G. F. Bottazzo

Combined Analysis of Autoantibodies Improves Prediction of IDDM in Islet Cell Antibody-Positive Relatives

Prediction of insulin-dependent diabetes mellitus (IDDM) is still largely based on islet cell antibodies (ICAs), but it may be improved by combined analysis with other humoral markers. We examined autoantibodies to insulin (IAAs), glutamic acid decarboxylase (GAD), and Mr 37,000 and Mr 40,000 fragments of islet antigens (37 and 40 kDa) together with ICA subtypes in 101 family members with ICAs ≥10 Juvenile Diabetes Foundation units (JDF U) followed for up to 14 years, of whom 18 have developed IDDM. Life-table analysis showed a 43% risk of IDDM within 10 years for those with ICAs ≥10 JDF U, rising to 53% for those with ICAs ≥20 JDF U. The risk for ICAs ≥10 JDF U was 62% in the family members in the youngest age quartile (<13.2 years) and fell with increasing age to 4% in those >40.7 years of age (P = 0.03). ICAs ≥10 JDF U combined with IAAs gave a risk of 84% (P = 0.03 compared with IAA−), and ICAs ≥10 JDF U combined with GAD antibodies gave a risk of 61% (P = 0.018). The risk for ICAs >10 JDF U with antibodies to 37-kDa antigen was 76% (P < 0.0001). Risk increased with the number of autoantibodies, from 8% for ICAs alone to 88% with >3 autoantibodies (14 cases detected) (P < 0.0001). The increased risk associated with multiple antibodies was observed independent of age. The median time to diagnosis in those with antibodies to 37- and/or 40-kDa antigen was 1.5 years, compared with 7.2 years in those with IAAs and GAD antibodies in the absence of antibodies to 37/40 kDa. The intensity and range of the autoantibody response offers better overall prediction of diabetes than any single autoantibody specificity, although antibodies to 37-/40-kDa antigens may prove to be useful markers of early clinical onset. We found that 78% of future cases of IDDM in ICA+ relatives came from the 27% with multiple autoantibodies and estimate that 88% of individuals within this category will need insulin treatment within 10 years. We propose a simple predictive strategy based on these observations.

Quantification of islet-cell antibodies and prediction of insulin-dependent diabetes

The sensitivity and predictive value of islet-cell antibodies (ICA) for the future onset of insulin-dependent diabetes mellitus (IDDM) were determined in 719 first-degree relatives of IDDM patients. ICA were quantified in Juvenile Diabetes Foundation (JDF) units, by indirect immunofluorescence in serum samples taken during prospective follow-up of up to 10.5 years. The threshold of ICA detection was 4 JDF units. ICA were detected in the first sample of 26 (3.3%) of the relatives, compared with 12 (2.2%) of 540 controls (298 blood donors and 242 healthy children). ICA were detected in follow-up samples from a further 14 relatives. IDDM developed in 14 (35%) of the 40 relatives with detectable ICA at any time and in 2 (0.3%) relatives without detectable ICA. In all 5 relatives with peak ICA levels above 80 JDF units IDDM developed within follow-up of 7 years; survival without IDDM at 10 years was 27% among relatives with peak ICA levels of 20-80 JDF units and 82% for peak ICA levels of 4-20. The predictive value for IDDM development within 10 years ranged from 40% (threshold 4 JDF units) to 100% (80 JDF units) and the sensitivity from 31% (80 JDF units) to 88% (4 JDF units).

Islet autoantibody markers in IDDM : risk assessment strategies yielding high sensitivity

SummaryIdentification of islet autoantigens offers the possibility that antibody tests other than islet cell antibodies may be used for assessing risk of insulin-dependent diabetes mellitus (IDDM). The aim of this study was to determine the combination of islet autoantibody markers that could identify most future cases of IDDM. Islet cell antibodies, antibodies to glutamic acid decarboxylase (GAD)65, 37,000/ 40,000 Mr islet tryptic fragments, carboxypeptidase-H, and islet cell autoantigen (ICA)69 were measured in sera from 100 newly-diagnosed IDDM patients, 27 individuals prior to onset of IDDM, and 83 control subjects. Islet cell antibodies were detected in 88 % of IDDM patients and 81 % with pre-IDDM, GAD65 antibodies in 70 % of IDDM patients and 89 % with pre-IDDM, and antibodies to 37,000/40,000 Mr islet tryptic fragments in 54 % of IDDM patients and in 48 % with pre-IDDM. The latter were found only in conjunction with islet cell antibodies and were more frequent in young onset cases. All 20 IDDM patients and the 3 pre-IDDM subjects who had islet cell antibodies without GAD65 antibodies had antibodies to 37,000/40,000 Mr islet tryptic fragments, and all but one had disease onset before age 15 years. No sera strongly immunoprecipitated in vitro translated ICA69 or carboxypeptidase-H; 4 % of patients had anti-ICA69 and 11 % anti-carboxypeptidase-H levels above those of the control subjects. The findings suggest that none of the single antibody specificities are as sensitive as islet cell antibodies, but that a combination of GAD65 antibodies and antibodies to 37,000/40,000 Mr islet tryptic fragments has the potential to identify more than 90 % of future cases of IDDM. Such a strategy could eventually replace islet cell antibodies in population screening for IDDM risk assessment.

Insulin autoantibodies in the pre-diabetic period: Correlation with islet cell antibodies and development of diabetes

SummaryIgG and IgM class insulin autoantibodies were measured by an enzyme-linked immunosorbent assay in sera from members of the Barts-Windsor-Middlesex prospective family study for Type 1 (insulin-dependent) diabetes. One hundred and twelve individuals from 28 families were selected for study on the basis of a clearly defined islet cell antibody status. IgG insulin autoantibodies were found to be significantly associated with islet cell antibody positive (n=30) versus islet cell antibody negative (n= 57) first degree family relatives (p=0.002), with increased significance (p=0.0003) if complement-fixing (CF)-islet cell antibody individuals (n=20) only were considered. In addition, a significant association of IgG insulin autoantibodies with subsequent development of diabetes was observed within the CF-islet cell antibody positive group (p<0.0003). No such associations were found for IgM insulin autoantibodies, but a higher prevalence of these autoantibodies was observed in islet cell antibody negative first degree relatives (n=57) compared with a control group of 73 Blood Bank donors (p=0.00007), and they were significantly associated with siblings (n=48) rather than parents (n=39), (p=0.001). We conclude that the presence of IgG insulin autoantibodies and CF-islet cell antibodies confer more risk for future development of diabetes than the presence of either marker alone.

Distinct cytoplasmic islet cell antibodies with different risks for type 1 (insulin-dependent) diabetes mellitus.

SummaryThe cytoplasmic islet cell antibody patterns of sera from islet cell antibody positive non-diabetic and diabetic endocrine autoimmune patients, and newly-diagnosed Type 1 (insulin-dependent) diabetic patients were characterised using four layer immunofluorescence with monoclonal antiproinsulin or anti-glucagon antibodies. Two distinct islet cell antibody types were identified. One gave a diffuse cytoplasmic staining in both Beta and Alpha cells (‘whole’ islet pattern), and was not affected by pre-incubation with rat brain homogenate. The other had a granular appearance with staining restricted predominantly to Beta cells (‘selective’ islet pattern) and was completely inhibited by pre-incubation with rat brain homogenate. Some sera appeared to have a ‘mixed’ islet pattern, in which glucagon-positive cells gave a weaker cytoplasmic staining than proinsulin-positive cells. The granular ‘selective’ pattern was found in sera from 19 (79%) of 24 non-diabetic endocrine autoimmune patients, in two (22%) endocrine autoimmune patients who developed Type 1 diabetes (p<0.0001 vs non-diabetic endocrine autoimmune patients), and in none of 19 newly-diagnosed diabetic patients. The ‘whole’ islet pattern was found only in sera from patients who had, or who subsequently progressed to, Type 1 diabetes. This study has identified a novel islet cell antibody specificity and demonstrates that in islet cell antibody positive endocrine autoimmune patients, only islet cell antibodies which stain both Beta and Alpha cells are associated with progression to Type 1 diabetes.

Analysis of apoptosis in relation to tissue destruction associated with Hashimoto's autoimmune thyroiditis

The level of apoptosis has been investigated in thyroid tissue from eight patients with Gravess disease, one with Hashitoxicosis, three with Hashimotos thyroiditis, and five patients with multinodular goitre, using flow cytometry and an in situ immunofluorescence technique. Cryostat sections have also been studied for Bcl‐2 and APO‐1/Fas expression in the thyrocytes and infiltrating lymphocytes, to determine their susceptibility to apoptosis. An increased level of apoptosis was detected in Hashimotos glands. This was associated with decreased Bcl‐2 staining and a patchy APO‐1/Fas reactivity on thyrocytes. In addition, APO‐1/Fas expression was noted within the germinal centres of lymphoid follicles. It is suggested that the dysregulation of apoptosis‐related genes could be an important factor in the progression of destructive thyroid autoimmune disease.

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

SummaryForty-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 <50% in three assays. Low specificity was strongly associated with poor precision. The detection limit of assays ranged from <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.

Genetic prediction of type 1 diabetes in a population with low frequency of HLA risk genotypes and low incidence of the disease (the DIABFIN study)

To develop a sensitive, specific screening strategy for predicting genetic risk for type 1 diabetes mellitus (T1DM) in the low‐incidence continental Italian population, and to define with this tool, a cohort of high‐to‐moderate risk infants for an immunological follow‐up study aimed at identifying environmental risk factors for T1DM.

Slow metabolic deterioration towards diabetes in islet cell antibody positive patients with autoimmune polyendocrine disease

SummaryWe studied metabolic progression to IDDM in a cohort of adults who are ICA-positive and have associated autoimmune endocrine disease or circulating organ-specific autoantibodies (the Polyendocrine Study). Of the 186 individuals recruited 27 developed overt diabetes after a median follow-up of 4.5 years (range 0.4–12). Of these, eight patients did not require insulin treatment until at least 6 months after clinical diagnosis, with an interval of 1.8 years (1.2–5.7). An IVGTT was performed in 38 subjects and 23 had sequential studies. Of the initial 38 subjects six developed diabetes and only three showed a loss of FPIR to glucose (below the first percentile of a normal control group) before clinical onset of the disease. An additional three subjects showed a loss of the FPIR, and all still have normal glucose tolerance after median follow-up of 28 months (22–95). A “whole” or “mixed” pattern of islet cell staining was found in five of the six patients who developed diabetes and antibodies against an islet 37 k-antigen were detectable in four patients, all of whom required insulin soon after diagnosis. A beta-cell “selective” ICA staining pattern was seen in 14 of 17 subjects who did not develop diabetes and the “mixed” pattern in only three. None of this group had detectable 37k-antibodies. We conclude that metabolic deterioration is slow in polyendocrine patients, and that the IVGTT has less prognostic significance in this group than in first degree relatives of patients with IDDM. In contrast, the presence of the “whole” or “mixed” ICA staining pattern or of 37k-antibodies can identify a high risk of progression to IDDM within this polyendocrine population and may indicate the rate of metabolic deterioration.

Early T-cell defects in pre-type 1 diabetes

Alterations of lymphocyte subsets have been recently reported in pre-type 1 diabetes but the relation with other immunological markers, in particular islet cell antibodies (ICA), is still unknown. In the present study, we have investigated prospectively changes of lymphocyte subsets in 86 first-degree relatives of patients affected by type 1 diabetes and correlated such modifications with ICA titres. Among individuals with persistent ICA, 8 had ICA titres of more than 20 JDF units, 14 had ICA titres between 5 and 20 JDF units and 64 had ICA titres between 0 and 5 JDF units. First-degree relatives with ICA titres of more than 20 JDF units had significantly decreased proportions of CD3 cells. This reduction was predominantly in the CD4 subset, giving rise to a decreased CD4/CD8 lymphocyte ratio. Those with ICA titres between 5 and 20 JDF units showed abnormalities in both CD3 and CD4 lymphocytes, but not in CD4/CD8 lymphocyte ratio. Further characterization of the CD4 cell subset was performed using three other monoclonal antibodies, CD45RO (UCHL1), CD45RA and CD29, phenotyping memory T-cells, the inducer cells of suppressor function and helper-inducer cells, respectively. The proportions of total CD45RO and CD45RA were not significantly different among first-degree relative with distinct ICA titres in a cross-sectional studt, whereas a trend towards a reduced proportion of CD4/ CD45RA cells was observed. The longitudinal study demonstrated that individuals potentially susceptible to the development of type 1 diabetes and who possess high titres of ICA have impairment of CD4/CD8 lymphocyte ratio, mainly due to a reduction in the CD4 subset. This alteration may contribute to the initial generation of the autoimmune response towards beta cells. All the calculations were made using minimum median and maximum value analyses.