Prevention of recurrent autoimmune diabetes in BB rats by anti-asialo-GM1 antibody

BB rats exhibit a syndrome of spontaneous diabetes that has clinical and pathological characteristics analogous to those found in human insulin-dependent diabetes mellitus (IDDM). Islet tissue transplanted into spontaneously diabetic BB rats is uniformly destroyed by a recurrence of the autoimmune response that destroyed the diabetic subject's native islets. To examine recurrent autoimmune destruction of transplanted islets, it is necessary to exclude islet damage that might result from allograft rejection. We utilized neonatal tolerance induction to prevent rejection of Wistar-Furth (WF) (RT1u) islet allografts by spontaneously diabetic BB recipients. We determined that islet-recipient treatment with anti-asialo-GM1 (anti-AGM1) antibody prevents recurrent autoimmune diabetes that would otherwise destroy transplanted WF islet grafts. Anti-AGM1 therapy significantly decreased peripheral blood natural killer (NK) cell activity. These data suggest a role for NK cells in the pathogenesis of recurrent diabetes in neonatally tolerant BB rats.     

Intrathymic islet transplantation in the spontaneously diabetic BB rat.

Recently it was demonstrated that pancreatic islet allografts transplanted to the thymus of rats made diabetic chemically are not rejected and induce specific unresponsiveness to subsequent extrathymic transplants. The authors report that the thymus can also serve as an effective islet transplantation site in spontaneously diabetic BB rats, in which autoimmunity and rejection can destroy islets. Intrathymic Lewis islet grafts consistently reversed hyperglycemia for more than 120 days in these rats, and in three of four recipients the grafts promoted subsequent survival of intraportal islets. In contrast intraportal islet allografts in naive BB hosts all failed rapidly. The authors also show that the immunologically privileged status of the thymus cannot prevent rejection of islet allografts in Wistar Furth (WF) rats sensitized with donor strain skin and that suppressor cells are not likely to contribute to the unresponsive state because adoptive transfer of spleen cells from WF rats bearing established intrathymic Lewis islets fails to prolong islet allograft survival in secondary hosts.     

Noninvasive diagnosis of recurrent autoimmune type 1 diabetes after islet cell transplantation

Islet cell transplantation is curative therapy for patients with complicated autoimmune type 1 diabetes (T1D). We report the diagnostic potential of circulating transplant islet–specific exosomes to noninvasively distinguish pancreatic β cell injury secondary to recurrent autoimmunity vs immunologic rejection. A T1D patient with hypoglycemic unawareness underwent islet transplantation and maintained normoglycemia until posttransplant day 1098 before requiring exogenous insulin. Plasma analysis showed decreased donor islet exosome quantities on day 1001, before hyperglycemia onset. This drop in islet exosome quantity signified islet injury, but did not distinguish injury type. However, analysis of purified transplant islet exosome cargoes showed decrease in insulin‐containing exosomes, but not glucagon‐containing exosomes, indicating selective destruction of transplanted β cells secondary to recurrent T1D autoimmunity. Furthermore, donor islet exosome cargo analysis showed time‐specific increase in islet autoantigen, glutamic acid decarboxylase 65 (GAD65), implicated in T1D autoimmunity. Time‐matched analysis of plasma transplant islet exosomes in 3 control subjects undergoing islet cell transplantation failed to show changes in islet exosome quantities or intraexosomal cargo expression of insulin, glucagon, and GAD65. This is the first report of noninvasive diagnosis of recurrent autoimmunity after islet cell transplantation, suggesting that transplant tissue exosome platform may serve as a biomarker in islet transplant diagnostics.     

Low-dose streptozocin-induced autoimmune diabetes in islet transplantation model.

An islet transplant model was used to gain further insight into the immunologic mechanisms involved in low-dose streptozocin (STZ)-induced diabetes. As shown by others, male C57BL/KsJ mice developed diabetes and insulitis after five daily injections of STZ (40 mg.kg-1.day-1). Syngeneic islet transplants beneath the renal capsule developed insulitis when the islets were transplanted 10-14 days before the daily injections of STZ. In contrast, insulitis of the grafts did not occur when the syngeneic transplants were done 3 days after the five daily injections of STZ. If the donor islets were incubated in vitro with 0.5 mg/ml STZ for 1 h at 37 degrees C and then transplanted after the low-dose STZ administration of the recipients, then a definite insulitis was present in the syngeneic transplants. These findings indicated that this brief exposure to STZ in vitro was sufficient to permit immunologic recognition of the grafts. In vitro STZ-exposed islets transplanted into high-dose STZ-induced diabetic mice also developed insulitis, whereas STZ-exposed islets transplanted into alloxan-induced diabetic mice did not. Donor islets incubated in vitro with STZ and transplanted into unexposed mice did not develop insulitis in the grafts. Thus, preexposure of the recipient to STZ is essential to the development of insulitis within in vitro STZ-exposed islet grafts. This is also specific to islets because in vitro STZ-exposed thyroid tissue transplanted into low-dose STZ recipients failed to exhibit a thyroiditis. These data are consistent with the idea that STZ immunologically alters the beta-cell by inducing an antigenic change.     

Prevention of vascular complications of diabetes by pancreatic islet transplantation.

Isolated pancreatic islets from Wistar-Lewis rats were transplanted into the liver of diabetic allogeneic recipients to assess ability to prevent diabetic renal and ophthalmic complications. At nine months, the diabetic animals without transplants showed significant increase in PAS-positive material in the renal glomerular mesangium and thickening of glomerular arterioles as compared with normal nondiabetic animals. New vessel formation was also significant in the retina and retinal capillary dilation. Animals in which diabetes had been corrected by early pancreatic islet transplantation were completely protected from these changes, showing no significant pathologic change when compared with normal animals.     

新生猪肾包膜下胰岛移植动物模型构建的初步研究

目的  构建新生猪肾包膜下胰岛移植模型,探讨其可行性和潜在应用价值。 方法  选取9只野生型同胎新生健康杜洛克猪,其中1只为对照（p6307）,6只为胰岛移植供体,2只为胰岛移植受体（p6210、p6207）。新生猪胰岛分离、体外培养分化后,行猪肾包膜下胰岛移植术,术后给予他克莫司（Tac）联合西罗莫司免疫抑制治疗。术后监测移植受体的体质量、血糖、血清肌酐水平,术后4周和8周分别处死受体新生猪p6210和p6207、对照新生猪,获取猪肾包膜下胰岛移植物进行病理学染色和胰岛素免疫荧光染色。 结果  猪肾包膜下胰岛移植术后,受体新生猪的体质量增长相对于对照新生猪明显减慢,并间断性伴有纳差、腹泻等症状;而受体新生猪的血糖、血清肌酐水平相较于术前及对照新生猪无明显变化。受体新生猪p6210肾包膜下可见明显的胰岛团块,病理学染色和胰岛素免疫荧光染色证实胰岛团块具有分泌胰岛素的功能,而受体新生猪p6207肾包膜下未见明显的胰岛团块,病理学染色未见明显胰岛团块,考虑该受体新生猪发生胰岛移植排斥反应导致胰岛定植失败。 结论  新生猪肾包膜下胰岛移植是可行的,为下一步构建用于异种移植治疗糖尿病终末期肾病的猪胰岛-肾复合供体器官提供前期铺垫。     

Encephalomyocarditis virus-induced diabetes mellitus treated by islet transplantation.

There is considerable evidence that at least some cases of juvenile onset diabetes mellitus in humans are a result of viral infection. Viral-induced diabetes in mice may provide an experimental counterpart more similar to the clinical situation than chemical-induced diabetes. Our experiments in such mice indicate that islet transplantation is effective in ameliorating viral-induced diabetes and is encouraging for ultimate clinical application of islet transplantation to juvenile onset insulin-dependent diabetics. In addition, our results show that islets in ectopic sites outside of the pancreas are resistant to damage induced by primary viral infection. The mechanism of this resistance is obscure and will be the subject of future investigations.     

Successful islet transplantation in spontaneous diabetes.

Optimism for islet transplantation is based on reversal of diabetes artificially induced in animals by pancreatectomy or beta cell toxins. In naturally occurring diabetes, implanted islets might be destroyed by the etiologic agent of the original disease; e.g., virus infection, genetic factors, or autoimmunity. Genetically determined diabetes in obese mice, in fact, is resistant to islet transplantation. Since these mice are hyperinsulinemic and not similar to human juvenile onset diabetes (JOD), more appropriate models were sought, "BB" rats spontaneously develop a syndrome remarkably similar to human JOD. We have studied 279 BB rats. In 31 rats the sudden onset of severe hyperglycemia was observed. Sinc BB rats proved to be AgB2 on serological typing, WF (AgB2) donors were selected. Six hundred Wistar-Furth isolated islets were transplanted intraportally in 10 BB diabetic rats immunosuppressed with antilymphocyte serum. All 10 recipients became normoglycemic, remaining so for 1 to 6 monts. An additional animal model studied was virus-induced diabetes in mice, since viral etiology of human diabetes seems likely. DBA mice receiving encephalomyocarditis virus became severely and persistently diabetic. Eight received syngeneic fetal pancreas to the renal subcapsule and became normoglycemic. Removal of the graft 30 days later demonstrated viable islets histologically and resulted in recurrent diabetes. That virally induced murine diabetes and one spontaneous syndrome in rats which is similar to human JOD responded to beta cell implantation argues that this treatment will be effective in man.     

Effects of streptozocin diabetes and diabetes treatment by islet transplantation on in vivo insulin signaling in rat heart.

The insulin signaling cascade was investigated in rat myocardium in vivo in the presence of streptozocin (STZ)-induced diabetes and after diabetes treatment by islet transplantation under the kidney capsule. The levels of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit, insulin receptor substrate (IRS)-2, and p52(Shc) were increased in diabetic compared with control heart, whereas tyrosine phosphorylation of IRS-1 was unchanged. The amount of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the level of PI 3-kinase activity associated with IRS-2 were also elevated in diabetes, whereas no changes in IRS-1-associated PI 3-kinase were observed. Insulin-induced phosphorylation of Akt on Thr-308 was increased fivefold in diabetic heart, whereas Akt phosphorylation on Ser-473 was normal. In contrast with Akt phosphorylation, insulin-induced phosphorylation of glycogen synthase kinase (GSK)-3, a major cellular substrate of Akt, was markedly reduced in diabetes. In islet-transplanted rats, the majority of the alterations in insulin-signaling proteins found in diabetic rats were normalized, but insulin stimulation of IRS-2 tyrosine phosphorylation and association with PI 3-kinase was blunted. In conclusion, in the diabetic heart, 1) IRS-1, IRS-2, and p52(Shc) are differently altered, 2) the levels of Akt phosphorylation on Ser-473 and Thr-308, respectively, are not coordinately regulated, and 3) the increased activity of proximal-signaling proteins (i.e., IRS-2 and PI 3-kinase) is not propagated distally to GSK-3. Islet transplantation under the kidney capsule is a potentially effective therapy to correct several diabetes-induced abnormalities of insulin signaling in cardiac muscle but does not restore the responsiveness of all signaling reactions to insulin.     

Immunologic studies of the prediabetic stage in the spontaneous autoimmune diabetes mellitus of the BB rat

Twenty-eight BB diabetes-prone rats underwent prospective analysis to determine levels of circulating Ia antigen-bearing "activated" T lymphocytes prior to 50 days of age using mouse antirat monoclonal antibodies OX6 (anti-Ia) and W3/13 (anti-T-cell) and a fluorescence-activated cell sorter. Pancreatic biopsies were obtained within 48 hrs of lymphocyte sampling and histologically examined for the presence of a lymphocytic infiltrate. Elevated levels of "activated" T lymphocytes (above 4.00%) accurately identified a prediabetic stage and predicted which BB rats would subsequently become diabetic (93% sensitivity). The insulitis lesion occurs in the latter portion of this immunologically defined prediabetic stage just prior to the development of hyperglycemia. This observation suggests that islet cell destruction represents the final focus of a chronic immune response.     

Prevention by tetrandrine of spontaneous development of diabetes mellitus in BB rats.

Treatment of BB rats with the plant alkaloid tetrandrine (20 mg.kg-1.day-1), a novel anti-inflammatory compound, from 35 to 120 days of age reduced the cumulative incidence of spontaneous diabetes from 75.5 to 10.9% (P less than 0.001). Dose-response studies with 0, 5, 10, and 20 mg.kg-1.day-1 of tetrandrine from 35 to 130 days resulted in spontaneous diabetes in 84.2, 63.1, 31.6, and 5.3% of the rats, respectively. When the start of treatment with 20 mg.kg-1.day-1 tetrandrine was delayed until 70 days of age, there was a significant reduction of the incidence of diabetes from 63.1 to 28.6% (P less than 0.01). Histological examination of the pancreases from tetrandrine-treated rats showed a lesser degree of insulitis than controls (P less than 0.01). Drug toxicity was not seen in the rats, as assessed by appearance, behavioral change, organ histology, and blood chemistry. These results provide some hope that tetrandrine may be of value in preventing diabetes and treating newly diagnosed diabetic subjects, either by itself or in combination with a more potent immunosuppressive agent.     

Evidence that cyclosporine prevents rejection and recurrent diabetes in pancreatic transplants in the BB rat

Cyclosporine prevents the development of diabetes in spontaneously diabetic BB rats and NOD mice. However, islet transplants have been shown to be subject to immunologic destruction in hosts treated with CsA and anti-CD4 antibody or those rendered tolerant to donor antigens. This study determines whether the minimum dose of CsA necessary to prevent rejection of pancreatic transplants will also prevent recurrent diabetes in pancreas transplants in BB rats. Lewis recipients promptly reject BN (n = 13, MST 9.2 +/- 0.7 days) and Fisher (n = 6, MST = 11.3 +/- 0.8 days) pancreatic transplants. Treatment with CsA 5 mg/kg/day 0-50 days posttransplant and 2 mg/kg/day 51-100 days (low-dose CsA) produced indefinite survival of BN (n = 5) but not Fisher (n = 4) allografts. Fisher allografts were uniformly successful if maintained on 5 mg/kg/day (n = 4). Treatment with CsA 5 mg/kg/day for 14 days had no deleterious effect on glucose tolerance in Lewis isografts (control [K = 1.81 +/- 0.24, n = 8] vs. CsA treated [K = 1.76 +/- 0.20, n = 8, P = NS]). Low-dose CsA ensured permanent survival of MHC-compatible WF (n = 7, MST greater than 115 days) and MHC-incompatible BN (n = 9, MST greater than 117 days, P = NS) pancreatic transplants in spontaneously diabetic BB/Wor hosts. Three of 11 recipients surviving greater than 100 days enjoyed seemingly permanent acceptance of their allografts after discontinuation of CsA. Immunocompetence was demonstrated by rejection of their pancreas transplants when challenged with donor skin. Vascularized pancreatic allografts treated with CsA appear to be less vulnerable to recurrent diabetes than are islet transplants. Low-dose CsA protects vascularized pancreatic allografts in BB rats from both rejection and recurrent diabetes.     

Complement-fixing islet cell antibodies in the spontaneously diabetic BB rat

Fourteen rats of the spontaneously diabetic BB line were bled from the retroorbital sinus approximately every 10 days. Sera taken from an early age up to 20 days after the onset of overt diabetes were assayed for complement-fixing antibodies against antigens of the surface of islet cells (CFA). Dispersed islet cells from normal Wistar rats prelabeled with 3H-leucine were used as targets. Target cells in suspension were incubated with heat-inactivated rat sera and then, after washing, exposed to guinea pig complement. Cytolytic "injury" was measured by the percentage of labeled cellular proteins released into the medium. Sera from sequential bleedings from eight normal Wistar rats and three rats from a nondiabetic BB subline were assayed to establish basal control cytolytic activity. The mean response +/- SD obtained with all control sera was 7.7 +/- 1.7%. A response exceeding the mean + 3 SD (12.8%) was considered significantly different from the basal value. Thirteen of the fourteen BB rats developed strongly positive sera. The cytolytic activity preceded the onset of overt diabetes. In several rats CFA appeared 4-8 wk preceding diabetes while in other rats CFA appeared 1-2 wk preceding the manifestation of the disease. These results indicate that CFA may contribute to the destruction of pancreatic islets directly or by attracting mononuclear cells.     

Spontaneous diabetes in the gnotobiotic BB/W rat.

To determine the influence of infectious agents on the initiation of diabetes in the spontaneously diabetic Bio-Breeding/Worcester (BB/W) rat, susceptible rats were raised in a germ-free environment. Between 2 and 3 mo of age, 3 of 12 pups became diabetic. Histologic examination of the pancreas revealed insulitis or end-stage islets. Culture and smears from various tissues were negative for bacteria or parasites. Serum vital antibody titers for all known rat viruses were undetectable. These data suggest that the diabetic syndrome of the BB/W rat is not dependent on recognized infectious agents.