Antibody response to islet antigens in anti-CD4/prednisolone immune intervention of type 1 diabetes.

Cytoplasmic islet cell antibodies, glutamic acid decarboxylase autoantibodies, spontaneous insulin autoantibodies, and insulin-induced antibodies were analyzed in a 1-year follow-up study of 12 newly diagnosed patients with insulin-dependent diabetes mellitus aged 14 +/- 2 years (range 7-20 years) who had been initially treated with either multiple injections of insulin alone (control group) or, in addition, anti-CD4 monoclonal antibody/prednisolone (treatment group). Despite individual variations in islet cell antibody titers, there were no significant differences in the prevalence or changes in the mean titers between the two groups. Glutamic acid decarboxylase autoantibodies remained almost unchanged, but correlated with levels of islet cell antibodies. While at initiation of treatment only 50% of the patients from both groups had spontaneous insulin autoantibodies, all patients developed insulin-induced antibodies upon conventional insulin therapy during the course of follow-up. This was not related to islet cell antibody or glutamic acid decarboxylase antibody levels. The insulin requirement was markedly reduced through the period of follow-up, but did not significantly differ between the two groups. A correlation between islet cell antibody levels and insulin requirement was observed in the control group but not in the treatment group. Plasma levels of the antibodies were not associated with changes in stimulated C-peptide or hemoglobin A1 concentrations. Activated T-lymphocytes persisted in both groups of patients, but their mean levels were not significantly different. The reason for the absence of statistically significant differences between treatment and control groups could be due to the small number of patients in the study. In conclusion, short-term immune intervention with anti-CD4 monoclonal antibody in addition to insulin therapy did not suppress autoimmune reactions towards the beta cells.

Autoantibodies to insulin and to proinsulin in type 1 diabetic patients and in at-risk probands differentiate only little between both antigens.

To answer the question whether insulin or proinsulin would be the true antigen for both insulin and proinsulin autoantibodies, displacement experiments of 125I-insulin and -proinsulin binding with both unlabeled antigens were performed in sera of four groups of antibody-positive probands: first-degree relatives of Type 1 diabetic patients, pre-Type 1 diabetic persons, recent-onset Type 1 diabetic patients, insulin-treated Type 1 diabetic patients. In subjects who were primarily screened to constitute these groups, prevalences of insulin and proinsulin autoantibodies were nearly identical. In antibody-positive sera, 125I-insulin and -proinsulin binding values in general were closely correlated to each other with regression coefficients near 1.0. In all groups of probands, mean values of 125I-insulin and -proinsulin binding did not significantly differ. With the exception of a few sera, insulin and proinsulin antibodies differentiated only little between both antigens. Epitopes of the insulin molecule are therefore preferred. Nevertheless, insulin and proinsulin autoantibodies are not completely identical nor are insulin autoantibodies merely a subgroup of proinsulin autoantibodies: In each group, in the mean, insulin antibodies as well as proinsulin antibodies reacted somewhat (but significantly) stronger with their respective antigen. In some cases a distinct (relative) specificity for either antigen of insulin and proinsulin autoantibodies were observed, the latter being still present after some months of insulin treatment. In conclusion, despite detectable differences in antigen specificity, insulin and proinsulin autoantibodies seem to be equally potent markers of Type 1 diabetes mellitus.

The detection of autoantibodies to pancreatic islet cells by immunoenzyme histochemistry.

Using horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies, an immunohistochemical assay was established for the detection of islet cell cytoplasmic antibodies (ICA). Determination of end-point titers showed a significant correlation between the conventional immunofluorescence and either immunocytochemistry assay. The assays with the enzyme-conjugated antibodies were more sensitive than the indirect immunofluorescence assay. Because of its simplicity, specificity, and easy microscopic evaluation of the chromogenic reaction product at the site of ICA binding, the indirect immunoperoxidase technique proved to be most suitable. This technique detected frequencies of ICA positives among newly diagnosed insulin-dependent (IDDM), noninsulin-dependent, and at-risk subjects that were comparable with previous studies. Preabsorption of ICA-positive sera with either rat or porcine brain extracts, containing the glutamate decarboxylase antigen, differently blocked, reduced or did not affect ICA reactivity with human or porcine pancreas sections. Testing of sera on human, bovine, and porcine pancreas sections demonstrated heterogeneity in ICA-binding with a high proportion of ICA false-positives on bovine pancreas. The results demonstrated that immunohistochemical techniques for detecting ICA are, in several aspects, preferable to indirect immunofluorescence and that individual serum ICA identify various antigens on pancreas from different species. However, bovine or porcine pancreas could not substitute for human pancreas in the ICA assay.

The frequency of diabetes in children of type 1 diabetic parents.

There is little information in the literature about the actual diabetes risk of children of Type 1 diabetic parents. In a follow-up study of 48 children born from 35 Type 1 diabetic parents between 1955 and 1989, we have recorded all data relating to the incidence of Type 1 diabetes and of appearance of signs of subclinical diabetes by June, 1992. Information about the state of health was obtained during repeated stays of the patients in our hospital or by interviewing them by letter. In 21 children aged between 4 and 29 years who had not developed diabetes, we were able to investigate oral glucose tolerance, first phase insulin secretion, islet cell antibodies and insulin autoantibodies. HLA-typing was performed in 69 of the 118 study subjects (children and their parents). Type 1 diabetes developed in 19 out of 48 offspring of Type 1 diabetic parents. Frequency is also 25% in those 24 offspring born before 1972. The children developed the disease significantly earlier than the parents (8.2 +/- 6.0 versus 15.9 +/- 8.3, p < 0.01). Diabetes became manifest in 4 single children, in 2 HLA-identical pairs of siblings, and in 4 children with non-diabetic siblings. In 5 out of 21 non-diabetic offspring investigated we found impaired glucose tolerance, diminished first-phase insulin secretion, and/or Islet Cell Antibodies between 5 and 80 JDF units. The frequency of diabetes in the offsprings of Type 1 diabetic parents was lower than expected, but in addition 24% of the non-diabetic children displayed signs of subclinical diabetes.

Proinsulin autoantibodies are more closely associated with type 1 (insulin-dependent) diabetes mellitus than insulin autoantibodies.

The disease association of autoantibodies to proinsulin and insulin was compared in patients with Type 1 (insulin-dependent) diabetes mellitus and first-degree relatives. Following the recommendation of the Fourth International Workshop on the Standardization of insulin autoantibodies, autoantibodies were determined by fluid-phase radioimmunoassay using equimolar concentrations of mono-125I-A14-insulin or -proinsulin to detect insulin or proinsulin autoantibodies, respectively. A higher prevalence of proinsulin autoantibodies vs insulin autoantibodies was found in 97 patients with Type 1 diabetes prior to insulin treatment (34.0% vs 22.7%, p less than 0.05) and in 16 islet cell antibody-positive relatives (43.8% vs 31.3%, NS). There was only one serum positive for insulin and proinsulin autoantibodies in 110 islet cell antibody-negative first degree relatives (0.9%). None of 88 normal sera contained proinsulin autoantibodies or insulin autoantibodies. There was a close correlation of proinsulin autoantibody and insulin autoantibody titres in individual sera (r = 0.95, p less than 0.01) due to crossreaction of all insulin autoantibodies with proinsulin. However, some proinsulin autoantibodies did not crossreact with insulin. Background binding in normal sera was lower for proinsulin autoantibodies. We conclude that proinsulin autoantibodies have a higher association to acute Type 1 diabetes than insulin autoantibodies.

Mathematical modelling of competitive labelled-ligand assay systems. Theoretical re-evaluation of optimum assay conditions and precision data for some experimentally established radioimmunoassay systems.

A mathematical theory of competitive labelled-ligand assays was developed with the intention of theoretically re-evaluating the optimal assay conditions and precision data of assay systems established by experiment. Our theory is based upon the assumptions of a simple bimolecular reaction mechanism, homogeneous reactants, as well as kinetically indistinguishable labelled and non-labelled ligands. The general case of two-step (non-equilibrium) assay was considered including the one-step (equilibrium) assay as a special case. The solution of the system of corresponding kinetic differential equations was used to mathematically construct standard curves. Furthermore, intraassay precision profiles and indices as well as detection limits were calculated considering solely the pipetting error, epsilon, as a source of experimental error. A procedure was outlined to mathematically determine the optimal incubation conditions for any assay system targeted to a given analyte concentration, P, at which the standard deviation of assay results is to be minimized. Estimates of both the content of binding sites and the equilibrium constant, K, of the specific binding agent are necessary, and these can be derived from Scatchard plots. For six RIA systems, of which three were one-step and three were two-step assays, experimental assay conditions and precision data were compared with theoretical predictions. Experimentally determined antibody binding site concentrations agreed fairly well with those independently evaluated by mathematical optimization. Mean precision indices, defined as constituting an average over the complete precision profile, were found to be within the theoretically predicted range, i.e. two- to threefold the pipetting error.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose tolerance behaviour before the onset of type I (insulin-dependent) diabetes in young people as a predictor of the further course of the disease: a retrospective analysis of 33 cases.

A study was made of glucose tolerance and insulin secretion in 33 persons who later developed insulin-dependent diabetes (aged 4-24 years) and observation continued further in the first years after manifestation. Patients who developed the typical labile type of diabetes were of normal weight and had either normal glucose tolerance tests before diagnosis or had impaired glucose tolerance (IGT) for a short interval of 2-16 months. Subjects with IGT over a significantly (p less than 0.01) longer period of 32.30 +/- 6.25 (normal body weight) or 94.71 +/- 20.62 (obese) months developed a milder form of diabetes with retarded insulin dependency in obese subjects. The severe and mild form of IDDM are distinct with respect to insulin requirement (0.75 +/- 0.03 or 0.28 +/- 0.04 U/kg b.w., P less than 0.01) and glucagon stimulated C-peptide (0.18 +/- 0.05 or 1.41 +/- 0.27, P less than 0.01) in the first 2.5-3.5 years after onset. The two forms were not different regarding HLA-DR antigens. Islet cell surface antibodies investigated in 15 probands at 27 occasions before diabetes onset had no prognostic value. The development of a mild form of IDDM may be expected in cases with pre-existing IGT for more than one year. The insulin secretion is of low predictive value under these conditions. The observation is of practical use and theoretical interest.

[Enzyme immunoassay for the determination of albumin in urine]. / Enzymimmunoassay zur Bestimmung von Albumin im Urin.

Two modifications of a double antibody enzyme immunoassay for the determination of urinary albumin content are described. The method is simple, rapid and precise and can be carried out in test tubes and on microtiter plates as well. In 1:10 diluted urine samples albumin concentrations of 1.25 to 20 mg/l (corresponding to the normal range) can be determined. For a control sample with 0.3 mg/l albumin the intra- and interassay coefficients of variation were 4.9% (n = 11) and 10.4% (n = 21), respectively, on microtiter plates.

A sensitive sandwich enzyme immunoassay for measurement of insulin on microtiter plates.

A sensitive enzyme immunoassay for the measurement of insulin in human sera on microtiter plates was established. The assay is based on the sandwich technique with guinea pig anti-insulin IgG adsorbed at microtiter plate wells, human insulin as standard and the same anti-insulin IgG labeled with horseradish peroxidase. Standards used cover a range from 0 to 1200 pmol/l with a detection limit of 10 pmol/l. Coefficients of variation between 3-7% for intraassay precision and 5-11% for interassay precision were obtained over the concentration range of 80-1000 pmol/l. The correlation of EIA-data with those of a commercially available double antibody radioimmunoassay (r = 0.98) could be expressed by the equation: EIA = 0.97 RIA - 57 pmol/l. Normal fasting serum insulin concentrations in healthy subjects ranged from 11-165 pmol/l. In subjects with potentially diminished basal values concentrations of 10-79 pmol/l were determined. The insulin response in oral glucose tolerance tests of children was discussed, who had a constitutional tall stature or Turner's syndrome, respectively.

Influence of polyethylene glycol (PEG) extraction on the C-peptide determination in sera from insulin-dependent diabetic patients with circulating insulin antibodies.

To investigate whether the unexpectedly high C-peptide levels in some insulin-dependent diabetic (IDDM) patients are due to co-determination of proinsulin bound to circulating insulin antibodies, 36 randomly selected sera from IDDM patients were assayed for C-peptide immunoreactivity (CPR) after polyethylene glycol (PEG) extraction, preceding incubation with proinsulin binding antibodies (LAB + PEG) or without pretreatment of the sera. Recovery of proinsulin was checked by addition of 1 nmol/l proinsulin to all sera. Recovery was found to be 101.5 +/- 4.0%. The mean values of concentrations were significantly lower (p less than 0.001) after treatment with PEG and IAB + PEG compared to the untreated sera. There was also a significant difference (p less than 0.05) between sera extracted with PEG alone or after IAB + PEG-treatment. However, no correlation (p greater than 0.1) was found to bound insulin (total minus free insulin) or to insulin binding capacity (IBC) of the sera. If an antiserum is not available with very low cross-reactivity with proinsulin to determine human C-peptide then sera should not be extracted with PEG alone but after additional incubation with a proinsulin binding antiserum. In spite of the extraction in some cases unexplicably high C-peptide levels may still be expected.

Autoantibodies against insulin (IAA), C-peptide (CAA), and glucagon (GAA) in new-onset type 1 diabetic patients.

Autoantibodies against insulin, C-peptide, and glucagon were determined by radio-binding assay in 63 new-onset Type 1 (insulin-dependent) diabetic patients as well as in 70 controls. Plasma peptide binding was determined by means of 125I-labeled peptides and charcoal-dextran separation technique. Binding values exceeding the mean plus three standard deviations of the controls were considered as antibody-positive. Sixteen patients (25%) were positive for IAA, as 6 (10%) were positive for CAA and 2 (3%) for GAA. Of all control subjects, none were positive for either IAA or CAA, whereas 2 (2%) had GAA. The mean 125I-glucagon binding in the patients' group was, however, slightly enhanced and could be suppressed to normal values by excess unlabeled glucagon. The presence of IAA and/or CAA was significantly associated with more severe symptoms at diabetes manifestation. These results indicate that in new-onset Type 1 diabetics autoimmunity arises against all the insular peptides tested but is predominantly directed against those antigens secreted from the beta cells. Nevertheless, extremely low-binding GAA seem to be common in these patients. The determination of IAA/CAA might be useful in detecting a possible heterogeneity of Type 1 diabetes with regard to its clinical mode of manifestation.

Radioiodination of peptide hormones and immunoglobulin preparations: comparison of the chloramine T and iodogen method.

Following optimization of the reaction conditions, e.g. concentration of oxidizing agents, reaction time, volume of reaction mixture, and pH, chloramine T and the new iodination reagent, Iodogen, were compared for their effectiveness in radioiodination of insulin, glucagon, human growth hormone (hGH), and rabbit anti-mouse IgG. The radioactive peptide hormones prepared were analyzed for the presence of aggregate and breakdown products by polyacrylamide gel electrophoresis (PAGE) at pH 8.9, the rabbit anti-mouse IgG was tested for the presence of low molecular weight damage products by gel filtration on Sephadex G-50. The results demonstrate that with respect to iodine incorporation, specific activity, and immunological reactivity either method can be used to prepare under carefully controlled conditions a wide range of tracers with high specific activity at minimal oxidation damage. These tracers are shown to be highly suitable in radioimmunoassays after previous purification by PAGE and gel filtration, respectively.

Purification of mouse monoclonal antibodies by chromatography on sepharose 6 B.

By using Sepharose 6 B, a simple procedure for purification of mouse monoclonal antibodies (mcAbs) of the IgM and IgG class from ascites has been developed. The procedure which was applied to purify mcAbs against insulin and pancreatic islet cells permits either direct chromatographic separation from ascites protein components or after precipitating the immunoglobulins with ammonium sulphate. Recovery of the immunoglobulins was found to be approximately 80%, and the immunological reactivity, as tested by indirect immunofluorescence and ligand binding assay, was almost completely retained. For purification of IgG from ascites, precipitation with ammonium sulphate is recommended prior to chromatography on Sepharose 6 B, whereas IgM can directly be subjected without any pretreatment.

Diabetogenic effects of N-nitrosomethylurea in rats.

The influence of a single i.v. injection of 75 or 175 mg N-nitrosomethylurea (NMU)/kg into Wistar rats on plasma glucose, i.p. glucose tolerance, plasma insulin and glucagon, glucose stimulated insulin secretion and release as well as on the biosynthesis of insulin in isolated pancreatic islets have been investigated at set intervals between the 3rd day and the 6th week post injectionem. At the end of the experimental period all NMU-treated animals were alive but had significantly reduced body mass. All animals remained normoglycemic even after the glucose load. 3 days after drug application plasma insulin decreased only insignificantly in the 175 mg NMU-group, whereas plasma glucagon was significantly reduced in both drug-groups; after 3 and 6 weeks the reduction of plasma glucagon was still about 30 percent. In isolated islets the glucose stimulated insulin secretion, the percent stimulation of insulin release and insulin biosynthesis were significantly decreased.

Screening monoclonal islet cell surface antibodies (ICSA) by radioimmunoassay--detection of crossreactivity with ICSA from insulin-dependent (type 1) diabetic patients.

A radioimmunoassay for the detection of monoclonal islet cell antibodies was developed using rat insulinoma cells as antigen carriers and 125I-labeled affinity-chromatographically purified anti-mouse Ig antibodies for detecting cell-bound mouse Ig. Prior to the assay cells had been attached to glass tubes by poly-dimethyl-diallyl ammonium chloride thus allowing to perform the assay as easy as a solid-phase immunoassay. Incubation protocol and cell number were chosen to ensure a high sensitivity of the assay. Results compared well with immunofluorescence findings. Of seven monoclonal islet cell antibodies tested for crossreactivity only one was displaceable by islet cell surface antibodies from diabetic sera. This antibody was induced by immunization with human islets whereas all others were from mice which had been autoimmunized with streptozotocin and complete Freund's adjuvant.

[Production and use of monoclonal glucagon and insulin antibodies--reduction of pancreatic insulin in rats by treatment with complete Freund's adjuvant]. / Erzeugung und Anwendung monoklonaler Glucagon- und Insulinantikörper--Reduktion des Pankreasinsulins bei Ratten durch Behandlung mit komplettem Freundschen Adjuvans.

Murine monoclonal antibodies against glucagon and insulin were generated by somatic cell hybridization and partially characterized. The monoclonal glucagon antibody K79bB10 exhibited no cross-reaction with gut glucagon. This antibody and the insulin antibody K36aC10 were found of very high concentration in ascites. An ascites dilution of 1:5,000 was used for immunohistochemical staining of insulin and glucagon on Bouin-fixed pancreatic tissue sections. By indirect immunofluorescence technique we could demonstrate a reduction of pancreatic insulin in Lewis rats after treatment with complete Freund's adjuvant. The glucagon staining was not altered. The results were confirmed by radioimmunoassay analysis of pancreatic insulin and glucagon content.

Measurement of insulin in human sera using a new RIA kit. 1. Insulin determination in the absence of insulin antibodies--conventional assay and micro modification.

A sensitive and versatile radioimmunoassay (RIA) for insulin was established using human insulin standard, a specific guinea pig anti-insulin antiserum and rabbit anti-guinea pig serum. Radioiodination was performed according to a modified chloramine T method. Tracer preparations were used for as long as 6 weeks after iodination. The standard curve ranges from 0.044 to 1.2 nmol/l. The intra-assay coefficient of variation (CV) was 3-5% and the inter-assay CV was 6-9% in the optimal range between 0.4 and 0.9 nmol/l. The average recovery of human insulin added to plasma or serum samples was 100.2 +/- 2.0% (n = 38) and 100.1 +/- 1.9% (n = 42), respectively. In addition to human insulin, porcine, canine, rabbit and bovine insulin can also be determined but not rat or mouse insulin. The cross-reactivity of the antiserum with porcine proinsulin was found to be 40% on the molar basis. The range of mean fasting plasma insulin concentrations in healthy subjects and under various pathological conditions were estimated.

Measurement of insulin in human sera using a new RIA kit. 2. Determination of free and total insulin--correlations to insulin antibody levels.

Using the micro-scale modification of a newly developed RIA kit for insulin, we established methods for the determination of free and total insulin in serum of insulin-treated diabetics. Precipitation with polyethylene glycol 6000 or acid alcohol extraction of sera was carried out to remove or to dissociate antibody-bound insulin. Both assays permit precise and accurate measurement of either serum insulin fraction. In 50 diabetic sera with tracer insulin binding of 0-97%, free (after equilibration of the sera at 37 degrees C) and total insulin levels as well as insulin antibody binding parameters were determined. There was a good correlation of free to total insulin levels with maximally 10-fold higher values of total insulin. Both free and total insulin were found to be correlated with the ability of the serum to bind insulin. In detail, binding affinities (i.e. the reciprocal of equilibrium dissociation constants) and binding site concentrations were evaluated which were shown to be positively correlated with free and total insulin levels as well. From these data we conclude that insulin antibodies in the serum may accumulate therapeutic insulin and function as a depot for delivering insulin in insulinopenic episodes (Keilacker et al., 1982 and 1986).