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.     

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.     

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.     

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.     

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)     

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.     

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.     

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 culturing on the functioning of major-histocompatibility-complex-compatible and incompatible islet grafts in diabetic mice

Culturing B10.BR (H-2k) islets promotes their survival in major histocompatibility complex (MHC)-incompatible BALB/c (H-2d) mice, but not in MHC-compatible CBA (H-2k) animals. These results provide further evidence that MHC restriction is involved in transplantation immunity--i.e., that allografts are only recognized as foreign if they possess donor macrophages (or cells of this family), or if antigen-presenting cells MHC-compatible with the graft can be provided by the host.     

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.     

Mitogen responsiveness of lymphocytes from the BB/W rat

The response of BB diabetes-prone (DP) and W-line non-diabetes-prone rats to the T-cell mitogen concanavalin A (ConA) was measured. The W line was a good responder to ConA, whereas the DP was relatively unresponsive. This unresponsiveness could not be reversed with exogenous interleukin 2 (IL-2). The response of DP rats was enhanced by removing adherent cells. To directly test the response of BB T-cells, they were isolated by flow sorting. These experiments demonstrated that BB T-cells could mount a normal ConA response. The normal function of isolated BB T-cells suggested that they were under suppression. Suppressor activity could not be found in the OX8+ population but was found in the DP-adherent cell population. Adherent cells from the W line were not suppressive at the concentrations used. These results showed that the decreased mitogen responsiveness of BB T-cells was not due to an intrinsic T-cell abnormality but was due, in part, to suppression by adherent cells.     

冷缺血时间对大鼠胰岛结构和功能的影响

目的 探讨胰腺的冷保存时间对胰岛的获得量、活性、纯度及功能的影响.方法 采用Wistar大鼠,切取其胰腺,然后保存于4℃UW液中,分别于保存3 h(3 h组)、6 h(6 h组)、9h(9 h组)、12 h(12 h组)、15 h(15 h组)、18 h(18 h组)和21 h(21 h组)取出胰腺,采用明尼苏达大学改良方法分离、纯化胰岛,并设不经过冷保存的对照组.分离、纯化所得的胰岛以双硫腙染色,分析胰岛数量与纯度;丫啶橙/溴化乙啶染色分析胰岛活性;测定胰岛在葡萄糖刺激下的胰岛素分泌量.结果 对照组每个大鼠胰腺平均叮获得560当量胰岛,形态完整.纯度达到88%,活性达到94%.3 h组和6 h组的胰岛获得量略有下降,但与对照组相比,差异无统计学意义.9 h组、12 h组、15 h组、18 h组和21 h组的胰岛获得量较对照组、3 h组和6 h组明显下降(P＜0.01,P＜0.05),各组间相比较,差异也有统计学意义(P＜0.01),且随保存时间的延长,各组的胰岛活性和纯度也逐渐下降.9 h组、12 h组和15 h组胰岛细胞形态基本完整,但可见少数细胞被膜不完整,内分泌颗粒外溢.18 h组和21 h组胰岛形态不规则,被膜破裂,大量内分泌颗粒外溢.对照组、3 h组和6 h组对高糖刺激反应良好,三者问的胰岛素释放指数(SI)的差异无统计学意义,保存9 h以后,各组SI均较对照组、3 h组和6 h组明显下降(P＜0.01,P＜0.05),且各组间的差异也有统计学意义(P＜0.01),18 h组和21 h组尤其明显.结论 采用UW液低温保存大鼠胰腺6 h以内,获得的胰岛的数量较多,质量较好;保存6～15 h,获得的胰岛数量及质量有所下降,但仍然可以用于移植;保存时间超过15 h,获得的胰岛数量及质量明显下降,不宜用于移植.     

Urinary bladder function 6 months after the onset of diabetes in the spontaneously diabetic BB rat.

Urinary bladder function was examined in the spontaneously diabetic BB rat six months after the onset of diabetes. Diabetes caused significant decreases in rat weight and increases in bladder body weights and in vivo bladder capacities compared to age-matched controls, but no changes in the weights of bladder bases. The absolute contractile responses of urinary bladder body and base strips to nerve stimulation, carbachol, 5-hydroxytryptamine, ATP, phenylephrine, and KCl were unaltered by diabetes. However, when the data were corrected for tissue mass, there were slight but not significant decreases in contractile responses of strips from diabetic rats. There were increases in total muscarinic receptor numbers and calcium channel binding sites in bladder bodies from BB rats as a result of the increases in tissue mass. The data indicate that the six month-diabetic BB rat differs from the streptozotocin-diabetic rat in the sensitivity of the urinary bladder to the complications of diabetes, probably as a result of the insulin treatment required to keep BB rats alive.     

Time course of islet cell antibodies in diabetic and nondiabetic BB rats

The BB rat develops a spontaneous type I diabetic syndrome with anti-islet autoimmunity. Sera from diabetic and nondiabetic BB rats (from diabetes-prone litters), nondiabetic BB rats (from low-risk lines), and nondiabetes-prone Sprague-Dawley rats were collected twice a week from age 40 days to 160 days. Sera were tested for: (1) complement-dependent toxicity to 51Cr-labeled islet cells in vitro; (2) immunoglobulin binding to RIN-5 F insulinoma cells; and (3) ability to selectively suppress insulin secretion from normal islets in vitro. All sera from rats that subsequently became diabetic or glucose-intolerant were toxic to islet cells from various rat strains in the presence of complement. They were toxic neither to hepatocytes nor to fibroblasts. The toxic potency was associated with the globulin fraction. It was, in most cases, maximal either before or immediately after the onset of the disease. Sera from the nondiabetes-susceptible BB rats and the rats which, in diabetes-prone litters, died too early to be classified tended toward greater toxicity to islets. Immunoglobulins from diabetic sera bound to RIN-5 F cells more than did the serum globulins from other groups, their maximal binding capacity occurring after the onset of diabetes. Furthermore, BB diabetic sera were capable of selectively inhibiting the insulin secretion from normal rat islets in vitro either in the presence or, in some cases, in the absence of complement. The A- and D-cell functions were not suppressed. The combination of such results suggests the presence of one or more antibodies capable of binding to beta cells, inhibiting their function, and inducing their lysis.(ABSTRACT TRUNCATED AT 250 WORDS)