Search

This study attempts to assess the prevalence of diabetes-associated autoantibodies in a general population in the northeastern part of Germany, with emphasis on autoantibodies against glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA-2A), and insulin (IAA) by radioassays >/= 98th percentile, and AAbs binding on pancreatic sections (ICA) by immunofluorescence >/= 10 Juvenile Diabetes Foundation units. From a total of 11,840 schoolchildren tested for all four AAbs, 821 (6.9%) children were positive for single AAbs, whereas 83 (0.7%) had multiple AAbs. If the primary screening were performed by testing GADA/IA-2A/IAA, 94% of probands with single AAbs and all with multiple AAbs would be identified. The combinations of GADA/IA-2A, GADA/IAA, and IA-2A/IAA would identify 97.6, 98.8, and 85.5% of probands with multiple AAbs, respectively. Thus, combined AAb screening in the general population identifies those probands at risk for diabetes.

The study aimed to compare the HLA specificities of AAb-positive healthy schoolchildren with those of patients with type 1 diabetes (T1D). HLA-DRB1 and DQB1 alleles were determined in 178 AAb-positive and 339 AAb-negative schoolchildren aged 6-17 years without first-degree relatives with T1D and in 274 patients with T1D. AAbs against glutamic acid decarboxylase (GADA), protein tyrosine phosphatase (IA-2A), and insulin (IAA) were determined by (125)I-antigen binding, and islet cell cytoplasmic antibodies (ICAs) immunohistochemically. Here, 82.6% (147/178) of AAb-positive schoolchildren had single AAbs and 17.4% (31/178) had multiple AAbs. In both groups, GADA occurred with highest and IAA with lowest frequency. In children with single AAbs at levels between the 99th and 99.9th percentile, frequencies of the diabetes-associated DRB1 (*03, *04) and DQB1 (*02, *0302) alleles and the protective DRB1 (*15) and DQB1 (*0602) alleles did not differ from those of controls. In patients, the positive associations were confirmed for DRB1*04 (OR = 5.39) and DQB1*0302 (OR = 9.05), whereas DRB1*15 (OR = 0.05) and DQB1*0602 (OR = 0.06) were negative-associated (p < 0.001). The same association was found in schoolchildren with multiple AAbs for DRB1*04 (OR = 3.84), DQB1*0302 (OR = 4.95), and DRB1*15 (OR = 0.1; p < 0.001-0.014), and with high-titer single AAbs (>/=99.9th percentile), but none of them had DQB1*0602. The highest risk genotype DQB1*02/*0302 occurred in 36.5% of patients (OR = 21.07) and in 19.3% of children with multiple AAbs (OR = 8.8; p<0.001). It is concluded that probands with multiple and high-titer single AAbs in the general population have the same genetic predisposition for T1D as patients and are therefore at highest risk for the disease.

The intent of this study was to analyze the prevalence of diabetes-associated autoantibodies (AAbs) at or above the 99(th) percentile as well as their association with human leukocyte antigen (HLA)-DQB1 alleles in a normal population of 6,337 schoolchildren. AAbs against glutamic acid decarboxylase (GADA), tyrosine phosphatase IA-2 (IA-2A), and/or insulin (IAA) were detected by (125)I-antigen binding and islet cell antibodies (ICA) immunohistochemically in 181 (2.86%) schoolchildren. HLA-DQB1 alleles were analyzed in 178/181 children and subsequently compared with 119 controls. 2.37% (150/6,337) possessed only one AAb, whereas 0.49% (31/6,337) had multiple AAbs but at increased levels (P < 0.001). Subjects with GADA, IA-2A, or IAA revealed an increased frequency of the diabetes-associated HLA-DQB1 alleles *0302 and/or *02 (P = 0.001-0.006) as well as a decreased frequency in the protective allele *0602 (P < 0.001-0.022). DQB1*0602 was completely absent within children with multiple AAbs or with GADA, IA2-A, or IAA at or above the 99.9(th) percentile. In comparison to children with single AAbs, the frequency of associated/protective alleles of children with multiple AAbs was enhanced/diminished (P = 0.004-0.009). The study shows that also in the general population the multiple AAbs or high level single AAbs predict rather certainly a HLA-DQB1-mediated diabetes susceptibility as shown for first degree relatives of type 1 diabetic patients.

We evaluated 6 batches of a solid phase enzyme-linked immunosorbent assay (ELISA) Isletest-ICA kit commercially available for the determination of autoantibodies to pancreatic islet cells, and compared the results with those obtained by a standardized immunohistochemical method. Following the immunohistochemical determination of autoantibodies to pancreatic islet cells, sera from patients with insulin-dependent diabetes mellitus, both positive and negative for autoantibodies to pancreatic islet cells, were randomly selected and analysed by ELISA. Sera from healthy control subjects, as well as standards recommended by the International Diabetes Workshop (IDW) ICA (Autoantibodies to Pancreatic Islet Cells) Proficiency Program, were included. Of the sera testing positive for autoantibodies to pancreatic islet cells in the immunohistochemical assay, only 14 +/- 5% were found to give a positive reaction in the ELISA. Among the sera from healthy control subjects and pancreatic islet cell autoantibody-negative insulin-dependent diabetes mellitus patients, 25 +/- 7% and 1 +/- 1%, respectively, yielded false-positive readings for autoantibodies to pancreatic islet cells. These results clearly show that the ELISA test presently available does not reliably detect autoantibodies to pancreatic islet cells, even qualitatively. Thus, it cannot be used for screening subjects at risk of developing diabetes.

Autoantibodies (AAb) to glutamate decarboxylase (GAD) occur with a high prevalence in sera of newly diagnosed type I (insulin-dependent) diabetic patients. The aim of this study was to establish a GAD-AAb radioimmunoassay using 125I-labelled GAD65 and to evaluate this assay in a cross-sectional study with newly diagnosed type I diabetic patients (diabetes duration < 6 weeks). Furthermore, subjects at high risk of developing type I diabetes and individuals suffering from other autoimmune diseases were examined in this assay. For GAD-AAb detection, 125I-labelled GAD65 was incubated with 10 microliters of human serum overnight on ice. Thirty of 51 (59%) type I diabetic patients but none of the 54 healthy blood donors tested were found to be positive. A displacement step using 100,000 g supernatant from rat brain containing or not containing GAD showed the specificity of the binding of 125I-GAD65. Concerning the individuals at high risk of developing diabetes. 9/12 (75%) islet cell antibody (ICA)-positive non-diabetic and 4/34 (12%) ICA-negative subjects with metabolic abnormalities were GAD-AAb positive. These results show the association between type I (insulin-dependent) diabetes mellitus and the occurrence of GAD65-AAb, which possibly predicts a risk of developing the disease.

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.

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.

In a T lymphocyte and macrophage-depleted mononuclear cell population of the peripheral venous blood of 10 of 41 first degree relatives of insulin-dependent diabetic individuals who had or had had disturbed glucose tolerance adenine uptake rates were significantly increased, the relative adenine incorporation rates into the adenine nucleotides, however, were diminished. Values were compared with those of 30 controls. In 7 of 9 investigated individuals with increased adenine uptake rates antibody-dependent cellular cytotoxicity against rat Langerhans islets (ADCC) was increased in the same cell population. In these individuals the number of diabetes manifestations was relatively high. Adenine uptake rates, ADCC and glucose tolerance changed with time.

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.

In 868 insulin-treated diabetic children and adolescents with onset of IDDM under age 20 we investigated the frequency of IDDM and NIDDM in all first-degree relatives. On the basis of the National Diabetes Register of the GDR the age-corrected lifetime risk for the development of IDDM and NIDDM was calculated for the general population and for parents and siblings of diabetic children. The age-corrected risk for IDDM, but not for NIDDM, is statistically significantly higher in fathers (10.26 +/- 1.75%) than in mothers (5.28 +/- 1.49%) and is about equal in brothers (30.71 +/- 6.07%) and sisters (35.54 +/- 6.28%) of children with IDDM. Among general population the age-corrected life-time risk for IDDM is equal for males (4.35 +/- 0.02%) and females (4.91 +/- 0.02%), but is significantly higher for NIDDM in females (27.49 +/- 0.04% contrary to 24.07 +/- 0.05%). In comparison with the data of Tillil and Köbberling (1987) our lifetime risk estimates show a shifting of risk for IDDM and NIDDM into older age groups.

The autologous mixed lymphocyte reaction (AMLR) represents activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Deteriorations in AMLR have been reported in many autoimmune diseases and in diseases with a derangement in T cell regulatory function. We have studied AMLR in 23 newly diagnosed Type-1 diabetic patients and 32 healthy subjects. T and non-T cells were purified by rosetting mononuclear cells with sheep erythrocytes and separating the rosetted T cells from the nonrosetted non-T cells by density gradient centrifugation. Purity of T-lymphocytes isolated was 90% as determined by indirect immunofluorescent analysis with monoclonal antibodies. Proliferation of lymphocytes was measured in response to phytohaemagglutinin and of concanavalin A in a lymphocyte transformation test. In the present study, a deficient AMLR is demonstrated in patients with newly diagnosed Type-1 diabetes. Our data provide evidence for an aberrant immune regulation at the time of diabetes manifestation. The deficient AMLR may represent the in-vitro expression of an in-vivo process against pancreatic cells.

HLA DQ beta restriction fragment length polymorphisms (RFLP's) were compared in 43 patients with insulin dependent diabetes mellitus (IDDM), 51 healthy first grade relatives of IDDM patients and 27 controls without IDDM heredity in their families. We were able to demonstrate an association between the presence of a 12 kb BamHI restriction fragment (p less than 0.001) and 12 kb/4 kb (p less 0.01) or 12 kb/4.4 kb (p less than 0.001) BamHI fragment combinations and IDDM. But for these fragments and fragment combinations we also found increased frequencies in the healthy first grade relatives of IDDM patients. That means for the evaluation of the importance of the characterised "risk fragments" in practice it is necessary to follow up the manifestation of IDDM in this risk group.

A modification of the original method of R.A. Johnson et al. for nonenzymatically glycated serum proteins (fructosamine test) is described. Problems of performance and optimization of the method as well as of calibration and the influence of protein composition on the results are discussed. Measuring is manually performed after a 8 min preincubation interval over a 1 min's period using the spectrophotometer "Spekol 220". Interserial precision was 3.2%. First clinical results demonstrate the possibility to assess glycemic control in type 1 diabetic patients over a integrated time interval.