Autoantibodies in the BB/W rat

The BioBreeding/Worcester (BB/W) rat is a model of spontaneous autoimmune diabetes mellitus and lymphocytic thyroiditis. Additional features supporting an immunologic pathogenesis of the BB/W syndrome include the protective action of antilymphocyte serum, neonatal bone marrow transfusions, and neonatal thymectomy. To evaluate other manifestations of immune dysregulation, the BB/W colony was surveyed for the presence of autoantibodies to a variety of tissue and cell constituents. Anti-smooth muscle and anti-thyroid colloid antibodies were present with great frequency in diabetic animals as well as in normoglycemic offspring of diabetic parents. Anti-parietal cell antibodies were less frequent and islet cell cytoplasmic and adrenal antibodies were not detected. These data suggest that the underlying defect in the BB/W rat is more likely to be an abnormal immune regulatory system than antigenically altered target tissues ("altered self") under attack by a normal immune surveillance system.     

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.     

Intratesticular islet allografts in the spontaneously diabetic BB/W rat

Thirty-three male BB/W rats with diabetes of 11-145 days duration were divided into 3 groups: six received abdominal, intratesticular islet allografts and no immunosuppression posttransplantation; 15 were similarly grafted and in addition were given four injections of ALS regularly at 10-day intervals for 30 days after transplantation; and 12 rats received scrotal, intratesticular islet allografts and four injections of ALS. The results: in the absence of immunosuppression all six of the rats with abdominal, intratesticular islet allografts became normoglycemic within 2 days after transplantation but in none did graft survival exceed 17 days; a marked prolongation of graft survival of greater than 65-441 days occurred in 13 of the 15 animals with identical intratesticular allografts; sustained immunosuppression was not needed for prolonged islet allograft survival in rats with cryptorchid islet allografts; and (4) only one of the 12 rats with scrotal, intratesticular allografts became normoglycemic whereas 11 remained severely glycosuric. However, on surgical translocation of the grafted testes from the scrotum into the abdominal cavity, the rats promptly became normoglycemic in the absence of any additional therapy.     

Extended survival of MHC-compatible islet grafts from diabetes-resistant donors in spontaneously diabetic BB/W rat

Transplantation of a large inoculum of incubated islets of MHC-compatible donors led to an extended survival of the grafts to an average of greater than 86 days in 71% of male diabetic BB/W recipients. Identical results were obtained whether the immunologically privileged abdominal testis or the nonimmunologically favored renal subcapsular space was used as the organ site for the injection of the islets. Survival of the islet grafts was also independent of the duration of diabetes in the BB/W rats at the time of transplantation. These results showed that under our experimental conditions the grafted islets were able to become established and survive for extended periods in nonimmunosuppressed spontaneously diabetic BB/W hosts.     

Total lymphoid irradiation prevents diabetes mellitus in the Bio-Breeding/Worcester (BB/W) rat

Total lymphoid irradiation (TLI) at doses of 2200 rads or greater prevented diabetes in susceptible BB/W rats. Two of 29 (7%) treated rats became diabetic compared with 23 of 39 (59%) controls (P less than 0.001). TLI did not, however, prevent insulitis or thyroiditis in nondiabetic rats, nor did it restore the depressed concanavalin-A responsiveness of BB rat lymphocytes. T-lymphocyte subset proportions were the same in both groups. TLI was associated with significant radiation-related mortality, and nondiabetic TLI-treated rats weighed significantly less than controls. We conclude that TLI is effective in the prevention of BB rat diabetes. However, TLI fails to correct the subclinical immunologic abnormalities of the model and is associated with significant morbidity.     

Interleukin 2 activates BB/W diabetic rat lymphoid cells cytotoxic to islet cells

We compared the cytotoxic effects to islet cells of lymphoid cells from diabetic and diabetes-resistant (DR) BioBreeding/Worcester (BB/W) rats with a 51Cr-release assay to detect lysis of normal rat islet cells. Splenic lymphoid cells from diabetic rats were more cytotoxic to islet cells (11.3 +/- 3.8%) than were lymphoid cells from DR rats (4.0 +/- 2.6%). This difference was amplified by incubating the lymphoid cells for 20 h with 5 micrograms/ml concanavalin A (ConA); islet cell lysis was 39.3 +/- 4.5% by ConA-activated diabetic cells and 9.6 +/- 2.7% by ConA-activated DR cells. The cytotoxic lymphoid cells were identified as natural killer (NK) cells, because treatment of diabetic lymphoid cells with anti-asialo GM1 serum and complement selectively removed a monoclonal antibody-defined subset of NK cells (OX8 +), and the NK-depleted lymphoid cells were not cytotoxic to either islet or NK-sensitive YAC-1 cells, even after culture with ConA. Of several lymphokine products of ConA-stimulated lymphoid cells, interleukin 2 (IL-2), but not interleukin 1 or interferon-gamma, significantly activated splenic lymphoid cells cytotoxic to islet cells, and the lymphoid cells from diabetic rats were more sensitive to IL-2 (3 U/ml) than were the cells from DR rats (30 U/ml). This study reveals the presence of ConA- and IL-2-responsive islet cytotoxic NK cells in the diabetic BB/W rat and suggests that IL-2 activation of NK cells may contribute to islet beta-cell destruction and diabetes in this animal.     

Functional and morphological studies of long-term islet allografts in the renal subcapsular space of the BB/W rat

Our primary objective in this study was to determine whether transplanted pancreatic islet B cells display normal glucose-stimulated insulin secretory responses. Since transplanted islets are deinnervated and are located in a potentially unfavorable hormonal environment, it is possible that transplanted islets can maintain blood glucose levels but still not be completely normal. Since immune mechanisms may alter secretory responses but fail to cause overt islet necrosis (rejection), we used the BB/W spontaneously diabetic rat as the recipient in these studies. Islets harvested from inbred Lewis rats were transplanted beneath the renal capsule with minimal ALS immunosuppression posttransplantation. The transplanted animals showed a normal response to a glucose tolerance test. After 122-155 days of normoglycemia, the islets were retrieved and subjected to 2.8 and 16.7 mM glucose. The results indicate that the islet allografts maintain their secretory response to glucose when compared to donor Lewis islets acutely isolated from the pancreas. Furthermore, the transplanted islets maintained their morphologic integrity.     

Influence of postdiabetic onset time and immunosuppressive treatment on islet grafts in the spontaneous diabetic BB/W rat

1000 islets from one donor, isolated with a modified neutral red method, were intraportally transplanted from BB/Wistar or Lewis (RT1) donors to short- and long-term diabetic BB/Wistar recipients. Recipients received immunosuppression with cyclosporine i.m.--either postoperative 4 X 25 mg/kg body weight or pre- and postoperative 3 X 10 mg and 4 X 25 mg/kg body weight. Graft survival in short-term diabetic recipients was significantly shorter than in long-term diabetics, whether they were immunosuppressed or not. Especially in recent onset recipients, the additional preoperative immunosuppression with cyclosporine provided longer graft survival than postoperative cyclosporine alone. Histological examinations of islet grafts showed eosinophilic cells in all transplanted islets, in both functioning and clinically rejected grafts of allogenic and "pseudoisogenic" transplanted recipients. a coincidence of eosinophilic cells with the reenactment of the original autoimmune disease in islet grafts seems possible.     

Activation of smooth muscle cell outgrowth from BB/Wor rat aortas

An essential event in atherogenesis is the migration and proliferation of vascular smooth muscle cells (VSMCs), which contributes to the fibrocellular component of the atherosclerotic intimal thickening. We have been modeling this process by studying the outgrowth of VSMC from aortic explants onto tissue-culture plastic substrate. Our hypothesis is that certain risk factors for atherosclerosis favor increases in subpopulations of cells with enhanced responsiveness to a variety of migratory and proliferative stimuli, in vivo and in vitro. As a known risk factor for atherosclerosis, diabetes might be reasonably postulated to induce such cell populations with altered susceptibility to additional noxious stimuli. We have tested this hypothesis with aortic explants from rats of the spontaneous autoimmune diabetic BB/Wor group, including diabetes-resistant, diabetes-prone, and treated acutely (less than 2 wk after onset of hyperglycemia) and chronically (greater than 2 wk) diabetic strains. As a group, the VSMCs from BB/Wor animals showed enhanced outgrowth in 0.1% serum (23-48%) compared with VSMCs from ordinary Wistar rats (less than 10%). Within the BB/Wor group, however, the rank order of enhanced outgrowth was diabetes-resistant greater than chronically diabetic greater than acutely diabetic subjects. When the outgrowth assay was performed in the presence of supplemental insulin (10 mU/ml), outgrowth was increased, especially for the diabetes-prone animals, giving a new rank order of diabetes-prone greater than acutely diabetic greater than diabetes-resistant greater than chronically diabetic subjects. These data suggest a genetic predilection in the entire BB/Wor rat group for increased VSMC responsiveness to migratory and proliferative stimuli, a sensitivity of these VSMCs to insulin, and the dissociation of this vascular cell effect from other manifestations of diabetes, e.g., hyperglycemia.     

Mitogen responses of lymphocytes from lung transplant recipients--correlation with rejection and infection.

Proliferative responses to nonspecific mitogens were analyzed for 119 bronchoalveolar lavages and 108 concurrent peripheral blood samples from 35 lung transplant patients. The patients were classified at each time as normal, rejecting, or infected on the basis of trans-bronchial biopsy, culture results, clinical signs, and pulmonary function. During rejection episodes the bronchoalveolar lavage responses to concanavalin A and phytohemagglutinin were significantly increased (P less than 0.004 and P less than 0.006, respectively). The differences were less pronounced when rejection occurred within 30 days after bolus immunosuppressive therapy, either as immunoprophylaxis or as treatment for a previous rejection episode, and were not significantly different from normal. Differences in response during rejection were limited to the graft; analysis of circulating T cells was not helpful (P = NS). In contrast, markedly depressed responses to Con A and PHA were seen during infection. Significant differences were observed both in the graft (P less than 0.007) and in circulating lymphocytes (P less than 0.02), suggesting that global depression of mitogen response is associated with immunocompromise. Sequential analysis of 6 patients showed that individual changes in mitogen response paralleled those seen in the population (P less than 0.046, normal vs. rejection and P less than 0.043 normal). These findings suggest that mitogen assays of bronchoalveolar lavage lymphocytes and, to a lesser extent, PBL, are clinically useful in assessing intragraft immunocompetence and in distinguishing rejection from infection in lung transplant patients.     

Normotensive diabetic BB/W rats show enhanced reflex tachycardia.

Spontaneously diabetic BB/W rats were compared with age-matched regular Wistar and nondiabetic BB/W rats to determine whether the presence of diabetes would alter cardiovascular regulation appreciably. Systolic and mean blood pressures measured with the tail-cuff method from 12 to 26 wk of age tended to be slightly higher in diabetic than nondiabetic BB/W rats, but the differences were not significant. Mean pressures recorded from indwelling catheters in the same rats at 28 wk of age also did not differ significantly, thereby verifying that the diabetic rats were not hypertensive. To measure baroreflex sensitivity, heart-rate responses were elicited reflexly by elevating blood pressure with phenylephrine or lowering it with sodium nitroprusside. Although reflex bradycardia elicited with phenylephrine was the same, reflex tachycardia elicited with sodium nitroprusside was more pronounced in diabetic BB/W than other rats. Underlying autonomic mechanisms were then assessed by repeating the baroreflex tests after either cholinergic blockade with methylatropine or beta-adrenergic blockade with propranolol. Magnitude of reflex bradycardia after inhibition by either cholinergic or beta-adrenergic blockade still did not differ between rat groups but that of reflex tachycardia remained significantly stronger in diabetic BB/W than other rats. These results collectively show that, although diabetic BB/W rats remained normotensive, they had enhanced reflex tachycardia that persisted even after efferent autonomic blockade. The failure to develop higher pressures with time further indicates that without additional manipulation, these rats cannot be used experimentally to simulate the simultaneous presence of hypertension in diabetic patients.     

Histological and immunopathological analysis of recovered encapsulated allogeneic islets from transplanted diabetic BB/W rats.

Allogeneic islets encapsulated in an alginate/poly-L-lysine membrane and transplanted into diabetic BB/W rats resulted in graft failure within 2 weeks of transplantation. Graft failure was associated with a dense pericapsular infiltrate (PCI) that resulted in necrosis of the encapsulated islets. The PCI could be inhibited by immunosuppressive agents, including cyclosporine and dexamethasone, and this resulted in a significant increase in graft survival. Immunopathological characterization of the PCI indicated that there was a predominance of macrophages. T helper cells also appeared to be present in this PCI. Empty capsules were also found to induce a similar PCI that was identical in composition to that found around encapsulated islets. Thus alginate/poly-L-lysine capsules do not appear to be biocompatible and may account for the variable results in islet graft survival found with these capsules.     

Transplantation of encapsulated allogeneic islets into diabetic BB/W rats. Effects of immunosuppression

Allogeneic islets obtained from Lewis rats were transplanted into diabetic BB/W rats with or without cyclosporine. In addition, these islets were encapsulated in alginate-poly L-lysine membranes and then transplanted into diabetic BB/W rats with or without immunosuppressive and/or antiinflammatory agents. The agents used were cyclosporine, dexamethasone, indomethacin (Ind), or a combination of these. Our results show that islets alone survived for 7 days, with or without CsA therapy. Encapsulated islets survived for 14.2 days, and this was extended by CsA, Dex, or CsA + Ind. Loss of encapsulated graft functions was associated with formation of a dense pericapsular infiltrate, which was inhibited by CsA, Dex, CsA + Ind, or CsA + Dex. In addition, the infiltrate was reduced in animals that had diabetes for long periods of time (greater than 5 months versus less than 1 month). Empty capsules also provoked this cellular response. Thus, encapsulation of islets resulted in slightly prolonged islet survival, which was further enhanced by immunosuppression.     

Growth of neonatal islet transplants in the spontaneously diabetic BB/Wor rat.

We have previously shown that culture-isolated neonatal islets are able to survive both rejection and the recurrence of autoimmunity in the spontaneously diabetic BB/Wor rat. In trials designed to demonstrate the MHC restriction of the autoimmune response in this model, we discovered that neonatal islet grafts from diabetic BB rats appeared larger than grafts from nondiabetic controls. This study was undertaken to quantify the mass difference seen in this original study and to determine the characteristics of graft growth in more highly controlled trials. Grafts from diabetic animals in the original study were significantly larger than those from nondiabetic animals (81 +/- 36 vs. 238 +/- 216 micrograms, P = 0.01). These findings were supported by results from a second series of experiments, in which the mean growth index of grafts from diabetic animals was 7.25 +/- 4.91, whereas that from nondiabetic animals was 2.5 +/- 1.15 (P = 0.011). Three animals in this study were reversed of hyperglycemia: two had normal and one had a subdiabetic ip GTTs. These three rats received 97, 317, and 408 micrograms of islet tissue that increased in mass to 1790, 3270, and 4107 micrograms, respectively. Nuclear/total cell area percentages were the same in diabetic and nondiabetic grafts (P = 0.76), suggesting that the increase in mass was attributable primarily to proliferation rather than hypertrophy. Limited studies that use BrDU incorporation support this conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Introgression of F344 rat genomic DNA on BB rat chromosome 4 generates diabetes-resistant lymphopenic BB rats

Failure to express the Gimap5 protein is associated with lymphopenia (lyp) and linked to spontaneous diabetes in the diabetes-prone BioBreeding (BBDP) rat. Gimap5 is a member of seven related genes located within 150 Kb on rat chromosome 4. Congenic DR.(lyp/lyp) rats, where BBDP lyp was introgressed onto the diabetes-resistant BBDR background (BBDR.BBDP.(lyp/lyp)), all develop diabetes between 46 and 81 days of age (mean +/- SE, 61 +/- 1), whereas DR.(lyp/+) and DR.(+/+) rats are nonlymphopenic and diabetes resistant. In an intercross between F1(BBDP x F344) rats, we identified a rat with a recombination event on chromosome 4, allowing us to fix 33 Mb of F344 between D4Rat253 and D4Rhw6 in the congenic DR.lyp rat line. Gimap1 and Gimap5 were the only members of the Gimap family remaining homozygous for the BBDP allele. Offspring homozygous for the F344 allele (f/f) between D4Rat253 and D4Rhw6 were lymphopenic (85 of 85, 100%) but did not develop diabetes (0 of 85). During rescue of the recombination, 102 of 163 (63%) rats heterozygous (b/f) for the recombination developed diabetes between 52 and 222 days of age (88 +/- 3). Our data demonstrate that introgression of a 33-Mb region of the F344 genome, proximal to the mutated Gimap5 gene, renders the rat diabetes resistant despite being lymphopenic. Spontaneous diabetes in the BB rat may therefore be controlled, in part, by a diabetogenic factor(s), perhaps unrelated to the Gimap5 mutation on rat chromosome 4.     

Diet can prevent diabetes in the BB rat

When compared with laboratory chow, a defined, semipurified diet prevented diabetes, reduced the frequency of insulitis, increased thymus weight and total white blood cell count, and doubled thymus T-helper/T-suppressor cell ratios in diabetes-prone BB rats. These data show that the diabetic syndrome in BB rats may be prevented or delayed by changes in diet, which may occur through alteration of pathogenic defects in the immune system.     

Lymphoid cells of BB/W diabetic rats are cytotoxic to islet beta cells in vitro

Assays were developed to detect cell-mediated immune destruction of pancreatic islet beta cells by lymphoid cells isolated from diabetic BioBreeding/Worcester (BB/W) rats. Splenic lymphoid cells from diabetic (D), diabetes-prone (DP), and diabetes-resistant (DR) BB/W rats were incubated for 2 days with monolayer cultures of major histocompatibility complex (MHC)-compatible Wistar-Furth (WF) rat islet cells or a rat islet cell line (RIN), and islet beta cell survival was determined by measuring insulin content in the cultures. D splenic lymphoid cells significantly decreased insulin content in WF rat islet (-32%) and RIN cultures (-77%). DP cells also significantly reduced the insulin content in WF rat islet (-20%) and RIN cultures (-63%), whereas DR cells had no significant effect. Islet-directed cytotoxicity was detected also by the release of 51Cr from RIN cells incubated for 8 h with BB/W spleen cells. Cytotoxicity was linearly related to the number of effector spleen cells. At a target: effector of 1:20, lysis (mean +/- SEM) of RIN target cells by spleen cells from D rats (21.6 +/- 2.0%) and DP rats (16.5 +/- 4.1%) was significantly greater than the effect of DR spleen cells (5.4 +/- 1.0%). D and DP splenic lymphoid cells activated in vitro for 2 days with concanavalin A exhibited a doubling of cytotoxicity to RIN islet cells. These results provide direct evidence for lymphoid cell-mediated immune damage to islet beta cells in diabetic BB rats.     

4-1BB在慢性肾炎患者外周血淋巴细胞的表达及其意义

目的：观察在慢性肾炎患者外周血中共刺激分子４－１ＢＢ的表达特点及其作用。方法：采用流式细胞仪，对３５例慢性肾炎患者外周血Ｔ淋巴细胞亚群和协同刺激分子４－１ＢＢ的表达进行了研究。结果：活动期慢性肾炎患者Ｔ淋巴细胞亚群明显失衡，表现为ＣＤ４减少，ＣＤ８增加，ＣＤ４／ＣＤ８比值显著降低。缓解期患者Ｔ淋巴细胞亚群失衡得到明显纠正。活动期慢性肾炎患者共刺激分子４－１ＢＢ显著高于正常对照组（４－１ＢＢ表达量为３０．５７±８．１２，比正常人０．７４±０．２８，Ｐ＜０．００１），缓解期患者４－１ＢＢ显著降低，而且４－１ＢＢ异常表达与ＣＤ４＋Ｔ细胞呈负相关（ｒ＝－０．８４，Ｐ＜０．０５）。结论：慢性肾炎中，Ｔ细胞活化所必需的共刺激分子４－１ＢＢ的异常表达可能为慢性肾炎发生和病变进展的免疫因素之一，为深入探讨慢性肾炎的免疫发病机理提供了重要价值。