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Two monoclonal Beta-cell surface antibodies M10H6 und K14D10 were obtained by fusion of spleen cells of Balb/c mice with the myeloma cell line P(3)0. The monoclonal antibody M10H6 was induced by immunization with rat insulinoma cells finally boostered with disintegrated rat islets, whereas the K14D10 was generated after immunization with porcine proinsulin. Both monoclonals belong to the IgG2A isotype and were screened with insulin-producing rat insulinoma cells by an indirect immunofluorescence test as well as by a cellular enzyme linked immunosorbent assay. In addition to the cell surface binding on living Beta cells the monoclonals react with islets on cryostat sections of rat pancreas. The anti-islet cytotoxic potential of these monoclonals was measured by 51Chromium-release in the presence of complement or Fc-receptor bearing leucocytes using 51Chromium-labelled rat islet cells as target. Both antibody secreting hybridomas were propagated in syngeneic mice resulting in high levels of islet cell surface antibodies in ascites and sera from the recipient. High anti-islet cytotoxicity was mediated by ascites fluid, but no mouse developed hyperglycaemia. Furthermore, the repeated injections of the monoclonals into rats did not exert a diabetogenic action and failed to reduce the pancreatic insulin content although the attraction of the K14D10 to the pancreatic islets in vivo could be demonstrated. We conclude that islet cell surface antibody-mediated Beta-cell lysis in vitro may not be relevant to Beta-cell destruction in vivo.

The effect of platelet-activating factor (PAF) antagonist BN 52021 (0.06-2.5 mM) on the cytotoxic activity of mononuclear cells (MNC) from newly diagnosed type 1 diabetic patients against 51Cr-labeled Langerhans islets from neonatal rats was investigated in a 6-hour cytotoxicity test. A dose-dependent inhibition of anti-islet cytotoxicity by BN 52021 was observed. The suppression of the islet lysis was significant at a concentration of 0.6 mM BN 52021. During a 4-day cell culture, BN 52021 had no inhibitory effect on the antigen-mediated triggering of immunocytes with anti-islet cytotoxicity. The results suggest that the drug is only effective during immunocytolytic reactions of MNC against pancreatic islets. A PAF-independent action of BN 52021 can not be excluded at present.

The offspring of mother animals with mild gestational hyperglycaemia exhibited basal hyperinsulinism, decreased glucose tolerance and increased susceptibility to streptozotocin diabetes. Following low-dose successive streptozotocin treatment a significantly increased spleen cell cytotoxicity against beta-cells was found in these animals as compared to the offspring of gestational normoglycaemic control mothers. Such increased cell-mediated cytotoxicity, considered as enhanced autoimmune reactivity, was positively correlated to blood glucose levels and negatively correlated to pancreatic insulin contents. Thus, hyperinsulinism, occurring during pre- and neonatal brain organization and produced by gestational hyperglycaemia, is a predisposing teratogenetic risk factor not only for the development of type II, but also of type I diabetes. Thus, it is comprehensible that the prevalence of type I diabetes in children could be decisively reduced by preventing gestational hyperglycaemia in their mothers (Dörner et al., 1985).

In this study the in vivo relevance of spleen cell anti-islet cytotoxicity measured in vitro was examined by transplantation of 1,200 syngeneic islets into the spleen of rats receiving 0.5 ml complete Freund's adjuvant (CFA) 24 h before 25 mg/kg body weight streptozotocin (STZ) was given. Control rats receiving CFA or STZ alone remained normoglycaemic whereas 12 out of 21 CFA/STZ-treated rats developed a severe hyperglycaemia after three combined treatments. After the first and second combined treatment splenocytes showed a significant cytotoxicity (p less than 0.01) against syngeneic islets measured by 51Cr-release. This cytotoxicity was not detectable after the third combined treatment. The CFA/STZ-induced diabetes with a residual pancreatic insulin content of only 5% was permanently reversed by intrasplenic islet isografts, but, surprisingly, syngeneic islets survived too, if transplanted at the time when an anti-islet cytotoxicity was measured in vitro. From our results we conclude that the polyclonal activation by complete Freund's adjuvant potentiates the beta cell-toxic effect of a low dose of streptozotocin and induced a transient splenocyte-mediated anti-islet cytotoxicity not recurrent after islet transplantation. Furthermore, our findings reveal a discrepancy between organ-specific immune reactions measured in vitro and those affecting the beta cells in vivo.

The intact pancreatic islet can be destroyed by antibody-dependent cell-mediated cytotoxicity (ADCC). T cell-mediated cytotoxicity (CMC) might additionally be important in the pathogenesis of IDDM. However, in vitro alterations of islets due to CMC have so far not been demonstrated. For the evaluation of the cytotoxic attack caused by ADCC and CMC against the islets in the development of IDDM we used a BB rat line with a high incidence of the disease (dp BB/OK). Cytotoxicity tests were carried out on autologous and on nonsyngeneic islets with mononuclear spleen cells and serum from non-diabetic BB rats of different ages. The cytotoxic action of the mononuclear cells was quantified by the specific 51Cr-release from the islets after pretreatment with serum. It was found that an anti-islet cytotoxicity appears with a peak incidence between 40 and 60 days of age. The frequency of cytotoxicity in vitro was related to the incidence of diabetes as normally observed in this rat line. Furthermore, it was shown that both autologous, allogeneic and xenogeneic islets can be destroyed by mononuclear spleen cells and serum of dp BB/OK rats.--The frequency and the strength of anti-islet cytotoxicity in vitro were higher in these dp BB/OK animals than in a control group of non-diabetes prone BB/PhiK rats. There was an association between cytotoxic 51Cr-release in the positive assays and a reduction in the hormone content of pancreatic islets.--This report provides evidence of cell-mediated immune damage of islets during the prediabetic state of BB rats suggesting that both CMC and ADCC are involved in islet cell killing.

ADCC (antibody-dependent cellular cytotoxicity) against xenogenic islets in vitro has frequently been found with mononuclear blood cells and heat inactivated autologous serum from newly diagnosed Type-1 diabetics. Anti-islet ADCC, as measured by enhanced 51Cr-release of islets after a 6h-incubation, leads to functional alteration of islets such as a decrease in insulin content and in leucine incorporation. In a follow-up investigation over at least three years it was demonstrated that anti-islet ADCC in vitro disappears, if there is no more C-peptide secretion in vivo. Furthermore, anti-islet ADCC has also not been found in long-term Type-1 diabetics who had no C-peptide secretion but an acutely stimulated immune system due to infectious diseases. An acute immunocytolytic process against pancreatic beta cells in vivo seems to be the precondition for anti-islet ADCC in vitro.

On the basis of the blood glucose increase during the capitivity sand rats born in the desert were classified as normals, protodiabetics and diabetics, indicating a different adaptation to the new environment within a definite period. Isolated islets of animals, which did not develop a hyperglycemia, enhanced their insulin content during the adaptation period. The absolute insulin secretion rates in response to 16.5 mM glucose were rather similar between the three investigated groups and not modified by the insulin as well as glucagon content of pancreatic islets. But, since islets of hyperglycemic sand rats could not increase the insulin content, a significantly enhanced fractional secretion (as % of the content) could be observed. The results let us assume that the B-cell reaction during the adaptation period can be modified by further factors additionally to the changed environment.

Antibody-dependent cell-mediated cytotoxicity against pancreatic islets was investigated in 13 newly diagnosed insulin-dependent diabetics, in 38 patients at high risk for the disease and in 20 age-matched healthy controls. For this purpose 51Cr-labeled neonatal rat pancreatic islets incubated with the specific anti-rat islet cell antiserum 339 or with serum of the lymphocyte donors were used as targets. The antibody-mediated cytotoxic activity of mononuclear cells was evaluated from the specific chromium release after 6 h exposure of pretreated islets to the effector cells. The specific cytotoxic effect of mononuclear cells from healthy controls on pancreatic islets pretreated with serum is weak. ADCC mediated by the specific antirat islet cell antiserum is significantly increased in 54% of newly diagnosed diabetics as well as in 32% of patients at high risk for insulin-dependent diabetes mellitus. 46% of the newly diagnosed diabetics were also ADCC-positive when their own serum was used for the pretreatment of islets, regardless of whether they were islet cell antibody- or islet cell surface antibody positive or not. Subsets of mononuclear cells (non E-rosette-forming cells, high affinity E-rosette-forming cells, low affinity E-rosette-forming cells) were prepared and their cytotoxic potential was analysed in patients with positive ADCC test results.(ABSTRACT TRUNCATED AT 250 WORDS)

The non-specific activation of the immune system by administration of complete Freund's adjuvant (CFA) was examined in two congenic Lewis rat strains LEW. 1A (RT1a) and LEW. 1W (RT1u) as a possible mean of amplification of the specific immune response, directed to pancreatic beta cells induced by multiple non-diabetogenic injections of streptozotocin (STZ). Rats were given intraperitoneally 0.5 ml CFA and 1 day later 25 mg/kg body weight STZ. This combined treatment was repeated twice at weekly intervals. Control groups received vehicle, STZ or CFA only with the same doses and at the same times. Only CFA/STZ-treated rats developed a persisting hyperglycaemia (greater than 15 mmol/l glucose) namely 3/18 (17%) LEW. 1W and 47/76 (62%) LEW. 1A rats. The pancreatic insulin content in these hyperglycaemic rats was reduced by 96.6% in LEW. 1A rats and by 93% in LEW. 1W rats measured 8 weeks after the last CFA/STZ treatment. The response to CFA indicated by an increase of number of peripheral leucocytes and relative spleen weight gain at 7 days after CFA administration, was higher in LEW. 1A rats compared with those of LEW. 1W rats. Spleen cells harvested 72 h/48 h after the first and second CFA/STZ administration showed a cytotoxic reaction to isolated syngeneic islets as measured by 51Cr-release in vitro. Control rats receiving vehicle, STZ or CFA only showed no cellular anti-islet cytotoxicity. The anti-islet cytotoxicity of spleen cells was only transient and disappeared after the third CFA/STZ administration. Anti-islet cytotoxic antibodies were not detectable in this short-term study.(ABSTRACT TRUNCATED AT 250 WORDS)

ADCC (Antibody-dependent cellular cytotoxicity) against xenogenic islets has frequently been found in newly diagnosed Type 1 (insulin-dependent) diabetics suggesting that when combined with autologous serum in vitro, the destruction of islet cells caused by mononuclear cells (MNC) reflects islet destruction in vivo. In this study the ability of Ciamexone to suppress the anti-islet ADCC in vitro was investigated. We selected both ADCC positive and ADCC negative subjects from a group of Type 1 diabetics. The targets, islets from neonatal rats, were incubated for one hour with the probands serum. The ADCC was then measured by the 51Cr-release of the serum-treated islets after a 6h-incubation with MNC of the same donor. Results both with and without co-incubation of Ciamexone were compared. Ciamexone does not influence the spontaneous 51Cr-release from neonatal rat islets treated with the probands serum but significantly suppresses the anti-islet ADCC. The fact that Ciamexone suppresses anti-islet ADCC may explain its possible effectiveness in Type 1 diabetes.

To evaluate the significance of ICSA as a prognostic marker for the development of type I diabetes we investigated 66 subjects with first degree relatives of type I (47) and type II (9) diabetes as well as subjects with anamnestical data suggestive of diabetes. Patients were studied for glucose tolerance (oGTT) and IRI-response, ICSA (indirect fluorescence of living rat islet cell suspensions), ADCC (specific 51Cr-release of serum pretreated neonatal rat islets elicited by mononuclear cells) and HLA-antigens. 23 subjects revealed normal glucose tolerance, 17 impaired glucose tolerance and 26 had a prevoius abnormality. The incidence of ICSA varied between 46 and 53 per cent, that of positive ADCC between 22 and 56 per cent, both being highest in subjects with IGT. 87 per cent of all patients revealed diabetes associated HLA-antigens. We found no correlation of ICSA with glucose tolerance, IRI-response, ADCC and HLA-antigens. In conclusion it can be said that ICSA are present in a high percentage in patients with high diabetes risk but their predictive role as a marker for the manifestation must be elucidated in follow-up studies.

In normoglycemic sand rats the insulin secretion of isolated islets was investigated dependent on the time of artificial nutrition in captivity. Whereas the islets of short-term adapted animals did not respond within 15 min with an enhanced insulin secretion after stimulation with 16.5 mM glucose or 10 mM arginine, prolongation of incubation time resulted in a significantly enhanced hormone release. An intraveneous glucose injection (1 g/kg body weight) 45 min prior to the excision of the pancreas caused an immediate insulin secretion of the isolated islets in response to glucose or arginine. Islets of sand rats adapted for more than 10 weeks to artificial nutrition responded immediately with an enhanced insulin release in contrast to the islets of short-term adapted animals. These differences could not be correlated to the insulin content, the A/B cell ratio, and ultrastructural alterations of the islets.

Electron microscopical investigations of islets of Langerhans are carried out in "protodiabetic" sand rats. In the B-cells and beta-Granules (2 to 5) aggregated. Mitochondrias, Golgi apparatus and rough endoplasmic reticulum are increased. These B-cells are stimulated. The A-cells are degranulated, and lipofuscin-granules are in the cytoplasm present. These cells were stimulated too. The D-cells are not stimulated.

Isolated pancreatic islets of normoglycemic sand rats do not respond to 2.5 mM glucose with an enhanced glucagon secretion, which could be observed in normal Wistar rats. Arginine stimulates glucagon release in the presence of 2.5 mM glucose in Wistar rats as well as in sand rats. The secretion pattern is not caused by insulin deficiency since sand rat islets are characterized by an increased insulin secretion rate in vitro. This paradoxical glucagon secretion is not caused by a changed glucagon content but might be related to this species which is able to develop a diabetic syndrome spontaneously.