Suppressive effect of antibodies to immune response gene products on the development of low-dose streptozotocin-induced diabetes.

Low-dose streptozotocin-induced diabetes in mice serves as a model of type I diabetes. Suppression of the development of diabetes (hyperglycemia) in C3H/He mice was achieved with in vivo administration of antibody reactive to Ir-gene products before streptozotocin treatment. A persistent effect was reached with two monoclonal antibodies directed against I-Ak gene products and, surprisingly, by an allo-antiserum to I-J determinants. These results suggest a role for I-A and I-J positive T-lymphocytes and/or macrophages in B-islet cell autoimmunity.

Administration of silica prevents diabetes in BB-rats.

Administration of silica to young BB-rats almost completely prevented the development of spontaneous diabetes. Only 1 of 31 silica-treated rats developed hyperglycemia, whereas 9 of 31 in the untreated group did so. Since silica is highly specific in its action against macrophages, our observations indicate an important role of these cells in the pathogenesis of the disease.

Low-dose streptozocin-induced diabetes in mice. Electron microscopy reveals single-cell insulitis before diabetes onset.

We investigated the morphology of mouse islets 5 days after completion of low-dose streptozocin treatment of C57BL/6 mice by electron microscopy. At this stage, mice were still normoglycemic and light microscopy did not reveal massive islet infiltration. The electron-microscopic investigation revealed two characteristics indicative of ongoing islet cell destruction. In all islets investigated, lysed islet beta-cells were recognized by disrupted plasma membranes and concomitantly decreased plasma contrast. Many of the lysed islet beta-cells still contained numerous insulin granules. We also found immunocytes scattered throughout the islets, most of which could be identified as macrophages. Some were found engaged in phagocytosis of islet beta-cell debris. This early stage of islet lesion termed single-cell insulitis is followed by the well-known later stage of massive infiltration easily recognized in light microscopy. Administration of silica particles to mice treated with low-dose streptozocin inhibited macrophage infiltration of islets as shown by immunocytochemistry with macrophage-specific monoclonal antibody F4/80. In parallel, the development of hyperglycemia was suppressed. The observations favor a pathogenic role of macrophages in islet destruction.

Induction of experimental autoimmune diabetes by low-dose streptozotocin treatment in genetically resistant mice.

We have studied the effect of suppressor cell elimination on the induction of experimental autoimmune diabetes in mouse strains which are normally low or intermediate responders to multiple low-dose streptozotocin treatment. BALB/c (low responder) and C57BL/6J (intermediate responder) mice received 70 mg cyclophosphamide/kg, 1 or 6 days before the onset of streptozotocin injections. Following cyclophosphamide treatment, BALB/c mice become susceptible to the diabetogenic effect of streptozotocin. Similarly the manifestation of diabetes in C57BL/6J mice is enhanced. Thus in both strains immunomodulation by cyclophosphamide treatment significantly increases the susceptibility towards the diabetogenic effect of streptozotocin. We therefore conclude that in mice of strains BALB/c and C57Bl/6J suppressor cells control the level of resistance towards the induction of experimental autoimmune diabetes by low-dose streptozotocin treatment.

Administration of silica or monoclonal antibody to Thy-1 prevents low-dose streptozotocin-induced diabetes in mice.

Multiple injections of low doses of streptozotocin induce an experimental diabetes in mice. We have analyzed in two inbred strains whether the development of hyperglycaemia can be influenced by administration of macrophage-toxic silica particles or by a monoclonal antibody to Thy-1.2. Mice received streptozotocin (30 or 40 mg/kg) on five consecutive days (day 0-day 4) and in addition either silica particles (starting at day 0) or anti-Thy-1.2 (starting at day -2 or -3). In both strains mice receiving streptozotocin alone became hyperglycaemic within two weeks. Additional treatment with silica almost fully prevented diabetes development. Anti-Thy-1.2 administration was similarly effective in C57B1/Ks and partially protective in C57BL/6 mice. Histological analysis of pancreatic islets showed that a large fraction of beta cells had been spared from destruction by this treatment. The data indicate a role for both macrophages and Thy-1 positive cells in the pathogenesis of low-dose streptozotocin-induced diabetes.

Autoimmune T-lymphocytes with specificity for pancreatic islet antigens.

The question is still unresolved whether in insulin-dependent (type I) diabetes T-lymphocytes mediate an autoimmune response towards pancreatic islets. We now present direct evidence in an animal model for autoimmune T-lymphocytes with specificity for islet antigens. Mice of strain C57BL/6 were immunized repeatedly with islet homogenates. Lymphocytes isolated from draining lymph nodes and the spleen show a specific proliferative response when challenged with mouse rat or islet antigens but do not respond to liver or spleen antigens. Islet antigen specific lymphoblasts have been maintained by in vitro culture for several weeks in the presence of T-cell growth factor containing medium.

HLA-DRB1,3,4,5 and -DQB1 allele frequencies and HLA-DR/DQ linkage disequilibrium of 231 German caucasoid patients and their corresponding 821 potential unrelated stem cell transplants.

Allelic matching within the HLA-DRB1 and -DQB1 loci significantly improves the clinical outcome of hematopoietic stem cell transplantation. Consequently, allelic typing of these loci is strongly recommended for the unrelated stem cell donor selection. In this study, the HLA-DRB1,3,4,5 and -DQB1 alleles of 231 patients and their corresponding 821 nonrandom potential stem cell donors were determined to define compatible donor/recipient pairs. Highly accurate HLA typing data were achieved by PCR-SSOP and a combination of group specific PCR-SSP and subsequent sequencing-based typing of nearly the whole second exon of each locus. The alleles DRB1*07, *09, and *10 were analyzed by PCR-reverse dot blot hybridization instead of sequencing. Additionally, DRB1 homozygosity was verified by temperature gradient gel electrophoresis. The identified 2104 HLA-DRB1 and HLA-DQB1 alleles as well as data on HLA-DRB3, -DRB4, and -DRB5 alleles were applied to a statistical program and absolute and relative delta values of DR/DQ linkages were calculated. The achieved data on the HLA-DRB1 allele distribution and on DR/DQ associations in terms of subtypes significantly ensure the typing reliability, since rare allele combinations will result in further investigations. Furthermore, detailed data on the DR/DQ allele associations allow estimations of the number of HLA-A, -B, and -DR matched unrelated stem cell donors necessary for the identification of DRB and DQB subtype identical donors.

The time course of insulin autoantibodies (IAA) in the diabetes prone BB rat.

The BB rat is a widely used animal model for the study of insulin dependent diabetes. An enzyme linked immunosorbent assay, using purified rat insulin, was used to measure serial insulin autoantibodies (IAA) levels in coded sera from the BB/W/D rat colony in order to establish the time course of IAA. The animals included 26 diabetes-prone BB rats, six diabetes-resistant BB rats and six Wistar controls. There was an increase in both IAA frequency and titre with time in the diabetes-prone group: none were positive at 45 days, 17/19 (89%) were positive by day 90 and all were positive thereafter. Similar results were observed in the diabetes-resistant BB group (0/6 positive at day 51, 6/6 positive at day 90). None of the Wistar controls were positive at 105 days, although occasional positive sera were observed at 120 days. IAA seem to be acquired early on in the majority of BB rats, both diabetes-prone and diabetes-resistant, and much later, if at all, in controls. A clear homology of the MHC genes exists in both BB rat sublines, thus IAA appear to be a strain related phenomenon rather than a marker for IDDM.

Donor work-up and transport of bone marrow--recommendations and requirements for a standardized practice throughout the world from the Donor Registries and Quality Assurance Working Groups of the World Marrow Donor Association (WMDA).

In October 1995 the World Marrow Donor Association (WMDA) was restructured in order to facilitate its primary function of establishing guidelines in relation to international bone marrow and blood stem cell transplants -- transplants in which the donor is in one country and the patient is in another country. Five new working groups were established -- Donor Registries, Ethics, Quality Assurance, Finances, and Stem Cells. This paper, prepared by members of the Donor Registries Working Group, in consultation with the Quality Assurance Working Group, provides recommendations for the 'donor work-up'. This term covers events that start when the definitive donor has been identified, includes the harvesting (collection) and transportation of the stem cell product and ends when the product reaches the transplant centre. The paper includes examples of the documentation intended to ensure compliance with the recommendations at all key points in the sequence.

Methimazole treatment aggravates low-dose streptozotocin-induced diabetes.

Treatment of mice with methimazole was found to modulate diabetes development following low-dose (5 x 40 mg/kg body weight) streptozotocin administration. The administration of 0.2 or 1 mg methimazole per kg body weight for 1-3 weeks significantly enhanced hyperglycemia. The enhancing effect of methimazole was also seen when administration began only after termination of streptozotocin injections. Methimazole treatment did not potentiate diabetes induced by a single high dose of streptozotocin (175 mg/kg). Serum thyroxin levels were not affected due to the short period of thyrostatic treatment. Semiquantitative immunocytochemistry of inflamed islets did not show a stronger influx of immune cells but rather a high activation state of infiltrated macrophages (M1/70 positive). We conclude that methimazole enhances the development of immune-mediated diabetes.

Suppression of low dose streptozotocin induced diabetes in mice by administration of a nitric oxide synthase inhibitor.

Nitric oxide has recently been identified as the primary toxic effector molecule in the lysis of islet cells by inflammatory macrophages. We show here that N-nitro-L-arginine-methylester (NAME), an inhibitor of endothelial and macrophage NO synthase partially suppresses diabetes development in the low dose streptozotocin induced diabetes model in C57BL/6J mice. Mean blood glucose levels were lower in the group receiving NAME throughout the observation period of 30d (p less than 0.05-0.001). Similar concentrations of NAME as expected in vivo were tested in vitro in macrophage-islet cell cocultures and were found to partially suppress NO production and islet cell lysis. We conclude that NO synthase activity is a pathogenetic factor in diabetes development.

Low-dose streptozotocin-induced autoimmune diabetes is under the genetic control of the major histocompatibility complex in mice.

In mice, an experimental autoimmune diabetes can be induced by multiple injections with low doses of streptozotocin. Since different mouse strains show a varying susceptibility towards this treatment, we have examined whether the experimental autoimmune diabetes is under the genetic control of the major histocompatibility complex (H-2 complex). Mice of five congenic resistant strains, differing in their genome only at the H-2 region, were identically treated on five consecutive days with 40 mg streptozotocin/kg body weight. Genes at the H-2 complex were found to determine the susceptibility towards the diabetogenic effect of streptozotocin: mice of H-2 haplotype k (B10.BR) developed persistent and strong hyperglycaemia (blood glucose approximately 17 mmol/l), mice of strain B10.A (H-2a), C57BL/10 (H-2b) and B10.D2 (H-2d) reacted with moderate hyperglycaemia (between 11.5 and 15.5 mmol/l), whereas mice of strain B10.S (H-2s) were resistant to the diabetogenic effect of low-dose streptozotocin except for a small and transient rise of blood glucose levels. It is concluded that genes within the major histocompatibility complex affect the diabetogenic response to multiple low-dose streptozotocin treatment.

Strain dependency of the transfer of experimental immune insulitis in mice.

Multiple treatment with low doses of streptozotocin induces hyperglycaemia with concomitant lymphocytic infiltrations into pancreatic islets (insulitis) in several mouse strains. The transfer of cellular immune reactions against islet cells by means of spleen cells was tested in two congeneic and five allogeneic strain combinations. Donor mice were treated on 5 consecutive days with 40 mg streptozotocin per kg body weight. Three weeks later, 5 x 10(7) live spleen cells were transferred into thymusless recipient mice. Insulitis which had developed in about 70% of the donors was only transferable from C57B1/6J to congeneic thymusless mice. In a second congeneic and in all allogeneic strain combinations, cellular immune reactions against pancreatic islets could not be transferred. In none of the recipients of spleen cells from diabetic donors was hyperglycaemia observed. As streptozotocin-induced cellular immune reactions against pancreatic islet cells were only transferable in one congeneic and in no allogeneic strain combinations, it is concluded that there is a genetic restriction both on the levels of donor and recipient mice.

Transfer of experimental autoimmune insulitis by spleen cells in mice.

We have tested whether experimental insulitis induced by multiple subdiabetogenic injections of streptozotocin can be transferred by lymphocytes to normal recipients. C57BL/6J mice were treated on 5 consecutive days with 40 mg streptozotocin/kg body weight. 5 X 10(7) nucleated spleen cells from 20 animals which had developed hyperglycaemia with concomitant insulitis three weeks after the first streptozotocin-injection, were transferred into congenic thymusless C57BL/6J-nu/nu mice. The cell transfer led to lymphocytic infiltrations of pancreatic islets in 75% of the recipients. Hyperglycaemia was not observed. It is concluded that low-dose streptozotocin treatment induces cellular immune reactions against pancreatic islets.

Spontaneous autoimmune reactions against pancreatic islets in mouse strains with generalized autoimmune disease.

The spontaneously autoimmune mouse strains NZB, NZB X NZW, MRL and BXSB have been examined for signs of autoimmune reactions against islet cells. Between 15 and 55 animals of each strain were tested. Infiltrates of lymphocytes and fibroblasts into pancreatic islets were found in more than 80% of NZB mice, in about 50% of MRL and NZB X NZW mice, and in less than 20% of BXSB mice. Infiltrates were not found in the exocrine portion of pancrea. All NZB mice had abnormal glucose tolerance. In the three other strains between 20 and 50% of animals had abnormal glucose tolerance. All mice had fasting normoglycaemia. The lesions in NZB mice were studied in more detail. It was found by ultrastructural analysis that in young mice pancreatic infiltrates consisted of lymphocytes and fibroblasts. Single lymphocytes were also seen outside the main infiltration area. After 2 to 5 months of age another type of infiltrate, consisting of lymphocytes and macrophages was observed. B-cell destruction by lymphocytes was apparent in both young and adult NZB mice. It is concluded that cellular autoimmune reactions against pancreatic islets may occur spontaneously as a consequence of immunological disorders in NZB, NZB X NZW and MRL mice.

Cellular immune reactions against pancreatic islets as a consequence of graft versus host disease.

We studied autoimmune reactions against pancreatic islets occurring as a consequence of defects of the immune system rather than after pathological changes in the endocrine organ itself. Immune dysregulation was induced by transfer of parental lymphocytes into semi-allogeneic F1 recipient mice (graft versus host reaction, GVHR). Recipients were killed 1 month after the induction of the disease. Pancreatic islets were screened by light microscopy for signs of lymphocytic infiltration (insulitis). Severe insulitis was found in all mice undergoing GVHR. The cytotoxic activity of lymphocytes was apparent in electron microscopic studies and affected only B cells. Infiltrations were not seen in the exocrine part of the pancreas nor in heart, liver or kidneys at this early stage of GVHR. It is concluded that non-specific disorders of the immune system induced by GVHR may lead to specific cellular autoimmune reactions against B cells of pancreatic islets.

Insulin binding and action in isolated cardiocytes from spontaneously diabetic BB rats.

Isolated cardiac myocytes from control and insulin treated diabetic BB rats were used to study cellular alterations related to partly controlled diabetes. Scatchard analysis of equilibrium binding data showed an unaltered affinity and number of insulin receptors in cardiocytes from both groups of animals. Moreover, insulin internalization was found to be identical under these conditions. Insulin action was determined by measuring the effect of the hormone on initial velocities of 3-0-methylglucose influx. Basal activity of the glucose transporter and maximal transport stimulation by insulin remained unaffected. In contrast, the sensitivity of the carrier towards stimulation by insulin was markedly reduced in cardiocytes from diabetic rats with a half-maximal action occurring at an insulin concentration of 3 X 10(-10) mol/l and 9 X 10(-9) mol/l in control and diabetic animals, respectively. The onset of insulin action was much slower in cells from diabetic BB rats exhibiting an increase in the coupling time by 400% from 5 to 20 min, respectively. The data suggest an association of partly controlled diabetes with myocardial alterations located at the postreceptor level.

Pathogenesis of low dose streptozotocin induced diabetes in mice: requirement for alpha 1-adrenoceptor activation and vasoactive amine release.

Pancreatic islet inflammation and subsequent diabetes was induced by multiple low doses of streptozotocin in male C57 Bl/6J mice. The development of hyperglycaemia was almost completely prevented by treating the animals with the alpha 1-adrenoceptor antagonist prazosin (20 mg.kg-1.day-1) as well as by the vasoactive amine antagonists methysergide (50 mg.kg-1.day-1), disodium cromoglycate (100 mg.kg-1.day-1), pizotifen (5 mg.kg-1.day-1) or cyproheptadine (20 mg.kg-1.day-1). Treatment with vasoactive amine antagonists largely inhibited infiltration of pancreatic islets by L3T4+-lymphocytes and to a lesser extent by Lyt2+-cells. The infiltration of macrophages was not affected except after pizotifen treatment. These results indicate that alpha 1-adrenoceptor activation is required for disease development and that vasoactive amine release is a prerequisite for lymphocytic insulitis but not for macrophage infiltration of islets.