Resistance to teratogenesis by F1 and F2 embryos of PAH-adapted Fundulus heteroclitus is strongly inherited despite reduced recalcitrance of the AHR pathway.

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Superfund site on the Elizabeth River (Portsmouth, VA, USA) are exposed to a complex mixture of polycyclic aromatic hydrocarbons (PAHs) from former creosote operations, but are resistant to the acute toxicity and cardiac teratogenesis caused by PAHs. The resistance is associated with a dramatic recalcitrance to induction of cytochrome P450 (CYP1) metabolism enzymes following exposure to aryl hydrocarbon receptor (AHR) agonists, along with an elevated antioxidant response and increased expression of several other xenobiotic metabolism and excretion enzymes. However, the heritability of the resistance in the absence of chemical stressors has been inconsistently demonstrated. Understanding the heritability of this resistance will help clarify the nature of population-level responses to chronic exposure to PAH mixtures and aid in identifying the important mechanistic components of resistance to aryl hydrocarbons. We compared the response of Atlantic Wood F1 and F2 embryos to benzo[k]fluoranthene (BkF), benzo[a]pyrene (BaP), 3,3',4,4',5-pentachlorobiphenyl (PCB-126), and a mixture of BkF and fluoranthene (Fl) to that of F1 embryos of reference site killifish. Resistance to cardiac teratogenesis and induction of CYP mRNA expression and CYP activity was determined. We found that both Atlantic Wood F1 and F2 embryos were highly resistance to cardiac teratogenesis. However, the resistance by Atlantic Wood F2 embryos to induction of CYP mRNA expression and enzyme activity was intermediate between that of Atlantic Wood F1 embryos and reference embryos. These results suggest that resistance to cardiac teratogenesis in Atlantic Wood fish is conferred by multiple factors, not all of which appear to be fully genetically heritable.

Expression of the enteroviral capsid protein VP1 in the islet cells of patients with type 1 diabetes is associated with induction of protein kinase R and downregulation of Mcl-1.

AIMS/HYPOTHESIS: Immunohistochemical staining reveals that the enteroviral capsid protein VP1 is present at higher frequency in the insulin-containing islets of patients with recent-onset type 1 diabetes than in controls. This is consistent with epidemiological evidence suggesting that enteroviral infection may contribute to the autoimmune response in type 1 diabetes. However, immunostaining of VP1 is not definitive since the antibody widely used to detect the protein (Clone 5D8/1) might also cross-react with additional proteins under some conditions. Therefore, we sought to verify that VP1 immunopositivity correlates with additional markers of viral infection. METHODS: Antigen immunoreactivity was examined in formalin-fixed, paraffin-embedded, pancreases from two different collections of type 1 diabetes and control cases: a historical collection from the UK and the nPOD (network of Pancreatic Organ donors with Diabetes) cohort from the USA. RESULTS: VP1 immunoreactivity was present in ~20% of insulin-containing islets of both cohorts under stringent conditions but was absent from insulin-deficient islets. The presence of VP1 was restricted to beta cells but only a minority of these contained the antigen. The innate viral sensor, protein kinase R (PKR) was upregulated selectively in beta cells that were immunopositive for VP1. The anti-apoptotic protein myeloid cell leukaemia sequence-1 (Mcl-1) was abundant in beta cells that were immunonegative for VP1 but Mcl-1 was depleted in cells containing VP1. CONCLUSIONS/INTERPRETATION: The presence of immunoreactive VP1 within beta cells in type 1 diabetes is associated with a cellular phenotype consistent with the activation of antiviral response pathways and enhanced sensitivity to apoptosis. However, definitive studies confirming whether viral infections are causal to beta cell loss in human diabetes are still awaited.

Immunohistochemical analysis of the relationship between islet cell proliferation and the production of the enteroviral capsid protein, VP1, in the islets of patients with recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: The enteroviral capsid protein, VP1, was recently shown to be present in some beta cells in more than 60% of patients with recent-onset type 1 diabetes but in very few age-matched controls. The rate of proliferation of islet cells was also markedly increased in the type 1 diabetic patients. As it has been suggested that enteroviruses replicate most efficiently in proliferating cells, we have investigated whether VP1 is preferentially present in proliferating beta cells in type 1 diabetes. METHODS: Combined immunoperoxidase and immunofluorescence staining was used to record the presence of enteroviral VP1, insulin and Ki67 in the islets of recent-onset type 1 diabetic patients. RESULTS: From a total of 1,175 islets, 359 (30.5%) contained insulin. VP1-producing endocrine cells were found in 72 islets (6.1% of total), all of which retained insulin. Ki67(+) endocrine cells were present in 52 (4.4%) islets, with 44 (84.6%) of these being insulin-positive. Overall, 28 of 1,175 (2.4%) islets contained both Ki67(+) cells and VP1(+) cells. Dual positivity of these markers accounted for 38.9% of the total VP1(+) islets and 53.8% of the total Ki67(+) islets. No individual islet cells were dual-positive for Ki67 and VP1. CONCLUSIONS/INTERPRETATION: Ki67(+) cells were frequently observed in islets that also contained VP1(+) cells, suggesting that the factors facilitating viral replication may also drive islet cell proliferation. However, in an individual cell, VP1 production does not require concurrent beta cell proliferation.

Evidence of increased islet cell proliferation in patients with recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: In adults, the rate of beta cell replication is normally very low, but recent evidence suggests that it may increase during insulitis. We therefore studied tissue from donors with recent-onset type 1 diabetes to establish whether islet cell proliferation is increased during the disease process. METHODS: Paraffin-embedded pancreatic sections from ten donors with recent-onset type 1 diabetes and a range of relevant controls were stained by immunohistochemical techniques with antibodies against the proliferation markers Ki67 and minichromosome maintenance protein-2 (MCM-2). A combination staining technique involving immunoperoxidase and immunofluorescence methods was developed to quantify the numbers of alpha and beta cells with Ki67-positive nuclei and to investigate the relationship between insulitis and islet cell proliferation. RESULTS: In non-diabetic control donors, only 1.1 +/- 0.3% (mean +/- SEM) of islets contained one or more Ki67(+) islet cells, whereas this proportion was increased markedly in recent-onset type 1 diabetes (10.88 +/- 2.5%; p < 0.005). An equivalent increase in Ki67(+) staining occurred in alpha and beta cells and was correlated positively with the presence of insulitis. A significant increase in the labelling of islet cells from type 1 diabetic donors was also seen when MCM-2 staining was employed. Increased islet cell proliferation was not evident in three donors with longer duration type 1 diabetes or in ten type 2 diabetic donors. CONCLUSIONS/INTERPRETATION: Alpha and beta cells undergo a marked increase in proliferation during the progression of type 1 diabetes in humans. The results imply that islet cell proliferation is re-initiated in response to the autoimmune attack associated with type 1 diabetes.

The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes.

AIMS/HYPOTHESIS: Evidence that the beta cells of human patients with type 1 diabetes can be infected with enterovirus is accumulating, but it remains unclear whether such infections occur at high frequency and are important in the disease process. We have now assessed the prevalence of enteroviral capsid protein vp1 (vp1) staining in a large cohort of autopsy pancreases of recent-onset type 1 diabetic patients and a range of controls. METHODS: Serial sections of paraffin-embedded pancreatic autopsy samples from 72 recent-onset type 1 diabetes patients and up to 161 controls were immunostained for insulin, glucagon, vp1, double-stranded RNA activated protein kinase R (PKR) and MHC class I. RESULTS: vp1-immunopositive cells were detected in multiple islets of 44 out of 72 young recent-onset type 1 diabetic patients, compared with a total of only three islets in three out of 50 neonatal and paediatric normal controls. vp1 staining was restricted to insulin-containing beta cells. Among the control pancreases, vp1 immunopositivity was also observed in some islets from ten out of 25 type 2 diabetic patients. A strong correlation was established between islet cell vp1 positivity and PKR production in insulin-containing islets of both type 1 and type 2 diabetic patients, consistent with a persistent viral infection of the islets. CONCLUSIONS/INTERPRETATION: Immunoreactive vp1 is commonly found in the islets of recent-onset type 1 diabetes patients, but only rarely in normal paediatric controls. vp1 immunostaining was also observed in some islets of type 2 diabetes patients, suggesting that the phenomenon is not restricted to type 1 diabetes patients.

Analysis of islet inflammation in human type 1 diabetes.

The immunopathology of type 1 diabetes (T1D) has proved difficult to study in man because of the limited availability of appropriate samples, but we now report a detailed study charting the evolution of insulitis in human T1D. Pancreas samples removed post-mortem from 29 patients (mean age 11.7 years) with recent-onset T1D were analysed by immunohistochemistry. The cell types constituting the inflammatory infiltrate within islets (insulitis) were determined in parallel with islet insulin content. CD8(+) cytotoxic T cells were the most abundant population during insulitis. Macrophages (CD68(+)) were also present during both early and later insulitis, although in fewer numbers. CD20(+) cells were present in only small numbers in early insulitis but were recruited to islets as beta cell death progressed. CD138(+) plasma cells were infrequent at all stages of insulitis. CD4(+) cells were present in the islet infiltrate in all patients but were less abundant than CD8(+) or CD68(+) cells. Forkhead box protein P3(+) regulatory T cells were detected in the islets of only a single patient. Natural killer cells were detected rarely, even in heavily inflamed islets. The results suggest a defined sequence of immune cell recruitment in human T1D. They imply that both CD8(+) cytotoxic cells and macrophages may contribute to beta cell death during early insulitis. CD20(+) cells are recruited in greatest numbers during late insulitis, suggesting an increasing role for these cells as insulitis develops. Natural killer cells and forkhead box protein P3(+) T cells do not appear to be required for beta cell death.

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.

Insulitis and mechanisms of disease resistance: studies in an animal model of insulin dependent diabetes mellitus.

Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease characterised by extreme insulin deficiency due to an overall decrease in the mass of properly functioning beta-cells. This reduction occurs as a result of insulitis. the outcome of which will depend upon the intensity of the cytotoxic attack and the ability of beta-cells to resist and repair immune mediated cell damage. To further elucidate the relationship between the insulitis process and beta-cell defence and repair mechanisms in the prevention of diabetes we have studied a unique subgroup of diabetes prone (DP) BB/S rats which have demonstrated an ability to recover from IDDM (BB/S-R). Animals were diagnosed as diabetic at 115 days of age, subsequently receiving insulin therapy (1.49+/-0.1 IU/day) for a total of 19.7 days during 1 to 4 episodes of IDDM. Following a prolonged symptom-free period of 90 days, an IPGTT revealed that BB/S-R rats possessed normal glycaemic control. Islets were isolated from the BB/S-R rats and their glucose-stimulated insulin response was shown to be comparable to Wistar control islets. Furthermore, control and BB/S-R islets showed both a similar structural integrity and insulin content. BB/S-R islets cultured for 24 hr in IL-1beta (10(-13) M) maintained a significant insulin secretory response to glucose in contrast to Wistar controls in which the response was completely inhibited. Nitrite production was induced by IL-1beta, in a dose-dependent manner, in control islets whereas there was no significant increase in production in the islets of BB/S-R rats. These findings suggest that previous immune directed beta-cell attack may induce a state of increased resistance to subsequent deleterious effects of cytokine-mediated cytotoxicity. Overall therefore, the present study shows how the "recovered" BB/S-R rat model provides a unique opportunity to assess the direct effects of insulitis on pancreatic islets and how this interaction may subsequently determine disease outcome.

Tumor necrosis factor-alpha and interferon-gamma inhibit insulin secretion and cause DNA damage in unweaned-rat islets. Extent of nitric oxide involvement.

Nitric oxide has been implicated as one possible mediator of interleukin-1 beta (IL-1)-induced inhibition of insulin secretion and islet cell damage. The aim of this study was to define the effects of tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN) on nitric oxide production, insulin secretion, and DNA damage in islets from unweaned rats. Treatment of islets with 0.5-500 U/ml of either TNF or IFN on their own inhibited glucose-stimulated insulin secretion in a dose-dependent manner (minimum effective dose 5 U/ml). In combination, the cytokines exerted a pronounced synergistic inhibitory effect on secretion and were equipotent at causing a significant and concentration-dependent increase in culture medium nitrite levels, islet cyclic GMP formation, and DNA damage. Used alone or in combination, TNF and IFN significantly enhanced the activity of inducible nitric oxide synthase as determined by measuring the conversion of 14C-labeled arginine to 14C-labeled citrulline and nitric oxide. Use of arginine-free medium, without or with NG-monomethyl-L-arginine, resulted in inhibition of nitrite formation by 5-1,000 U/ml IFN+TNF and partial restoration of the insulin secretory response to glucose. Treatment of rat islets with increasing doses of TNF+IFN (5, 50, and 500 U/ml) resulted in a progressive increase in DNA damage, as shown by the comet assay, which detects DNA strand breaks in individual islet cells. The DNA damage caused by an intermediate concentration (50 U/ml) of TNF+IFN was comparable to that generated by IL-1 when used at 20 U/ml. We conclude that TNF and IFN induce nitric oxide formation, which partially inhibits glucose-induced insulin secretion and causes significant DNA strand breakage, but that as cytokine concentrations increase, non-nitric-oxide-mediated events predominate.

Serum biopterin--a novel marker for immune activation during pre-diabetes in the BB rat.

Tetrahydrobiopterin (BH4) is a pteridine product which is released by rodent macrophages on activation by cytokines. We have used serial pancreatic biopsy, and measurement of serum biopterin at 30, 60, 90 and 120 days in the BB/S rat to relate histological change to macrophage activation during the course of pre-diabetes. Using immunohistochemistry, and an arbitrary scoring system read blind and standardised against day 30, we found that pancreatic MHC class I, MHC class II and infiltrating macrophage staining were up-regulated in the BB/S diabetes-prone rats (n = 17) at day 60, markedly so at day 90, and less so at day 120. Staining for resident pancreatic macrophages remained unchanged throughout in diabetes prone, diabetes resistant and Wistar (n = 28) control animals. Serum biopterin fell progressively and identically with age in BB diabetes resistant rats (n = 11) and Wistar controls. No change in weight gain or biopterin levels was observed in the biopsied animals. Mean serum biopterin levels in diabetes prone rats (of which 13 of 17 became diabetic at median 85 days) were the same as in diabetes resistant and Wistar rats at days 30, 60 and 120, but showed a striking and highly significant elevation (p < 0.001) at day 90. Although macrophages infiltrate the islet early in pre-diabetes, the timing of their activation is unknown. The rise in biopterin we observed is a potentially important immunological event which occurred late in the progression of pre-diabetes. This acute terminal event has not been reported previously, and may modify current concepts concerning the tempo of cell destruction during pre-diabetes.

Metabolic control in streptozotocin diabetic rats following transplantation of microencapsulated pancreatic islets.

Microencapsulated islet grafts implanted into the peritoneal cavity of a variety of animal models of diabetes have been shown to reverse hyperglycaemia over prolonged periods without immunosuppression. Here, effects of these grafts on intermediary metabolites, diurnal blood glucose and glycated haemoglobin were studied in streptozotocin-diabetic Wistar rats. Following transplantation (approximately 3000 islets) glucose and the ketone 3-hydroxybutyrate fell significantly (glucose: 19.1 +/- 0.6 (SD) to 9.2 +/- 4.3 mmol/l, p < 0.01; 3-hydroxybutyrate: 1.51 +/- 0.48 to 0.55 +/- 0.38 mmol/l, p < 0.02) and remained within/close to the normal range for at least four weeks. In control diabetic animals, values remained abnormally elevated. There was no difference in lactate, alanine or glycerol between the two groups. In transplanted animals there was a marked variation in blood glucose over a 24h period, values being low during daylight hours but with nocturnal peaks (up to 25 mmol/l) during the animals' normal feeding time. Glycated haemoglobin was also lower in transplanted animals but did not return to normal and the difference was not significant. In conclusion, microencapsulated islet grafts ameliorated the diabetic state. However, normal metabolic homeostasis was not achieved. The intraperitoneal site precludes direct graft vascular access and this may be a contributory factor. Additionally, daytime blood sugar values in murine models of diabetes may be a poor guide to graft function and glucose tolerance.

Expression of an islet regenerating (reg) gene in isolated rat islets: effects of nutrient and non-nutrient growth factors.

The expression of a novel regenerating (reg) gene has been reported previously in the regenerating islets of a surgical model of diabetes in rats. We exposed collagenase-isolated rat islets for three days to nutrient and non-nutrient growth factors in minimally supplemented RPMI medium (2.7 mmol/l glucose, 2% fetal calf serum), and investigated the relationship between reg gene expression and islet cell replication. RNA was prepared from half of the islets by homogenisation in guanidinium isothiocyanate followed by phenol/chloroform extraction. Northern/dot blot analyses were used to semi-quantify reg mRNA. Islet cell replication was estimated by culturing the remaining islets in radiolabelled thymidine to determine de novo DNA synthesis. Thymidine uptake was stimulated by the following factors: 11 mmol/l glucose (50% increase); 10% amino acids (126% increase); 10% fetal calf serum (39% increase); 100 ng/ml insulin (45% increase); 250 ng/ml growth hormone (65% increase); 1.5 nmol/l aldosterone (29% increase); 2 U/ml platelet derived growth factor (116% increase). The results are expressed as a percentage of the thymidine incorporated into control islets cultured in minimal RPMI (1118 +/- 100 (SD) cpm/microgram protein, n = 15). Increased islet cell replication was paralleled in each case by a clear rise in reg mRNA expression compared to controls. Furthermore, the rank order for reg gene expression was the same as that for thymidine uptake (r = 0.90). The present findings suggest a clear association between reg gene expression and islet cell replication in vitro, and are the first to demonstrate reg gene expression in response to individual growth factors.

Insulin-mimicking anti-idiotypic antibodies in development of spontaneous autoimmune diabetes in BB/E rats.

BB/E rats spontaneously develop a form of autoimmune diabetes resembling insulin-dependent diabetes mellitus (IDDM) in humans. IDDM results from central destruction of the insulin-producing beta-cells of the pancreatic islets. Herein, we report that the outbreak of IDDM in BB/E rats is preceded by the spontaneous development of an anti-idiotypic antibody to a particular antibody to insulin made by the rats. This anti-idiotype, designated anti-DM-id, behaves as an antibody to the insulin-hormone receptor. Thus, a spontaneous anti-idiotypic antibody network whose products can affect the peripheral utilization of insulin seems to accompany the central destruction of beta-cells in developing IDDM.

Comparison of an enzyme-linked immunosorbent assay (ELISA) with a radioimmunoassay (RIA) for the measurement of rat insulin.

A recently developed competitive enzyme-linked immunosorbent assay (ELISA) was compared with a conventional competitive radioimmunoassay (RIA) for the measurement of rat insulin in culture medium. Fifty-six samples were analysed by both assays. There was a correlation coefficient of r = 0.783 between results obtained using the two assay systems. The binding curves of the two assays were differently shaped, so that the ELISA gave good reproducibility over the concentration range 5-50 microU/ml insulin with inter- and intra-assay coefficients of variation less than 14%, but poor reproducibility at higher concentrations. Conversely, the RIA showed excellent reproducibility at concentrations greater than 50 microU/ml insulin, but poor sensitivity and high coefficients of variation below this level. The ELISA procedure offers practical advantages over the RIA, and performs well when measuring physiological concentrations of insulin.

Increased preproinsulin mRNA in pancreatic islets incubated with islet cell-stimulating antibodies from serums of type I diabetic patients.

We recently described autoantibodies that stimulate the release of insulin from pancreatic beta-cells both in vitro and in vivo. The aim of this study was to establish whether islet cell-stimulating antibodies (ICSTAs) also increase islet cell preproinsulin mRNA content. Wistar rat islets, isolated by collagenase digestion, were exposed to 2.7 and 11.1 mM glucose. Insulin release increased 10-fold in response to the higher glucose concentration, and dot-blot analysis of islet mRNA with a rat preproinsulin cDNA probe showed a concomitant increase in mRNA levels. The globulin fractions of four test serums, three from patients with type I (insulin-dependent) diabetes and one from a patient with the insulin autoimmune syndrome, showed clear (5- to 8-fold) stimulation of insulin release. The nonglobulin fractions of these serums and both fractions of three control serums failed to stimulate secretion of insulin. The insulin mRNA content of islets incubated with the ICSTA globulin fractions was greatly increased compared with levels observed in islets treated with control serum globulin fractions. We conclude that ICSTAs not only can stimulate the release of insulin but also increase the preproinsulin mRNA content of islet cells.

HbA1 in assessment of metabolic control in diabetic BB/E rats.

Estimations of HbA1 levels have been used to assess long-term glycaemic control in spontaneously diabetic BB/E rats. The degree of metabolic control achieved by once daily insulin injections and continuous insulin infusion by osmotic minipump was compared. Citrate gel electrophoresis of lysed erythrocytes, previously washed and incubated in 0.9% NaCl, gave accurate HbA1 values without interference from either abnormal Hb variants or labile glycosylation products. Over a 12 week period there was no significant difference in the mean random weekly plasma glucose concentrations between diabetic rats maintained on insulin injections or continuous infusion therapy. The HbA1 values in the injection-treated animals remained unchanged throughout the study period (mean +/- SEM = 5.1 +/- 0.1%). Diabetic rats treated by osmotic minipump showed a steady decline in values over the same period (4.1 +/- 0.1%; p less than 0.001 vs injected rats) but levels remained higher than those recorded in non-diabetic control rats (2.9 +/- 0.01%; p less than 0.001 vs pump-treated rats). These differences in HbA1 were reflected in the plasma glucose values obtained during a 30 h glucose profile performed after six weeks of insulin therapy. Diabetic rats on injection therapy showed considerable diurnal variation in plasma glucose concentration (5.5-11.2 mmol/l; mean 8.9 +/- 0.5) but continuous insulin infusion eliminated the fluctuations giving a significantly lower mean glucose level over the 30 h period (7.3 +/- 0.1 mmol/l; p less than 0.005). HbA1 levels show a poor correlation with random plasma glucose estimations (r = 0.43) but provide a simple and accurate assessment of long-term glycaemic control without the need for multiple 24 h glucose profiles.

Effect of cyclosporin on pancreatic events and development of diabetes in BB/Edinburgh rats.

The effect of cyclosporin administered from 30 to 100 days of age on pancreatic events and the development of insulin-dependent diabetes has been studied by serial pancreatic biopsy of individual diabetes-prone BB/Edinburgh rats. Cyclosporin completely prevented the development of diabetes up to 150 days of age and reduced the incidence to 50% of controls at 452 days of age. Islet cell surface antibodies paralleled the development of diabetes. Insulin autoantibodies were unrelated to diabetes and not affected by cyclosporin. Immunohistochemical analysis of pancreatic biopsies from untreated control diabetes-prone rats with monoclonal antibodies specific for rat MHC molecules and T- and B-lymphocyte and macrophage subsets showed that the first abnormality seen in rats that subsequently developed diabetes was hyperexpression of MHC class I molecules on vascular endothelium and islet cells. This was followed by accumulation of ED1+ macrophages at perivascular and periductal sites adjacent to noninfiltrated islets. Increased expression of MHC class II molecules on vascular endothelial cells was also noted. Most cells infiltrating the islets initially were also ED1+ macrophages, followed by increasing numbers of other activated effector cells including helper and cytotoxic-suppressor T lymphocytes and natural killer cells. Obliteration of insulin-containing cells was associated with regression of the infiltrate. Treatment with cyclosporin had no effect on pancreatic hyperexpression of MHC class I molecules but markedly inhibited accumulation of ED1+ cells at extraislet sites, the subsequent recruitment of immune effector cells, and islet infiltration. This resulted in a delay of the onset of diabetes in some rats and prevention of diabetes in others.