mice, the development of diabetes was dependent on homozygosity for the NOD mouse's unique major histocompatibility region.     

Mitotic division in pancreatic beta cells

Successful expansion of the islet cell mass occurs in genetically diabetic mice (C57 Bl/Ks-dbdb) following a period of dietary restriction, in the absence of a population of precursor cells. Differentiated cells that synthesize insulin retain the capability of undergoing mitotic division.     

Three recessive loci required for insulin-dependent diabetes in nonobese diabetic mice

A polygenic basis for susceptibility to insulin-dependent diabetes in nonobese diabetic (NOD) mice has been established by outcross to a related inbred strain, nonobese normal (NON). Analysis of first and second backcross progeny has shown that at least three recessive genes are required for development of overt diabetes. One, Idd-1s, is tightly linked to the H-2K locus on chromosome 17; another, Idd-2s, is localized proximal to the Thy-1/Alp-1 cluster on chromosome 9. Segregation of a third, Idd-3s, could be shown in a second backcross. Neither Idd-1s nor Idd-2s could individually be identified as the locus controlling insulitis; leukocytic infiltrates in pancreas were common in most asymptomatic BC1 mice. Both F1 and BC1 mice exhibited the unusually high percentage of splenic T lymphocytes characteristic of NOD, suggesting dominant inheritance of this trait. The polygenic control of diabetogenesis in NOD mice, in which a recessive gene linked to the major histocompatibility complex is but one of several controlling loci, suggests that similar polygenic interactions underlie this type of diabetes in humans.     

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。     

H—Y单克隆抗体的制备方法

用Ｂａｌｂ／ｃ雄小鼠的脾细胞和皮肤组织对同系雌小鼠脾脏作一次直接免疫和５次腹腔加强免疫，雌小鼠产生良好的免疫应答。其抗血清用未经免疫的雌小鼠脾细胞吸收后，经细胞毒性法检测，Ｈ－Ｙ抗血清的效价可达１：１０２４；选择免疫应答最好的２只雌小鼠，取其脾细胞与ＮＳ－１骨髓瘤细胞进行融合，融合率１００％，经３次有限稀释克隆后，取４只阳性最强的杂交瘤细胞制备腹水。取其中一株的腹水采用胚胎细胞毒法进行特异性鉴定，结果其对２０个正常胚胎中的７个有杀伤溶解现象。４株细胞系分泌的抗体（腹水）以精子细胞毒法测定其效价分别为１：５１２、１：５１２、１：２５６和１：１２８。     

H－Y单克隆抗体的制备方法

用Ｂａｌｂ／ｃ雄小鼠的脾细胞和皮肤组织对同系雌小鼠脾脏作一次直接免疫和５次腹腔加强免疫，雌小鼠产生良好的免疫应答。其抗血清用未经免疫的雌小鼠脾细胞吸收后，经细胞毒性法检测，Ｈ－Ｙ抗血清的效价可达１：１０２４；选择免疫应答最好的２只雌小鼠，取其脾细胞与ＮＳ－１骨髓瘤细胞进行融合，融合率１００％，经３次有限稀释克隆后，取４只阳性最强的杂交瘤细胞制备腹水。取其中一株的腹水采用胚胎细胞毒法进行特异性鉴定，结果其对２０个正常胚胎中的７个有杀伤溶解现象。４株细胞系分泌的抗体（腹水）以精子细胞毒法测定其效价分别为１：５１２、１：５１２、１：２５６和１：１２８。     

Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes

Endoplasmic reticulum (ER) stress is a key link between obesity, insulin resistance, and type 2 diabetes. Here, we provide evidence that this mechanistic link can be exploited for therapeutic purposes with orally active chemical chaperones. 4-Phenyl butyric acid and taurine-conjugated ursodeoxycholic acid alleviated ER stress in cells and whole animals. Treatment of obese and diabetic mice with these compounds resulted in normalization of hyperglycemia, restoration of systemic insulin sensitivity, resolution of fatty liver disease, and enhancement of insulin action in liver, muscle, and adipose tissues. Our results demonstrate that chemical chaperones enhance the adaptive capacity of the ER and act as potent antidiabetic modalities with potential application in the treatment of type 2 diabetes.     

猪瘟基因疫苗免疫小鼠外周血细胞免疫动态变化研究

应用ＦＡＣＳ、ＭＴＴ法对猪瘟基因疫苗ｐｃＤＳＴ、ｐｃＤＳＷ免疫鼠外围血中ＣＤ４＾＋和ＣＤ８＾＋淋巴细胞数、淋巴细胞的转化功能等动态变化进行了观察。结果：ｐｃＤＳＴ、ｐｃＤＳＷ猪瘟基因疫苗免疫小鼠后,小鼠的外周血淋巴细胞对ＬＰＳ有强烈的反应性,ＣＤ４＾＋细胞数量增加,外周血淋巴细胞ＣＤ８＾＋细胞数量在一段时间内增加。ＣＤ４＾＋和ＣＤ８＾＋细胞数量在免疫后３２天达到高峰后开始下降。表明ｐｃＤＳＴ、ｐｃＤＳＷ质粒免疫小鼠后诱发免疫反应。     

Induction of donor-specific unresponsiveness by intrathymic islet transplantation

The application of isolated pancreatic islet transplantation for treatment of diabetes mellitus has been hampered by the vulnerability of islet allografts to immunologic rejection. Rat islet allografts that were transplanted into the thymus of recipients treated with a single injection of anti-lymphocyte serum survived indefinitely. A state of donor-specific unresponsiveness was achieved that permitted survival of a second donor strain islet allograft transplanted to an extrathymic site. Maturation of T cell precursors in a thymic microenvironment that is harboring foreign alloantigen may induce the selective unresponsiveness. This model provides an approach for pancreatic islet transplantation and a potential strategy for specific modification of the peripheral immune repertoire.     

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.     

Donor-derived cells in the central nervous system of twitcher mice after bone marrow transplantation

The twitcher mouse is an animal model of galactosylceramidase deficiency, comparable to Krabbe's disease, a lysosomal storage disease in humans. As in most lysosomal storage diseases, neurological deterioration is a prominent feature of the disease in these mice. Transplantation of enzymatically normal congenic bone marrow was earlier found to result in prolonged survival and increased levels of galactosylceramidase in the visceral organs of twitcher mice. It is now reported that bone marrow transplantation results in increased galactosylceramidase levels in the central nervous system (CNS). Concomitantly, the levels of psychosine, a highly toxic lipid that progressively accumulates in the CNS of untreated twitcher mice, stabilized at much lower levels in the CNS of treated twitcher mice. Histologically, a gradual disappearance of globoid cells, the histological hallmark of Krabbe's disease, and the appearance of foamy macrophages capable of metabolizing the storage product were seen in the CNS. By immunohistochemical labeling it was demonstrated that these foamy macrophages were of donor origin. The infiltration of enzymatically competent, donor-derived macrophages was accompanied by extensive remyelination in the CNS. It is concluded that after bone marrow transplantation, donor-derived macrophages infiltrate the affected brain tissue and are capable of inducing a partial reversal of the enzyme deficiency.     

H-2 antigen class: effect on mouse islet allograft rejection

Rejection of mouse pancreatic islet allografts occurred in a high percentage of donor recipient combinations identical for H-21-region antigens and differing at H-2K and H-2K + H-2D without I-region disparities. The results suggest that disparities in major histocompatibility complex antigens of class I (H-2K and H-2D) alone are capable of eliciting islet allograft rejection, and that lack of a stimulus from class II (I-region) alloantigens does not ensure permanent islet allograft survival.