Brigitte Ziegler

Antibody response in rats against non-toxic glucose sensor membranes tested in cell culture

Several glucose sensors have been developed, but none are commercially available. The most urgent in vivo problem is the drift of glucose sensor output with time, which may be caused by leakage or denaturation of glucose oxidase, and events at the body-sensor interface such as protein coating, encapsulation with cells, toxicity of the device and inflammation. In the present study, a specific immune response against the outer cellulose acetate membrane of a glucose sensor implanted into rats has also been proved. The immune response against polymeric membranes can be confirmed by detection of specific immunoglobulin G antibodies to cellulose acetate, polyurethane and regenerated cellulose after implantation of the respective membrane. The individually different antibody formation against polymers in rats was amplified by one application of complete Freunds adjuvant in combination with the first implantation. The cell culture results using the fibroblast cell line L-929 showed only a minor toxicity of regenerated cellulose, whereas the other polymers had no effect on cell growth and viability. From the results of this study, it is proposed to integrate immunogenicity as a further parameter for evaluation of the biocompatibility and biosafety of materials or medical devices which are provided for implantation.

Murine monoclonal glutamic acid decarboxylase (GAD)65 antibodies recognize autoimmune-associated GAD epitope regions targeted in patients with type 1 diabetes mellitus and Stiff-man syndrome

To study the immune response to glutamic acid decarboxylase (GAD) in insulin-dependent diabetes mellitus, monoclonal GAD antibodies after fusion of splenocytes from a nondiabetes-susceptible BALB/c mouse immunized with human recombinant GAD65 were generated. Of the 44 monoclonals, 35 are specific for the GAD65 isoform, whereas 9 also react with GAD67. Some 37 monoclonals, including all GAD65/67 reactive antibodies, react with GAD by Western blot analysis. The remaining 7 GAD65 monoclonals bind GAD only in an immunoprecipitation assay, which implies that they target epitopes dependent on the conformation of the GAD molecule. The125I-GAD binding of the GAD65 monoclonals reactive on Western blotting was significantly diminished by all 3 sera from Stiff-man syndrome patients but only by 3/30 (10%) sera from type 1 diabetic patients. In contrast, the 7 monoclonal antibodies reactive with a conformation-dependent GAD epitope were competitive with 83% of GAD-autoantibody-positive sera from these diabetic patients. Using chimeric GAD65/67 proteins, the epitope region targeted by these monoclonals was mapped to the middle of GAD65 (amino acids 221–442). This central conformation-dependent GAD region was also targeted by sera from patients with type 1 diabetes. In conclusion, our data show that evne after common immunization of a nondiabetes-susceptible mouse strain, monoclonals were obtained which preferentially react with the GAD65 linear amino-terminus (amino acids 4–17) and a conformation-dependent region located in the middle of GAD targeted by autoantibodies, indicating that this GAD region is not restricted to the autoimmune response associated with the Stiff-man syndrome and the bete-cell destruction in type 1 diabetes mellitus.

Impact of metabolic activity of beta cells on cytokine-induced damage and recovery of rat pancreatic islets

The influence of beta cell activity on cytokineinduced functional and structural impairments as well as the ability of those damaged cells to recover were investigated. Rat islets cultured for 4 days in the presence of 5, 10, and 30 mmol/l glucose were exposed to interferon-γ (IFN, 500 U/ml) and tumor necrosis factor-α (TNF, 250 U/ml) for the last 24 h. After cytokine removal islets were allowed to recover spontaneously in culture medium containing 10 mmol/l glucose for a further 7 days. Cytokines significantly inhibited insulin release into culture medium, insulin storage, glucose-stimulated insulin secretion, protein, and DNA synthesis. In the presence of cytokines there was a six- to eightfold increase in nitrite production by the islets. The functional impairments were more pronounced in metabolically stimulated beta cells. In addition, cytokines caused membrane alterations as indicated by increased spontaneous chromium-51 release. The cytokines specifically induced the synthesis of two proteins (72 and 88 kDa, respectively). By immunoblotting, the 72-kDa protein was identified as heat shock protein. After a 1-week recovery period, insulin storage and stimulated insulin secretion of cytokine-treated islets were still significantly diminished. However, protein and DNA synthesis of cytokine-exposed islets returned to pre-exposure levels. In conclusion, high beta cell activity increases islet susceptibility to TNF+IFN. Cytokine-induced, longlasting, inhibitory effects are primarily directed to betacell-specific functions, while general vital cell functions clearly recover after cytokine removal. The induction of certain proteins and the increased protein synthesis and replication rate after cytokine removal might reflect activated repair processes.

Sensitive Monoclonal Antibody-Based Sandwich Elisa for Determination of the Diabetes-Associated Autoantigen Glutamic Acid Decarboxylase GAD65

Although various methods for the detection of autoantibodies against glutamic acid decarboxylase (GAD65-AAb) are known, no sensitive method for the quantification of GAD65 as autoantigen is available. We describe a sandwich ELISA based on monoclonal GAD65 antibodies (Mc-GAD65-Ab) of different epitope specificities to quantify GAD65 in pancreatic islets and in different organ/cell extracts and during the preparation of GAD from brain extracts. GAD65 was captured via solid phase coated Mc-GAD65-Ab and detected via a second biotin-labelled Mc-GAD65-Ab recognizing a NH2-terminal epitope of the molecule. The detection limit was estimated to be 0.03 ng GAD65/ml using alkaline phosphatase (AP)-conjugated streptavidin. GAD65 contents in islets of neonatal BB/OK rats and Lewis rats amounted to 37.4 and 43.7 pg/islet, respectively. Furthermore, GAD65 was quantified in brain extracts of pig (55.1 ng/mg protein), mouse (39.5 ng/mg), rat (243.8 ng/mg) and pig cerebellum (514.8 ng/mg) and in different organ extracts of Lewis rat.

A Monoclonal Antibody-Based Characterization of Autoantibodies against Glutamic Acid Decarboxylase in Adults with Latent Autoimmune Diabetes

Autoantibodies to glutamic acid decarboxylase (GAD) are an important marker of the autoimmune-mediated beta-cell destruction in insulin-dependent (Type I) diabetes. However, these autoantibodies are also found in patients with Stiff-man syndrome (SMS) without onset of diabetes and some diabetic patients who initially present as non-insulin dependent (Type II) diabetes later becoming insulin-dependent, called as latent autoimmune diabetes in adults (LADA). To study the immune response to GAD in these LADA patients a competitive radiobinding assay based on murine monoclonal antibodies recognizing three different GAD regions was performed. The monoclonal antibodies against GAD recognize two different linear epitopes localized at the N- (amino acids 4-17) and C-terminus (amino acids 572-585) and one conformation-dependent epitope region (amino acids 221-442 IDDM-E1) known to be immunodominant for diabetes-associated autoantibodies. All LADA sera (20/20) reduced substantially the 125I-GAD binding of the monoclonal antibodies reactive with the conformation-dependent epitope region IDDM-E1 and only 20% of these sera additionally diminished the 125I-GAD65 binding by those monoclonals reactive with the both linear epitopes. The SMS sera completely abolished the GAD binding of all three monoclonals, reflecting a broader repertoire including an immune response against the IDDM-E1, a conformation-dependent GAD65 epitope region, also revealed if the SMS sera are diluted to equivalent antibody concentrations. In summary, our results show that diabetes-associated GAD autoantibodies even in adult patients with a late autoimmune process preferentially recognize a conformation-dependent middle GAD65 region. An immune response to all three GAD epitope regions is seldom in these LADA patients and only detectable in association with high antibody titres.

Paradoxical glucagon response after stimulation with glucose and arginine in isolated pancreatic sand rat islets

SummaryIsolated 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.

Eignung spezifischer Färbemethoden für die Bestimmung des β-Zellvolumens im Rattenpankreas mit normalem and reduziertem Insulingehalt

Zusammenfassung Die Untersuchungen wurden an mit Streptozotocin (SZ) behandelten, weiblichen Wistarratten (einmalige i.v. Injektion von 30 mg/kg Korpermasse) Bowie citratpuffer-behandelten Kontrolltieren durchgefuhrt. Nach Farbung der pankreatischen β -Zellen mit Aldehydfuchsin (AF), Viktoriablau nach Ivic bzw. FITC-markiertem Antiserum (indirekt) wurde das β -Zellvolumen nach einem point-sampling Verfahren ermittelt. Bei den Kontrolltieren ergeben alle 3 genannten β -zellspezifischen Farbemethoden das gleiche β -Zellvolumen. Mit Reduktion des Insulingehaltes im Pankreas and in den β -Zellen durch SZ-Applikation nimmt die Empfindlichkeit der histochemischen Farbemethoden im Vergleich zum immunhistochemischen Nachweis ab. Normoglykamische SZ-behandelte Tiere (Pankreasinsulingehalt ca. 30 % der Kontrollwerte) zeigen nach der AF-Farbung ein β -Zellvolumen von 0,22 %, nach der Ivic-Farbung von 0,14 %. Bei hyperglykamischen Tieren (ca. 6 % des Pankreasinsulingehaltes der Kontrolle) sind mit der AF-Farbung 0,08 % and nach Ivic 0,03 % β -Zellvolumen mesbar. Mit der Immunfluorescenzmethode Bind die β -Zellvolumine beider SZ-behandelten Gruppen identisch (0,34 %). Die nach SZ-Behandlung auftretende Abnahme des Pankreasinsulingehaltes ist auf die Reduktion des β -Zellvolumens, vor allem aber auf die starke Abnahme des Insulingehaltes pro β -Zelle zuruckzufuhren.

Prophylactic Insulin Treatment of Diabetes-prone BB/OK Rats by Application of a Sustained Release Insulin Implant

Normoglycemic diabetes-prone BB/OK rats aged 33, 45 or 75 days were subjected to prophylactic insulin treatment by means of a single subcutaneous application of a sustained release insulin implant. The single application of a sustained release insulin implant decreased the incidence of diabetes or delayed the onset of the disease in BB/OK rats of all treatment groups. Prophylactic insulin administration caused a transient hypoglycemic period accompanied by an inhibition of glucose stimulated insulin secretion and a decrease of the insulin content of Langerhans islets as detectable in vitro . Compared to islets of normoglycemic controls pancreatic islets isolated from hypoglycemic BB/OK rats within 7-21 days after the insulin application at 45 days of age displayed a decreased susceptibility of the cells to complement-dependent cytotoxicity of the monoclonal islet cell surface antibody (ICSA) K14D10 but not to the cytotoxic effect of the ICSA M3aG8. The appearance of complement-dependent antibody-mediated cytotoxicity to islet cells and pancreatic exocrine cells in serum regarded as a sign of immune dysregulation in BB/OK rats seems not to be affected by insulin prophylaxis and was detectable during hypoglycemia as well as in the subsequent normoglycemic state. In conclusion, BB/OK rats of different age can be protected from diabetes by a single application of a sustained release insulin implant. Insulin and/or hypoglycemia seem to influence the expression of cell surface antigens, thus render the islets of Langerhans less vulnerable to immune cytolysis, whereas the appearance of humoral immunological abnormalites is not affected.

No association between anti-bovine serum albumin antibodies and islet cell reactive antibodies in newly diagnosed type 1 diabetic patients

Serological findings have suggested that antibodies (Ab) to bovine serum albumin (BSA-Ab) are associated with type 1 diabetes mellitus. The aim of our study was to evaluate a competitive fluid-phase radioimmunoassay for detecting BSA-Ab using different incubation times and to study a possible association of these BSA-antibodies with autoantibodies (AAb) frequently detected in type 1 diabetic patients. For the overnight incubation time, there was an enormous overlap in the [125I]BSA binding by serum samples between 52 newly diagnosed type 1 diabetic patients (mean [125I]BSA binding 23.6 +/- 17.4%) and 54 healthy blood donors (mean [125I]BSA binding 10.2 +/- 15.7%). By an incubation time of only 3 min the BSA-antibody prevalence was found to be 15.4% (8/52) for type 1 diabetic patients and 3.7% (2/54) for control subjects. However, there was no association between BSA-Ab and type 1 diabetes-associated antibodies as cytoplasmic islet cell antibodies (ICA), or glutamate decarboxylase autoantibodies. Our results confirm that (i) BSA-Ab occur more frequently in newly diagnosed type 1 diabetic patients compared with a healthy control group and (ii) that the BSA-Ab detected by the fluid-phase radioimmunoassay with an incubation time of 3 min are more disease-associated than the [125I]BSA binding after an overnight incubation. The competitive BSA-Ab fluid-phase radioimmunoassay described is a simple and rapid method to detect antibodies specifically reactive with BSA. It is suggested that the humoral immune reactivity to BSA in type 1 diabetic patients probably reflects an unspecific defect of the immune system and gives no additionally diagnostic value about the type 1 diabetes.

Pregnancy-associated changes in the endocrine pancreas of normoglycaemic streptozotocin-treated Wistar rats

SummaryThe effect of pregnancy on pancreatic insulin content and relative B-cell volume has been studied in normoglycaemic Wistar rats treated with streptozotocin 14 days before mating. A single intravenous injection of streptozotocin (30 mg/kg body weight) caused a significant reduction of pancreatic insulin content and B-cell volume. The islet insulin content was 60% of control values. However, pregnancy-associated adaptation was preserved in these streptozotocin-treated animals. Plasma insulin levels, pancreatic insulin and B-cell volume were significantly enhanced compared with non-pregnant rats investigated on the same date. The incorporation of [3H]-thymidine into islets from pregnant rats (day 10.5) was higher than that in islets isolated from non-pregnant animals. After delivery insulin content and B-cell volume returned to pre-pregnant values. Also during a longer period after streptozotocin treatment (156 days), no measurable enhancement of B-cell volume and pancreatic insulin content was observed indicating the unresponsiveness of residual B cells to compensate spontaneously for the loss despite persisting normoglycaemia.