Low prevalence of GAD and IA2 antibodies in schoolchildren from a village in the southwestern section of the Netherlands.

Glutamic acid decarboxylase (GAD) and insulinoma antigen 2 (IA2) antibodies are increasingly used as a tool to predict type I diabetes in children and as a differential diagnostic tool to distinguish type I and type II diabetes in adults. However, the background frequency of these antibodies in the general population has not been extensively studied and may differ between countries. The current study aims to establish the frequency of GAD and IA2 antibodies in an unselected population of schoolchildren and confirm the previously reported low prevalence of islet cell antibodies (ICA) in the general Dutch population. The study population consisted of 1403 unselected schoolchildren. All children were tested for GAD antibodies, and 1085 children were analyzed for IA2 antibodies by radiobinding assay. Development of diabetes was recorded during a 7-year follow-up. Five children (0.4%) were positive for GAD antibodies, one child (0.1%) was positive for IA2 antibodies. Two children developed diabetes during follow-up, one was positive for GAD antibodies only, the second was positive for both GAD and IA2 antibodies. The frequency of GAD and IA2 antibodies in the southwestern part of The Netherlands is low. This observation is in concordance with earlier studies on ICA in Dutch schoolchildren. For future diabetes prediction and intervention trials it is important to establish the background frequencies and predictive power of antibody screening in different populations.

Islet cell cytoplasmic antibody reactivity in midgestational human fetal pancreas.

The reactivity of islet cell cytoplasmic antibodies (ICA)-positive and ICA-negative sera of recent onset type 1 diabetic patients was studied in human fetal pancreata of 12-18 weeks' gestation and compared with the reactivity of these sera in adult human control pancreata. The aims of the study were: (1) to observe the presence of ICA staining in human fetal islet cells; (2) to compare endpoint titres (in Juvenile Diabetes Foundation units) of ICA-positive patient sera in fetal pancreata and adult human control pancreata. Ten ICA-positive sera and eight ICA-negative sera from newly diagnosed diabetic patients and four sera from healthy controls were tested on three human adult and eight human fetal pancreata. As in the adult control pancreata. ICA-positive sera reacted to insulin-, glucagon-, and somatostatin-positive cells of fetal pancreata of all gestational ages. This was observed both in single cells and in cells in islet-like cell clusters. Dilution of a reference serum gave similar results in both adult and fetal pancreata. In contrast, the ICA-positive patient sera yielded a striking heterogeneity in fetal as well as in adult pancreata. However, end-point titres between adult and fetal pancreata did not differ significantly (P > 0.05). In conclusion, ICA-positive sera from recent onset diabetic patients show that the expression of molecules to which ICA react is present in all islet cells and starts before week 12 of gestation.

Value of antibodies to GAD65 combined with islet cell cytoplasmic antibodies for predicting IDDM in a childhood population.

The value of a test for islet cell cytoplasmic antibodies together with a test for GAD65 antibodies to predict the subsequent development of diabetes over a period of 11.5 years was assessed in an open childhood population comprising 2,805 individuals. A single serum sample was obtained from each individual between 1975 and 1977 and screened for islet cell cytoplasmic antibodies for which eight individuals were positive (0.29%). During the average follow-up period of 11.5 years, four of eight islet cell antibody positive and three islet cell antibody negative individuals developed clinical diabetes. Sera from all individuals, who were islet cell antibody positive and/or developed diabetes (total of 11) and from 100 randomly selected control subjects were analysed for GAD65 antibodies. Six of eight islet cell antibody positive individuals were GAD65 antibody positive including all four who subsequently developed IDDM. Furthermore, one of the three islet cell antibody negative individuals who developed IDDM was GAD65 antibody positive both in 1976 and in 1989. Thus, a positive test for GAD65 antibodies alone correctly predicted diabetes in five of seven children, who developed the disease. Only one of the children, who developed diabetes was positive for insulin autoantibodies and this individual was also positive for islet cell cytoplasmic antibodies and GAD65 antibodies. One of the 100 control subjects was positive for GAD65 antibodies (1%). The results suggest that a single GAD65 antibody test may have a higher sensitivity for predicting IDDM than a test for islet cell cytoplasmic antibodies, but that a combined positive test for both antibodies increases the specificity for predicting IDDM over a period of 11.5 years.

Cardiolipin antibodies and lupus anticoagulant in young patients with a cerebrovascular accident in the past.

To determine whether young patients who suffered a stroke in the past, have a higher prevalence of ACA of LAC as compared to healthy controls, we evaluated 44 stroke patients and 46 controls in a case-control study for the presence of ACA and LAC. All the patients had had a stroke under the age of 50 yr and the stroke date was less than 5 yr ago (mean 2.5 yr). Stroke was defined as an ischaemic cerebral infarction and was confirmed by angiography, CT-scan or MRI. An age- and sex-matched group of healthy volunteers served as controls. The mean age of the patients was 41.4 yr (range 22-52 yr), and of the controls 36.8 yr (range 24-50 yr). Serum and plasma from both groups was examined for IgM- and IgG-ACA and LAC. One patient was positive for both IgG- and IgM-ACA, whereas 3 controls were found positive for IgG-ACA. For 2 patients and 5 controls an equivocal result was obtained for IgG-ACA or IgM-ACA. None of the patients or controls were positive for LAC. The differences between the patient and control group were statistically not significant. In conclusion, no difference was found in the prevalence of cardiolipin antibodies in sera from patients with a stroke within the last 5 yr and an age- and sex-matched control group. There was no correlation either between the presence of lupus anticoagulant and the occurrence of a stroke in the past.

Improved specificity of ICA assays in the Fourth International Immunology of Diabetes Serum Exchange Workshop.

The goal of the Fourth International Workshop for Standardization of ICA Measurements was to determine the specificity of ICA assays and their ability to distinguish between control sera (n = 57) and sera from IDDM-related individuals--representing relatives of IDDM patients (n = 21), healthy individuals who later developed IDDM (n = 8), or newly diagnosed IDDM patients (n = 23). Results from 28 laboratories were analyzed. The mean specificity (percentage of control sera reported as negative) among 27 laboratories was 91%, including 6 laboratories with 100% specificity. Nevertheless, 78% of laboratories found at least one control sample > 0 JDF U. Among samples from first-degree relatives, the mean concordance was 86%, including three sera found negative (0 JDF U) by all laboratories. Among individuals who later developed diabetes, the mean concordance was 93%, with two sera found positive by 100% of laboratories. In sera from newly diagnosed IDDM patients, the mean concordance was 82%. Three sera were found positive and one serum negative by all laboratories. The JDF U of the sera considered to be positive were significantly greater than each laboratory's average for the controls. In conclusion, the results from laboratories participating in the Fourth International ICA Workshop demonstrated excellent specificity, good concordance, and an ability to separate control sera from defined, IDDM-related subjects.

Assessment of precision, concordance, specificity, and sensitivity of islet cell antibody measurement in 41 assays.

Forty-one assays were analysed at the 3rd International Workshop on the standardisation of islet cell antibodies. Analysis of precision demonstrated assays consistently detecting blind duplicates within one doubling dilution and capable of discriminating one doubling dilution differences in islet cell antibody concentration. Some assays, however, reported duplicates discrepantly by more than seven doubling dilutions, and consequently could not distinguish even large quantities of islet cell antibodies. Precision was best in assays from laboratories which had participated in all three Standardisation Workshops and was not dependent upon methodology. The use of the Juvenile, Diabetes Foundation reference islet cell antibody standard and standard curves reduced the scatter of results, and was best amongst assays with better precision. Twenty-seven assays reported all ten blood donor sera as negative. However, 14 assays did not, and specificity (negativity in health) was less than 50% in three assays. Low specificity was strongly associated with poor precision. The detection limit of assays ranged from less than 5 to 50 JDF units and was partially dependent upon methodology. Assays incorporating extended incubation had the lowest detection limits without a decrease in the specificity of the ten blood donor sera. Precise quantification is fundamental for the standardisation and comparability of islet cell antibodies. Precise quantitative assays have been identified and reference standards and common units established.

No evidence for the enhanced production of insulin autoantibodies after confrontation with common viral antigens in insulin dependent diabetes mellitus.

The production of insulin autoantibodies (IAA) was studied after common viral infections in 12 children with type 1 diabetes mellitus and in their 18 healthy siblings. In addition, the production of IAA was measured after influenza vaccination with booster in 39 patients with type 1 diabetes mellitus and in 39 healthy controls. In 7 of the 12 diabetic children 13 viral infections were serologically confirmed. Among the siblings 14 periods of infection were noted in 9 individuals. A significant rise in IAA antibody titre was demonstrated in patients twice (IgG both times) and in siblings 11 times (IgM 5x, IgG 6x, difference significant P less than 0.05). In only three cases the rise in antibody titres occurred 6-12 wk after documented infection. There was a significant inverse correlation with age in both patients (r = 0.89, P less than 0.0001) and siblings (r = 0.67, P less than 0.001) for IgM IAA. After influenza vaccination a significant increase in IAA was noted twice: IgM IAA in a patient with diabetes and IgG IAA in a healthy volunteer. A four-fold decrease in IgG IAA was demonstrated in one diabetic patient. From these results it is concluded that IAA formation is not a direct sequela of viral infection or vaccination.

Ten-year follow-up study of islet-cell antibodies and childhood diabetes mellitus.

To find out whether subclinical autoimmunity precedes onset of nonfamilial insulin-dependent diabetes mellitus (IDDM), 4806 schoolchildren aged 5-19 years from a township in Holland were followed-up for at least ten years after blood was sampled for measurement of islet-cell antibodies (ICA). ICA positivity conferred a relative risk of IDDM of 533 (95% CI 145-1955). In the 10 years of follow-up 4 of the 8 ICA-positive subjects became insulin dependent, whereas the probability of being free of IDDM was 99.9% for those who were ICA-negative at the start of the study. The findings suggest that, although chronic autoimmunity involving the pancreatic beta-cells may precede non-familial IDDM by many years, a positive ICA test on a single occasion predicts the development of IDDM in only 4 out of 8 subjects over a period of 10 years.

Recombinant gamma interferon induces class II major histocompatibility complex antigens on insulinoma cells.

Aberrant expression of major histocompatibility (MHC) antigens has been implicated as a factor contributing to organ-specific autoimmunity, such as progressive loss of pancreatic beta cells in type 1 diabetes. We investigated the potential of a rat beta cell tumour, RINM5F, to express enhanced levels of MHC antigens in vitro. To this purpose we treated RINM5F cells in vitro with recombinant rat gamma interferon (rIF gamma). We used monoclonal antibodies to RT1.A (class I) and RT1.B (class II) antigens of the rat MHC in conjunction with flowcytometry and immunoperoxidase techniques to analyse the expression of MHC antigens. Untreated RINM5F cells express low levels of RT1.A, whereas they are negative for RT1.B. Treatment with rIF gamma appeared to increase the expression of RT1.A antigens substantially. Most importantly, RT1.B antigens were newly expressed by rat insulinoma cells in vitro after treatment with rIF gamma. To our knowledge this is the first documentation of the potential of beta cells or their derivatives to express class II MHC antigens following IF gamma-treatment. This mechanism may play an important role in the augmentation and perpetuation of insulitis leading to type 1 diabetes mellitus.

Prediction of type 1 diabetes mellitus--a report on three cases.

In three children (patients 1, 2 and 3) insulin-dependency was predicted 28, 32 and 4 months, respectively before the disease became clinically manifest, by the finding of islet cell antibodies at that time. These retrospective findings support the evidence for a long pre-diabetic phase in childhood diabetes, marked by the presence of islet cell antibodies, as well as the linkage of HLA-antigens to the susceptibility to this disease. The possibility of detecting pre-diabetic states in children before the endogenous insulin secretion decreases to the point of producing clinical symptoms support efforts by basic scientists to develop techniques for immunological intervention early in the course of the disease.

Rheumatoid factors in rheumatoid arthritis and vasculitis.

To study the occurrence of rheumatoid factors (RF) in relation to the activity of rheumatoid arthritis and the occurrence of vasculitis, RF of IgM, IgA, and IgG classes were measured in sera from 35 patients with definite or classic rheumatoid arthritis (RA) using ELISA. For 26 patients, the RF levels were studied longitudinally and compared with changes in the articular index. Although IgM RF was occasionally found in patients without RA, IgA and/or IgG RF were almost exclusively associated with RA. The titers of IgM, IgA, and IgG RF were significantly higher in sera from patients with clinically diagnosed rheumatoid vasculitis than in sera from patients without vasculitis. No significant correlation between changes in the articular index and changes in titer of any class-specific RF could be found for the group of RA patients as a whole. However, in individual patients, increases or decreases in IgM and IgG RF titer were significantly correlated with an increase or decrease in the articular index.

Analysis of islet cell antibodies on frozen sections of human pancreas.

The sensitivity and specificity of the assay for islet cell cytoplasmic antibodies in human serum were examined using cryostat sections from fresh frozen pancreas. The specificity of the assay was close to 100% while the sensitivity was 40%-98% depending on the pancreas used. Inter-observer variation was 12-27%. End-point titres of islet cell antibodies varied with the sensitivity of each pancreas. End-point titration of the antibodies in two different laboratories using the same pancreas was significantly correlated (Spearman test p less than 0.001). We conclude that a reliable determination of islet cell antibody titres in human serum requires careful characterization of the sensitivity and specificity of each pancreas used as a source of frozen sections, in the indirect immunofluorescence assay.

Naturally occurring leukemia viruses in H-2 congenic C57BL mice. II. Antibody response to viral envelope antigens.

In C57BL mice milk-borne infection with B-tropic murine leukemia virus (V+ denoting positive for milk-transmitted B-tropic virus and V- denoting negative for milk-transmitted B-tropic virus) was accompanied by an antibody response against viral envelope antigens (VEA). Milk transmission of virus led to higher virus titers and lymphoma incidence in B10.A (H-2a) mice than in B10 (H-2b) mice, and the latter strain produced higher titers of anti-VEA antibodies than did the former. H-2 control of the antivirus-antibody response was confirmed in the (B10.A V+ X B10 V+)F2 cross. B10 V+ mice produced both IgM and IgG antibodies, whereas in the sera of B10.A V+ mice only IgM antibodies were demonstrable. The production of IgG and high-titer IgM antibodies to VEA was dominant in (B10.A V+ X B10 V+)F1 animals. The failure of B10.A V+ mice to produce IgG antibodies against VEA was not due to an intrinsic defect of helper T-cell function because these mice produced IgG antivirus antibodies after sc immunization with killed viral vaccine. Furthermore, in B10.A mice without milk-transmitted virus (B10.A V- subline), expression of genetically transmitted virus upon aging was associated with the production of IgG antibodies to VEA. The combined data were compatible with the existence of an H-2-linked dominant immune-response gene regulating the antibody response to milk-transmitted murine leukemia virus.

Spontaneous release of Fc receptor-like material from human lymphoblastoid cell lines.

In the culture medium of some human lymphoblastoid cell lines material is released with the following properties: (a) hemagglutination reaction of IgG-sensitized erythrocytes; (b) enhancement of precipitation of DNA-anti-DNA complexes; (c) inhibition of binding of C1q to immune complexes; (d) inhibition of immune complex binding to lymphocytes; (e) inhibition of antibody-dependent lymphocytotoxicity. The material is not identical with C1q or rheumatoid factor, it is heat resistant (30 min at 56 degrees C); the molecular weight is about 100 000 daltons and it is capable of inhibiting antibody production in vitro. It is suggested that this material consists of Fc receptors spontaneously shed from lymphocyte membranes.

Interaction of human lymphocytes with fluid phase human C3b detected by immunofluorescence.

Human lymphocytes obtained from tonsils and peripheral blood were found to bind human fluid phase C3b, obtained by trypsin treatment. This binding was detected by indirect immunofluorescence (IIF) using specific anti-C3 antisera. Lymphocytes isolated from thymus tissue scored low percentages in IIF, indicating that the main population of thymus-derived lymphocytes are T cells. The distribution pattern of C3b-binding cells was compared with that of cells forming rosettes with sheep erythrocytes coated with antibody and complement (EAC) and with sheep erythrocytes (E) only, as well as with that of Ig-bearing lymphocytes, as detected by direct immunofluorescence. It appeared that the distribution pattern of lymphocytes which can bind fluid phase C3b is similar to that of EAC rosette-forming and of Ig-bearing lymphocytes. Pre-incubation of the lymphocytes with C3b and pretreatment of the cells with trypsin decreased the capacity to form rosettes and to bind C3b to their surface. Human monocytes granulocytes and erythrocytes did not bind fluid phase C3b, as judged by IIF. Therefore, the selective binding of fluid phase C3b to lymphocytes provides a specific method for the detection of complement-reactive lymphocytes in lymphoid cell preparations.