Quantitative phenotypic and functional analyses of islet immune cells before and after diabetes onset in the BB rat

Summary Inflammatory cells invading islets are thought to be mediators of islet destruction in spontaneous autoimmune diabetes mellitus. Thus methods were developed to isolate and characterize in situ islet inflammatory cells from 75–95-day-old prediabetic and diabetic BB rats. Islet inflammatory cells were structurally examined using single- and double-colour flow cytometry. Functional studies consisted of cytolytic assays using normal rat islet target cells and in situ islet or spleen effector cells. Structural data reveal natural killer cells to be the major cell population (70%) of total immune cells present in inflamed islets during prediabetes. At diabetes onset, the natural killer cell population remained at a high level (47%), but an increasing population of T cells (40%) was noted also. Analyses of T-cell subsets before and after diabetes onset revealed CD4⁺ T cells as predominant (50–55% of total T cells) with double-negative (CD4^– CD8^–) T cells (25–30%) and CD8⁺ T cells (15–20%) also present in significant quantities. Activated T cells accounted only for a minority of T cells (<3%). Functional studies indicate that in situ islet-derived cytolytic effector cells are more potent killers (ten-fold) of normal islet target cells than are splenic effector cells. These data suggest that in situ islet inflammatory cells (a) can be quantitatively studied both structurally and functionally; (b) express structural phenotypes differing substantially from splenic mononuclear cell populations; (c) are considerably more cytolytic than splenic effectors; and (d) should prove informative in determining the most significant autoimmune functional events prior to and during islet beta-cell destruction.This work was reported in part at the European Association for the Study of Diabetes in Copenhagen, Denmark, in September, 1990 and at the International Diabetes Federation Congress in Washington DC, June, 1991     

Characterization of monoclonal islet cell reactive autoantibodies from the diabetic biobreeding (BB/OK) rat

Two fusion experiments using the heteromyeloma cell line CB-F7 and splenocytes from two diabetesprone BB (BioBreeding) rats at the onset of diabetes resulted in 128 islet cell reactive autoantibodies primarily detected with permeabilized insulin-producing rat insulinoma cells (RIN) by a cellular enzyme-linked immunosorbent assay. Seventy-nine (62%) of 128 RIN cell reactive supernatants exhibited a cross-reactivity with rat splenic lymphocytes. Six stable hybridomas secreting monoclonal ICSA (islet cell surface antibodies) were established, but only one monoclonal antibody, R4B10, showed preferential beta-cell binding. Six monoclonal antibodies showed a dual reactivity as ICA (islet cell cytoplasmic antibodies) detected by immunostaining of pancreatic islet cryosections and as ICSA on the surface of viable islet cells, whereas two reacted only with an ICA-like pattern. One monoclonal ICSA was specifically displaced from the RIN cell surface by sera of type 1 diabetic patients.     

Preservation of GLUT 2 expression in islet beta cells of Kilham Rat Virus (KRV) — infected diabetes-resistant BB/Wor rats

Summary Loss of GLUT 2, the glucose transporter isoform of pancreatic beta cells, has been reported to accompany the onset and perhaps contribute to the pathogenesis, of insulin-dependent and non-insulin-dependent diabetes mellitus in BB/Wor and Zucker fatty rats. In this study we investigated the effect of Kilham Rat Virus infection on GLUT 2 expression in diabetes-resistant BB/Wor rats. Viral antibodyfree diabetes-resistant rats do not develop spontaneous diabetes, but inoculation with Kilham Rat Virus induces autoimmune beta-cell destruction and hyperglycaemia. Pancreas sections from normoglycaemic diabetes-resistant BB/Wor rats were obtained 5, 7 and 25 days after inoculation with Kilham Rat Virus and stained for GLUT 2 using a rabbit polyclonal antibody. At all time points, beta cells displayed GLUT 2 expression comparable to uninfected diabetes-resistant controls. Immunostained insulin content of the beta cells also remained unchanged. Sections were also examined from Kilham Rat Virus inoculated diabetes-resistant rats with lymphocytic insulitis or diabetes. GLUT 2 and insulin immunostaining were unchanged in non-diabetic rats with early insulitis. GLUT 2 beta-cell staining was variably reduced in diabetic rats with established insulitis and reduced beta-cell insulin immunostaining. Hence, the initial stages of Kilham Rat Virus-induced diabetes in diabetes-resistant rats are not accompanied by a significant reduction in GLUT 2 expression. These results suggest that the loss of GLUT 2 does not play a significant role in the aetiology of diabetes in the Kilham Rat Virus-infected diabetes-resistant BB/Wor rat.Abbreviations KRV Kilham Rat Virus - NHS normal horse serum - BB/Wor Biobreeding/Worcester rats     

The ACE-inhibitor captopril improves myocardial perfusion in spontaneously diabetic (BB) rats

Summary The aim of this study was to examine the influence of inhibition on angiotensin converting enzyme (ACE) of myocardial function and perfusion of the rat impaired by diabetes. Spontaneously diabetic rats were treated with the ACE-inhibitor captopril for 4 months. Cardiac performance was analysed in the isolated heart perfused at constant volume. Epicardial perfusion was determined by measuring changes in epicardial fluorescence after injection of a bolus of fluoresceinisothiocyanate-dextrane (3 kDa) as described previously. As compared to untreated diabetic controls, captopril prevented the increase of end diastolic pressure, coronary perfusion pressure and vascular resistance. The intravascular volume was enlarged and the epicardial perfusion rate increased in hearts of diabetic rats treated with captopril as compared to diabetic controls. Treatment of diabetic rats with the ACE-inhibitor captopril (1) increases the number of perfused capillaries, and (2) can partly prevent the development of cardiac dysfunction in diabetes. Together with morphological data demonstrating an inhibition of interstitial and perivascular fibrosis in hearts of diabetic rats treated with captopril, our data suggest that ACE-inhibition is cardioprotective in diabetes. These observations are also compatible with the assumption that an accelerated generation of angiotensin II may be involved in the pathophysiological chain of events leading to diabetic cardiopathy.Abbreviations ACE Angiotensin converting enzyme - CVR coronary vascular resistance - EDP end diastolic pressure - LVP left ventricular pressure - CPP coronary perfusion pressure     

Antidiabetic effects of chitooligosaccharides on pancreatic islet cells in streptozotocin-induced diabetic rats

AIM： To investigate the effect of chitooligosaccharides on proliferation of pancreatic islet cells, release of insulin and 2 h plasma glucose in streptozotocin-induced diabetic rats. METHODS： In vitro, the effect of chitooligosaccharides on proliferation of pancreatic islet cells and release of insulin was detected with optical microscopy, colorimetric assay, and radioimmunoassay respectively. In vivo, the general clinical symptoms, 2 h plasma glucose, urine glucose, oral glucose tolerance were examined after sixty days of feeding study to determine the effect of chitooligosaccharides in streptozotocin-induced diabetic rats. RESULTS： Chitooligosaccharides could effectively accelerate the proliferation of pancreatic islet cells. Chitooligosaccharides （100 mg/L） had direct and prominent effect on pancreastic β cells and insulin release from islet cells. All concentrations of chitooligosaccharides could improve the general clinical symptoms of diabetic rats, decrease the 2 h plasma glucose and urine glucose, and normalize the disorders of glucose tolerance. CONCLUSION： Chitooligosaccharides possess various biological activities and can be used in the treatment of diabetes mellitus.     

Effects of a high protein diet on the evolution of diabetes in streptozotocin-induced and spontaneously diabetic “BB” wistar rats

Summary Recently, we demonstrated a reduction in the diabetogenic action of streptozotocin (STZ) in rats previously adapted to a high protein (HP) diet. These data suggested that amelioration of diabetes resulted from the combination of two effects of the HP diet: initial protection against the diabetogenic action of the drug at the time of exposure and subsequent improvement of the induced diabetic condition. The present study evaluated the effects of a HP diet on the evolution of the metabolic condition in rats with STZ-induced or spontaneous diabetes (BB Wistar rats). Two days after STZ injection, the animals were given isocaloric HP (70% protein, 8% fat) or control (66% carbohydrate, 16% protein, 8% fat) diets for 15 days. After 13 days, the STZ-treated rats fed HP diet showed an impressive decrease in severity of diabetes, as judged by rate of body weight change, plasma glucose, urine volume and glycosuria, serum and pancreatic insulin. The BB Wistar rats, already diabetic for 5 weeks before being transferred to the HP or control diet, were treated with daily injections of insulin. After 31 days on the HP diet, the BB rats showed reduced insulin requirement, reduced blood and urinary glucose levels, but no difference in body weight gain or pancreatic insulin content. The data show that short-term use of HP diets can greatly improve the diabetic condition in STZ-treated animals, but that the beneficial effects of the diet are much less marked in rats with chronic spontaneous diabetes. These data suggest that the ameliorating effect of HP diet is fully manifested only when the diabetic rats have a sufficient number of residual functioning B-cells.     

Production and characterization of a monoclonal islet cell surface autoantibody from the BB rat

Summary Islet cell surface autoantibodies are present in the serum of the spontaneously diabetic BB rat. The availability in large quantities of such autoantibodies should help us understand their significance in vivo. Fusions between BB rat lymphocytes and rat myeloma cells were screened by cellular enzyme linked immunosorbent assay and indirect immunofluorescence on rat living cells. They resulted in a stable hybridoma, called IC₂, secreting a monoclonal immunoglobulin M specific for the surface of rat islet cells. This monoclonal antibody was found to bind to the surface of 56% normal rat islet cells and 72% rat insulinoma cells. Protease treatment of rat islet cells resulted in a subsequent 72–100% binding inhibition of IC₂ to the surface of these cells, suggesting that IC₂ specific antigen is a protein.     

B cell lymphoproliferation in spontaneously diabetic BB Wistar rats

Summary Ninety-six spontaneously diabetic BB Wistar rats were maintained for their natural life span and, at death, were autopsied together with 86 age- and sex-matched non-diabetic BB control rats. A 15% incidence of abdominal B cell lymphoproliferative lesions was documented in the diabetic rats compared with 1% incidence in the non-diabetic rats (p<0.005). The B cell lymphoproliferative process included minute mesenteric and omental aggregates of plasma cells and small lymphocytes (one rat), atypical partially fibrotic lymphoproliferative mesenteric nodules (three rats), and malignant lymphoma with features of immunoblastic sarcoma (eight rats) or plasma cell lymphoma (two rats). Cytoplasmic immunoglobulin was demonstrated in two of the four lymphomas examined by the peroxidase-antiperoxidase technique, thus confirming their B cell derivation. The striking incidence of B cell lymphoproliferation in this diabetic population is additional evidence of altered immunity in this animal model of insulin-dependent diabetes mellitus.     

免疫印迹法检测老年2型糖尿病患者血清胰岛自身抗体的临床价值

目的 评价免疫印迹法检测老年2型糖尿病患者胰岛自身抗体(IAA)的临床价值. 方法 采用免疫印迹(IB)法和酶联免疫吸附(ELISA)法分别检测350例老年2型糖尿病患者和120例健康对照组血清谷氨酸脱羧酶抗体(GADA)、胰岛细胞抗体(ICA)和IAA,比较不同方法检测胰岛相关抗体的阳性率. 结果 在老年2型糖尿病患者中,IB法检测GADA阳性患者为56例,ELISA法检测阳性患者为38例,IB法检测的阳性率要明显高于ELISA法,而2种方法检测ICA、IAA的阳性率比较无明显差异.2种方法检测病程≥5年患者GADA、ICA的阳性率均低于病程＜5年患者,但IB法检测GADA的阳性率要高于ELISA法. 结论 IB法在检测老年2型糖尿病患者IAA尤其是GADA的敏感性要优于ELISA法.     

Effect of transplantation site on the results of pancreatic islet isografts in diabetic rats

Summary Isolated pancreatic islets were transplanted isogeneically into the subcutaneus tissue, peritoneum and portal vein of diabetic rats. Implantation of islets subcutaneously did not modify the diabetic state. Intraperitoneal islets ameliorated the effects of diabetes but normal urine volumes, blood glucose and urine glucose levels were not achieved. Direct injection of islets into the portal vein resulted in normal urine volumes, normoglycemia and abolition of glycosuria in the rats studied. These effects have been maintained 2 months later. — It is suggested that the portal environment may be the most effective site for transplanted pancreatic islets.     

Islet cell surface and lymphocyte antibodies often precede the spontaneous diabetes in the BB rat

Summary The diabetic syndrome of the BB rat shows many homologies with that of human insulin-dependent diabetes and evidence that the onset of the disease is associated with the presence of autoantibodies, including islet cell surface antibodies. In this study, sera were sampled serially from weaning to 157 days of age from 26 BB rats in two low-incidence litters, and 22 rats of three high-incidence litters. Clinical and metabolic variables were monitored concurrently with blood lymphocyte counts. Islet morphology was correlated at sacrifice. In the high-incidence litters, eight rats developed insulin-dependent diabetes, five impaired glucose tolerance, and the remaining nine all showed insulitis. In the low-incidence litters, only one animal showed impaired glucose tolerance and another insulitis. In the high-incidence litters 16 rats (73%) had islet cell surface antibodies compared with 4 out of 26 (15%) low-incidence controls (p<0.002). Antibodies reactive with Wistar rat spleen lymphocytes were present in all high-incidence rats compared with 19% (5 out of 26) among the control litters (p<0.002). Time courses of islet cell surface and lymphocyte antibody appearance and their peak values varied, but already at weaning the levels of both antibodies were increased among the high-incidence litter rats (p< 0.001). Islet cell surface and/or lymphocyte antibodies were therefore present in the majority of animals at an age where neither morphological nor metabolic evidence of the diabetic syndrome were yet detected. All rats that showed any form of the syndrome were lymphopenic. These findings suggest that BB rats have an abnormal immune response which predisposes to later development of insulin-dependent diabetes, often preceded by the presence of islet cell surface and/or lymphocyte antibodies.     

Studies on autoimmunity for initiation of Beta-cell destruction VIII. Pancreatic Beta-cell dependent autoantibody to a 38 kilodalton protein precedes the clinical onset of diabetes in BB rats

Summary Autoantibody to a rat islet cell-protein of 38 kilodalton was detectable at around 30 days of age in the sera of diabetes-prone Biobreeding (DP-BB) rats by both immunoprecipitation and differential Western blotting methods. Anti-38 kilodalton islet cell autoantibody was not, however, observed in the sera from 5- to 20-day-old DP-BB rats. Over 90% of DP-BB rats in which the antibody was detected, eventually developed Type 1 (insulin-dependent) diabetes mellitus. The antibody disappeared within 2 weeks after diabetes onset. However, it was preserved in the sera of DP-BB rats which had been treated with silica to prevent insulitis. The anti-38 kilodalton islet cell autoantibody was not detected in sera from control Wistar Furth (WF) rats. The autoantibody also cross-reacted with a rat insulinoma (RINm5F) cell protein of 38 kilodalton, but did not react with protein from mouse fibroblast (L-929 cells), rat pituitary cells (GH3 cells), or normal rat lymphocytes. The production of the autoantibody appears to be pancreatic Beta-cell dependent, since the autoantibody disappears after almost complete depletion of Beta cells, but is consistently present as long as Beta cells remain. Identification of the Beta-cell dependent anti-38 kilodalton islet cell autoantibody, which cross-reacts with a rat insulinoma cell protein of 38 kilodalton and precedes the onset of Type 1 diabetes in BB rats, will be invaluable for study of the molecular nature of a target islet cell autoantigen associated with the induction of autoimmunity in DP-BB rats.     

Antioxidant activity of chito-oligosaccharides on pancreatic islet cells in streptozotocin-induced diabetes in rats

AIM： To investigate the antioxidant activity of chitooligosaccharides （COSs） on pancreatic islet cells in diabetic rats induced by streptozotocin.
METHODS： The antioxidant effect of COSs on pancreatic islet cells was detected under optical microscopy and with colorimetric assay and gel electrophoresis. The activities of glutathione peroxidase and superoxide dismutase, total antioxidant capacity, and content of malondialdehyde in serum and tissue slices of pancreas were examined after 60 d to determine the effect of COSs in streptozotocin-induced diabetes in rats.
RESULTS： COSs can prohibit the apoptosis of pancreatic islet cells. All concentrations of COSs can improve the capability of total antioxidant capacity and activity of superoxide dismutase and decrease the content of malondialdehyde drastically. Morphological investigation in the pancreas showed that COSs have resulted in the reduction of islets, loss of pancreatic cells, and nuclear pyknosis of pancreatic cells.
CONCLUSION： COSs possess various biological activities and can be used in the treatment of diabetes mellitus.     

Apoptosis and disease progression in the spontaneously diabetic BB/S rat

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is an autoimmune disease culminating in pancreatic beta-cell destruction. A role for apoptosis in this destruction has been suggested, although controversy exists over the identity of the apoptotic cells and the time of onset of apoptosis. This study investigates the extent and timing of islet cell apoptosis in vivo in the spontaneously diabetic BB/S rat. Methods. Pancreatic biopsies were taken from 30 diabetes-prone and 6 diabetes-resistant BB/S rats matched for age. Animals were serially biopsied before, during and after development of diabetes and apoptotic cells analysed in serial sections. The diabetes-prone group included animals (n = 6) that had insulitis but did not develop diabetes. Results. Apoptosis was not detected in any pancreatic sections from diabetes resistant animals at any age investigated or from any animal before 50 days of age. By 68 days, apoptosis was, however, detectable in both the diabetes-prone group and in the group that had insulitus but did not develop diabetes and this correlated with a decrease in pancreatic insulin staining and a development of insulitis. There was a further increase in apoptosis in the diabetes-prone group at 85 days, which coincided with the time of onset of diabetes (84 days). In addition, there was a sixfold increase in intra-islet apoptosis between 68 and 85 days in the diabetes-prone group and at 85 days intra-islet apoptosis was threefold higher in the diabetes-prone group than in the group that had insulitus but did not develop diabetes. At 107 days, apoptosis (total and intra-islet) was higher in the group that had insulitus but did not develop diabetes (OND-DP) than in either the diabetes resistant (DR) or diabetes-prone (DP) groups. Conclusion/interpretation. We have shown significant islet cell apoptosis in the pancreas of diabetes-prone BB/S rats, which coincides with the appearance of insulitis and the onset of diabetes. We have also detected differences in the levels of apoptosis between diabetic and non-diabetic animals and suggest that such differences could be an important determinant of disease progression in this animal model of Type I diabetes. [Diabetologia (2001) 44: 320–324] Received: 15 May 2000 and in revised form: 31 October 2000     

A prospective analysis of antibodies reacting with pancreatic islet cells in insulin-dependent diabetic children

Summary Islet cell cytoplasmic and cell surface antibodies along with other autogenic tissue antibodies were determined prospectively from the day of diagnosis of insulin-dependent diabetes in a group of children and adolescents. Prior to the initiation of insulin therapy 30 out of 33 were antibody-positive, 67% having islet cytoplasmic antibodies and 67% islet cell surface antibodies. Among 74 age- and sex-matched non-diabetic individuals 1% had islet cell cytoplasmic antibodies and 3% had islet cell surface antibodies. A prospective analysis in 17 patients showed a diminishing prevalence of islet cell antibodies with increasing duration of diabetes. Islet cell cytoplasmic or cell surface antibodies were found independently of each other or in combination and with various patterns of persistence. The results indicate a strong association of islet cell antibodies with the onset of insulin-dependent diabetes in childhood and adolescence.     

Melatonin prolongs islet graft survival in diabetic NOD mice

Abstract:  Islet transplantation has been established as a potential therapy for type 1 diabetes. However, inflammation, allorejection, and on-going autoimmune damage contribute to early graft loss and failure of islet transplantation. Melatonin is the major secretory product of the pineal gland during the dark period of each day and displays multifunctional properties including the regulation of circadian and seasonal rhythms, antioxidation reactions and immune modulation. Based on the immunosuppressive properties of melatonin, we investigated whether melatonin treatment prolonged the survival of islet grafts in non-obese diabetic (NOD) mice. The mean islet graft survival time was 7.33 ± 1.51 and 7.75 ± 2.66 days in untreated controls and in the solvent-treated animals, respectively. Strikingly, the mean survival time of islet grafts in recipients treated with melatonin (200 mg/kg/bw) was 17 ± 7.76 days. Moreover, melatonin treatment reduced the proliferation of splenocytes in NOD mice. Using a T1 and T2 double transgenic mouse model, we found that T helper 1 (Th1) cells in mice treated with melatonin were significantly decreased. The reduction of Th1 cells and T cell proliferation may result from an increase in the immunosuppressive cytokine IL-10. Our results indicate that melatonin treatment suppresses autoimmune recurrence by inhibiting the proliferation of Th1 cells in NOD mice and thus prolongs the survival of syngeneic islet grafts.     

Human islet cell transplantation--future prospects.

BACKGROUND: Islet transplantation has the potential to cure diabetes mellitus. Nevertheless despite successful reversal of diabetes in many small animal models, the clinical situation has been far more challenging. The aim of this review is to discuss why insulin-independence after islet allotransplantation has been so difficult to achieve. METHODS: A literature review was undertaken using Medline from 1975 to July 2000. Results reported to the International Islet Transplant Registry (ITR) up to December 1998 were also analysed. RESULTS: Up to December 1998, 405 islet allotransplants have been reported the ITR. Of those accurately documented between 1990 and 1998 (n = 267) only 12% have achieved insulin-independence (greater than 7 days). However with refined peri-transplant protocols insulin independence at 1 year can reach 20%. CONCLUSIONS: There are many factors which can explain the failure of achieving insulin-independence after islet allotransplantation. These include the use of diabetogenic immunosuppressive agents to abrogate both islet allo-immunity and auto-immunity, the critical islet mass to achieve insulin-independence and the detrimental effects of transplanting islets in an ectopic site. However recent evidence most notably from the Edmonton group demonstrates that islet allotransplantation still has great potential to become an established treatment option for diabetic patients.     

Transplantation of isologous islets of langerhans in diabetic rats

Summary Isologous isolated islets of Langerhans were transplanted into the peritoneum and, via the portal vein, into the liver of diabetic rats. In both groups almost normal blood glucose and serum insulin levels were achieved for a period of three months. Glucose tolerance tests were markedly improved. Morphological examination of the transplanted islets and immunohistochemical tests for insulin and glucagon showed the liver to be a more suitable site for islet grafting than the peritoneum.     

Islet cell antibodies: variable immunostaining of pancreatic islet cells and carcinoid tissue

Abstract. Objectives. Islet cell antibodies (ICA) in sera of patients with autoimmune diabetes mellitus generally stain not only the insulin-producing beta cells but also the non-beta cells of the islets of Langerhans. The antibodies have been reported to react also with the chromaffin cells of carcinoid tissue. In the present study, we examined in detail the reactivity of 10 ICA-positive sera of patients with new onset insulin-dependent diabetes mellitus (IDDM) and two sera of patients with stiff-man syndrome. Design. The sera were analysed by immunofluorescence and by immunoperoxidase staining of human islets as well as by immunoprecipitations using ³⁵S-methionine labelled rat islet lysates. In addition, immunofluorescence analyses of carcinoid tissues were carried out. Results. Eight of the 10 IDDM-positive sera reacted with all islet endocrine cells, whereas two sera showed staining restricted to the beta cells, as did the two sera of the patients with stiff-man syndrome. All beta cell ‘selective’ sera, but only 6 of 8 ‘whole’ islet positive sera, immunoprecipitated the 64 kDa glutamic acid decarboxylase (GAD) antigen. The staining of carcinoid tissue was variable and did not correlate with the ‘whole’ or ‘selective’ staining pattern of islets. Conclusion. The data underline a heterogeneity of ICA and indicate the presence of a separate, non-GAD antigen in islet cells. It is possible that in future studies, a resolution of ICA titres with respect to different types of islet cellular reactivity might provide insights into the pathogenesis of IDDM and improve the prognostic implications of antibody determinations.